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Role of RhoA and Rho-associated kinase in phenotypic switching of vascular smooth muscle cells: Implications for vascular function

  • Tedy Sawma
    Affiliations
    Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Lebanon
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Abdullah Shaito
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Biomedical Research Center, Qatar University, Doha, Qatar
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    1 These authors contributed equally to this work.
    Nicolas Najm
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Lebanon
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    1 These authors contributed equally to this work.
    Munir Sidani
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Lebanon
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  • Alexander Orekhov
    Affiliations
    Laboratory of Cellular and Molecular Pathology of Cardiovascular System, Institute of Human Morphology, Moscow, Russia

    Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Moscow, Russia

    Institute for Atherosclerosis Research, Skolkovo Innovative Center, Moscow, Russia
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  • Ahmed F. El-Yazbi
    Affiliations
    Faculty of Pharmacy, Alamein International University, Alamein, Egypt

    Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
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  • Rabah Iratni
    Affiliations
    Department of Biology, College of Science, United Arab Emirates University, Al-Ain, United Arab Emirates
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  • Ali H. Eid
    Correspondence
    Corresponding author.
    Affiliations
    Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar
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    1 These authors contributed equally to this work.

      Highlights

      • Phenotypic switch of vascular smooth muscle cells (VSMCs) contributes to vascular disease such as atherosclerosis.
      • Modulation of RhoA/ROCK-mediated VSMC contraction process is an integral part of VSMC phenotype switching.
      • RhoA/ROCK modulates proliferation, dedifferentiation, apoptosis and migration of VSMCs.
      • RhoA/ROCK signaling is a key player in arterial stiffness and atherosclerosis.

      Abstract

      Cardiovascular disease (CVD) continues to be the primary cause of global mortality. Vascular smooth muscle cells (VSMCs) are integral components of vascular structure and function, evident by their vital roles in modulating blood flow and pressure. Such roles exist due to the differentiated contractile phenotype of VSMCs. However, VSMCs may switch to a dedifferentiated, proliferative synthetic phenotype in a phenomenon known as phenotypic switching. This switch involves dramatic changes in VSMC migration, proliferation, gene expression programs, differentiation, cellular stiffness and extracellular matrix (ECM) deposition. In this review, we explore the role of the small GTPase Rho and its effector, Rho-associated kinase (ROCK), in phenotypic switching as well as apoptotic pathways in VSMCs. We critically dissect how RhoA promotes cell migration and proliferation as well as its role in modulating the expression of a battery of VSMC marker proteins. We also discuss how RhoA modulates apoptosis, induces dedifferentiation, increases vascular stiffness, or modifies ECM accumulation. These alterations in VSMC phenotypes contribute to multiple vascular dysfunctions, including hypertension and atherosclerosis. Understanding the molecular underpinnings and the signaling pathways involved in these altered phenotypes may provide novel avenues of drug design and other therapeutic interventions for the management of CVDs.

      Graphical abstract

      Keywords

      1. Introduction

      Proper vascular smooth muscle cell (VSMC) activity is essential for physiologic functioning of the vasculature, such as regulation of vasotone and blood flow. In healthy vasculature, VSMCs are contractile, stationary, and quiescent. In addition, VSMCs are considered differentiated and express contractile proteins, like smooth muscle α-actin (SM-α-actin), SM myosin heavy chain, calponin, SM 22α, smoothelin, and SM myosin light chain (MLC), among others [
      • Chen Y.
      • Su X.
      • Qin Q.
      • et al.
      New insights into phenotypic switching of VSMCs induced by hyperhomocysteinemia: role of endothelin-1 signaling.
      ,
      • Gomez D.
      • Owens G.K.
      Smooth muscle cell phenotypic switching in atherosclerosis.
      ,
      • Rensen S.S.
      • Doevendans P.A.
      • van Eys G.J.
      Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.
      ]. Despite the differentiated phenotype of VSMCs, they still show plasticity. VSMCs may shift from the contractile phenotype in response to stimuli in the interest of maintaining homeostasis of the vasculature. Importantly, VSMC phenotype switching is necessary during development and vascular remodeling for the formation and maturation of vessels [
      • Gomez D.
      • Owens G.K.
      Smooth muscle cell phenotypic switching in atherosclerosis.
      ]. While this phenotype switching is primarily physiological, it may, when dysregulated, contribute to vascular disease processes. For instance, intimal hyperplasia, a process underlying the pathogenesis of several diseases such as atherosclerosis, aortic aneurysm, and hypertension, involves VSMC dysfunctional phenotypic shifts [
      • Chen Y.
      • Su X.
      • Qin Q.
      • et al.
      New insights into phenotypic switching of VSMCs induced by hyperhomocysteinemia: role of endothelin-1 signaling.
      ,
      • Gomez D.
      • Owens G.K.
      Smooth muscle cell phenotypic switching in atherosclerosis.
      ,
      • Rzucidlo E.M.
      • Martin K.A.
      • Powell R.J.
      Regulation of vascular smooth muscle cell differentiation.
      ,
      • Petsophonsakul P.
      • Furmanik M.
      • Forsythe R.
      • et al.
      Role of vascular smooth muscle cell phenotypic switching and calcification in aortic aneurysm formation.
      ,
      • Zhuge Y.
      • Zhang J.
      • Qian F.
      • et al.
      Role of smooth muscle cells in Cardiovascular Disease.
      ]. Notably, the process of VSMC phenotypic switching is often one of the earliest manifestations of vascular diseases. Small conditional RNA (scRNA) studies have been useful in examining the transcriptome of VSMCs that undergo phenotypic transformation, as well as the factors that contribute to this change [
      • Conklin A.C.
      • Nishi H.
      • Schlamp F.
      • et al.
      Meta-analysis of smooth muscle lineage transcriptomes in atherosclerosis and their relationships to in vitro models.
      ]. During a diseased vascular state and upon phenotype switching, VSMCs dedifferentiate, exhibit abnormal gene expression profiles, assume a synthetic and proliferative phenotype. In addition, they become more migratory - especially to areas of cellular injury, prone to apoptosis, and prone to clustering as per scRNA studies [
      • Rensen S.S.
      • Doevendans P.A.
      • van Eys G.J.
      Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.
      ,
      • Badran A.
      • Nasser S.A.
      • Mesmar J.
      • et al.
      Reactive oxygen species: modulators of phenotypic switch of vascular smooth muscle cells.
      ,
      • Fardon M.M.
      • Dehaini H.
      • Baydoun E.
      • et al.
      Estrogen attenuates phenotypic switch of human arteriolar smooth muscle cells: role of p53 and pRb.
      ,
      • Rensen S.S.M.
      • Doevendans P.A.F.M.
      • van Eys
      • Gjjm
      Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.
      ]. Contextually, scRNA sequencing reveals that VSMC-derived foam cells display a propensity to undergo multiple forms of cell death, such as apoptosis, autophagy, necroptosis, and pyroptosis, all of which contribute to the structure and physio-pathology of atherosclerotic plaques [
      • Gui Y.
      • Zheng H.
      • Cao R.Y.
      Foam cells in atherosclerosis: novel insights into its origins, consequences, and molecular mechanisms.
      ].
      Given the involvement of VSMC phenotypic switching in the initial stages of vascular disease, it is prudent to elucidate the molecular pathways that regulate this process. A better understanding of the molecular mechanisms of this phenotypic switching may help shed light on potentially new therapeutic targets in the management of CVD. In context, this review analyzes the involvement of the small GTPase RhoA and its effector kinase, Rho-associated kinase (ROCK), in the various aspects of VSMC phenotypic switching.

      2. RhoA and ROCK regulate VSMC contractility

      VSMC relaxation and contraction are determined by the dephosphorylation and phosphorylation of MLC by myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK), respectively. Initially, contraction is mediated by an elevation of intracellular calcium (Ca2+), which leads to calmodulin-mediated activation of MLCK. Vascular contraction is then maintained by mechanisms that sensitize MLCK to Ca2+ signaling (Fig. 1).
      Fig. 1
      Fig. 1Activation of RhoA/ROCK pathway sensitizes VSMCs to calcium-mediated contraction.
      Calcium or upstream vasoactive signals can activate MLCK leading to MLC phosphorylation, which then induces polymerized F-actin to form actomyosin, the machinery needed for VSMC contraction. Vasoconstrictors can activate Rho/ROCK signaling which then increases MLC phosphorylation and the consequent VSMC. ROCK can phosphorylate and inactivate MLCP, thus increasing levels of phosphorylated MLC and thereby actomyosin-mediated VSMC contraction. Active ROCK can phosphorylate CPI-17, thus inhibiting MLCP leading to contraction. ROCK: Rho-associated kinase; MLCK: myosin light chain kinase; MLC: myosin light chain; MLCP: myosin light chain phosphatase.
      RhoA is a member of the small GTPase family that includes RhoA, Rac1, and cdc42. RhoA acts as a molecular switch due to its GTPase activity, which cycles RhoA between an active GTP-bound state and an inactive GDP-bound state (Fig. 1). RhoA activity is also modulated by activating guanine nucleotide exchange factors (GEFs), as well as inactivating GTPase activating proteins (GAPs). Principal effectors of RhoA include Rho-associated kinases or ROCKs [
      • Matsui T.
      • Amano M.
      • Yamamoto T.
      • et al.
      Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.
      ,
      • Kureishi Y.
      • Kobayashi S.
      • Amano M.
      • et al.
      Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.
      ], which robustly induce VSMC contraction by directly phosphorylating and inhibiting the activity of MLCP, an enzyme that attenuates VSMC contraction by dephosphorylating MLC [
      • Crestani S.
      • Webb R.C.
      • da Silva-Santos J.E.
      High-salt intake augments the activity of the RhoA/ROCK pathway and reduces intracellular calcium in arteries from rats.
      ]. Active ROCK can also indirectly inactivate MLCP through CPI-17, a phosphorylation-dependent regulatory protein that, when phosphorylated, inhibits the phosphatase subunit of MLCP. This leads to heightened MLC activation, VSMC contraction, and stress fiber formation (Fig. 1) [
      • Eto M.
      • Kitazawa T.
      • Matsuzawa F.
      • et al.
      Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor.
      ,
      • Kitazawa T.
      • Eto M.
      • Woodsome T.P.
      • et al.
      Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility.
      ].
      ROCK can also enhance VSMC contractility through direct action on MLC. Indeed, ROCK phosphorylates, and consequently sensitizes, MLC to Ca2+ signaling, which eventually results in a potentiated and more sustained VSMC contractile response (Fig. 1) [
      • Zhuge Y.
      • Zhang J.
      • Qian F.
      • et al.
      Role of smooth muscle cells in Cardiovascular Disease.
      ,
      • Kureishi Y.
      • Kobayashi S.
      • Amano M.
      • et al.
      Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.
      ,
      • Crestani S.
      • Webb R.C.
      • da Silva-Santos J.E.
      High-salt intake augments the activity of the RhoA/ROCK pathway and reduces intracellular calcium in arteries from rats.
      ,
      • Wang Y.
      • Zheng X.R.
      • Riddick N.
      • et al.
      ROCK isoform regulation of myosin phosphatase and contractility in vascular smooth muscle cells.
      ,
      • Calò L.A.
      • Davis P.A.
      • Pagnin E.
      • et al.
      Increased level of p63RhoGEF and RhoA/Rho kinase activity in hypertensive patients.
      ]. Thus, RhoA/ROCK-mediated prolongation of MLC action underlies the increased contraction, and potentially heightened vasoconstriction [
      • Zhuge Y.
      • Zhang J.
      • Qian F.
      • et al.
      Role of smooth muscle cells in Cardiovascular Disease.
      ,
      • Crestani S.
      • Webb R.C.
      • da Silva-Santos J.E.
      High-salt intake augments the activity of the RhoA/ROCK pathway and reduces intracellular calcium in arteries from rats.
      ].
      Modulation of RhoA/ROCK-mediated VSMC contraction process is an integral part of VSMC phenotype switching. Indeed, VSMC contraction and the involved actin polymerization have been intrinsically shown to modulate VSMC migration and stiffness. Of prime importance to this review is the signaling which, downstream of ROCK, contributes to pathological VSMC phenotypic switching. We henceforth dissect the signaling molecules associated with RhoA/ROCK signaling in the context of pathological phenotypic switching, specifically in relation to VSMC migration, proliferation, gene expression, apoptosis, dedifferentiation, ECM deposition, and vascular stiffness.

      3. Mechanisms of RhoA/ROCK-mediated VSMC migration

      VSMC migration involves remodeling of the cytoskeleton in response to signals that emanate from cell surface receptors. During VSMC migration, reorganization of the actin cytoskeleton occurs and causes protrusion of the leading edge, along either a path of variable adhesion to the ECM or in the direction of a chemotactic cue [
      • Gerthoffer W.T.
      Mechanisms of vascular smooth muscle cell migration.
      ]. Instantly, new focal adhesions form in the vicinity immediate to the leading edge, resulting in enhanced adhesion of the cell membrane to the ECM [
      • Gerthoffer W.T.
      Mechanisms of vascular smooth muscle cell migration.
      ]. Actomyosin molecules within the cytoplasm cause contraction that, in addition to remodeling of the cytoskeleton and the disassembly of focal adhesions at the trailing edge, moves the cell toward its destination [
      • Gerthoffer W.T.
      Mechanisms of vascular smooth muscle cell migration.
      ].
      Migration of VSMCs is important for many physiological processes, prime of which are vascular development and the maintenance of vascular integrity [
      • Gerthoffer W.T.
      Mechanisms of vascular smooth muscle cell migration.
      ]. Dysregulation of VSMC migration is equally as important in the pathogenesis of vascular diseases, such as the response to vascular injury or atherogenesis [
      • Gerthoffer W.T.
      Mechanisms of vascular smooth muscle cell migration.
      ]. For instance, migration of VSMCs from the medial to intimal layers of vessels is at the heart of atherosclerosis and vascular injury or restenosis [
      • Gomez D.
      • Owens G.K.
      Smooth muscle cell phenotypic switching in atherosclerosis.
      ,
      • Chava K.R.
      • Karpurapu M.
      • Wang D.
      • et al.
      CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.
      ].
      The upstream signals that activate the process of VSMC migration, as well as the downstream effectors, have been extensively studied. Chemotactic factors involved include platelet-derived growth factor-BB (PDGF-BB), angiotensin II (Ang II), thrombin, and tumor necrosis factor-α (TNF-α), among others [
      • Gomez D.
      • Owens G.K.
      Smooth muscle cell phenotypic switching in atherosclerosis.
      ,
      • Chava K.R.
      • Karpurapu M.
      • Wang D.
      • et al.
      CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.
      ]. These molecules can stimulate VSMC migration not only during development, but also under physiological and pathological states. The Rho/ROCK pathway is a major player in the signaling implicated in these states. In order to reasonably limit the scope of this review, the discussion will be limited to upstream signaling by Ang II, PDGF-BB, and asymmetric dimethyl arginine (ADMA) that culminate in RhoA/ROCK-mediated VSMC migration.
      Angiotensin II is a vasoactive peptide involved in the homeostasis of vascular volume and pressure. Upon binding to and activating its receptor (AT1 receptor), Ang II triggers the activation of several downstream effectors, including Rho/ROCK (Fig. 2), which then results in polymerization of G (globular)-actin into F (fiber)-actin. Paradoxically, this polymerization will downregulate contractile proteins [
      • Qi Y.
      • Liang X.
      • Dai F.
      • et al.
      RhoA/ROCK pathway activation is regulated by AT1 receptor and participates in smooth muscle migration and dedifferentiation via promoting actin cytoskeleton polymerization.
      ]. Notably, contractile protein downregulation induces VSMC remodeling, an indispensable step in the initiation of VSMC migration. In this context, inhibitors of AT1 receptor, RhoA, or ROCKs have been shown to suppress migration [
      • Qi Y.
      • Liang X.
      • Dai F.
      • et al.
      RhoA/ROCK pathway activation is regulated by AT1 receptor and participates in smooth muscle migration and dedifferentiation via promoting actin cytoskeleton polymerization.
      ].
      Fig. 2
      Fig. 2Upstream signals that promote VSMC migration like include Ang II, PDGF-BB or ADMA, which act by stimulating the RhoA/ROCK pathway.
      ERK and JNK MAPKs can be activated by ROCK. These kinases can translocate to the nucleus to activate transcription factors like c-jun (activated by JNK), thereby modulating expression of specific genes implicated in cell migration. Ang II binding to the AT1R receptor also activates RhoA/ROCK which then activates the p38-Syk-Src kinase cascade, eventually promoting migration. In addition, RhoA/ROCK can act by phosphorylating and thus inhibiting MLCP. This renders MLC active, and facilitates actin polymerization and actomyosin assembly necessary for VSMC contraction and migration. In the urokinase pathway, uPA binds to uPAR at the leading fronts forming a complex along with TYK2 and PI3K. This complex then recruits RhoA to the leading fronts resulting in its activation. RhoA will then activate ROCK which, through MLCP inhibition, results eventually in actin polymerization and consequent migration. Finally, RhoA, independently of ROCK, can activate mDia1, leading to polymerization of actin filaments and filopodia formation, an initial step in VSMC migration. Ang II: angiotensin II; PDGF-BB: platelet derived growth factor-BB; ADMA: asymmetric dimethyl arginine; ROCK: Rho-associated kinase; ERK: extracellular signal-regulated kinase; JNK: c-Jun N-terminal kinase; MAPK: mitogen activated protein kinase; AT1R: angiotensin II receptor type 1; Syk: spleen tyrosine kinase, uPA: urokinase-type plasminogen activator; uPAR: uPA receptor; MLC: myosin light chain; MLCP: myosin light chain phosphatase; mDia 1: mammalian diaphanous protein 1.
      Ang II can also stimulate VSMC migration by a different mechanism. Ang II can activate p38 MAPK, a downstream target of RhoA/ROCK and a member of the MAPK family of kinases [
      • Muniyappa R.
      • Xu R.
      • Ram J.L.
      • et al.
      Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells.
      ]. The involvement of RhoA/ROCK/p38 in VSMC migration warrants further investigation, and the idea currently established is still an evolving model. For example, accumulating evidence points to RhoA/ROCK activation of p38 as one driver of VSMC migration. Current evidence for potential p38 involvement in VSMCs migration includes the fact that activated p38 can activate c-Src protein production through Syk (spleen tyrosine kinase), leading to cell migration (Fig. 2) [
      • Mugabe B.E.
      • Yaghini F.A.
      • Song C.Y.
      • et al.
      Angiotensin II-induced migration of vascular smooth muscle cells is mediated by p38 mitogen-activated protein kinase-activated c-Src through spleen tyrosine kinase and epidermal growth factor receptor transactivation.
      ]. Another piece of evidence shows that p38 activates Mitogen Activated Protein Kinase 2/3 (MAPKAPK 2.3), which then phosphorylates heat-shock protein 27 (HSP 27) leading eventually to VSMC migration [
      • Huang J.
      • Xie L.D.
      • Luo L.
      • et al.
      Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro.
      ] (Fig. 3). In addition, p38 can phosphorylate and activate paxillin, a key protein involved in focal adhesion dynamics [
      • Huang C.
      • Jacobson K.
      • Schaller M.D.
      MAP kinases and cell migration.
      ]. These pathways could possibly be Rho/ROCK dependent, since p38 activation is a direct downstream event of Rho/ROCK activation [
      • Muniyappa R.
      • Xu R.
      • Ram J.L.
      • et al.
      Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells.
      ]. Given that blocking p38 inhibits VSMC migration [
      • Chava K.R.
      • Karpurapu M.
      • Wang D.
      • et al.
      CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.
      ,
      • Chava K.R.
      • Karpurapu M.
      • Wang D.
      • et al.
      CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.
      ], by association, we can postulate that activation of p38 by Ang II, via the RhoA pathway, could promote migration of VSMCs. Nevertheless, this notion remains to be fully elucidated.
      Fig. 3
      Fig. 3Extracellular signals such as VEGF, FGF and fMLP can activate p38. RhoA/ROCK may possibly mediate this activation in VSMCs. Once activated, p38 can mediate cell migration via three different pathways.
      p38 can activate MAPKAPK 2/3 which then phosphorylates and inactivates HSP27, a heat-shock protein needed for cell migration. Phosphorylated HSP27 can no longer inhibit actin polymerization, thus blocking VSMC migration. MAPKAPK 2/3 may also activate p-16 Arc and 5 lipoxygenase leading to cell migration. In addition, p38 can also activate paxillin, a protein that is involved in focal adhesion dynamics facilitating the process of cell migration. Finally, p38 can activate caldesmon, which induces the urokinase pathway allowing VSMC migration. VEGF: vascular endothelial growth factor; FGF: fibroblast growth factor; fMLP: N-formyl-methionyl-leucyl-phenylalanine; ROCK: Rho-associated kinase; HSP27: heat shock protein 27; MAPKAP 2/3: mitogen activated protein kinase activated protein kinase 2/3; p-16 Arc: Arp2/3 complex 16 kDa subunit. RhoA/ROCK also activates the MEK/ERK pathway in VSMCs. Such activation may lead to the stimulation of four important mediators: MLCK, calpain, FAK and paxillin. Activated MLCK phosphorylates and activates MLC leading to actin polymerization, an important step in VSMC migration. MLCK is also directly involved in the process of focal adhesion turnover. FAK is phosphorylated and activated by MEK/ERK. Activated FAK can interact with activated paxillin, an interaction implicated in focal adhesion turnover. Also, activated FAK can interact with activated calpain. This will allow calpain to degrade cytoskeletal proteins at the focal adhesion facilitating for focal adhesion turnover. This continuous turnover is essential for appropriate cell migration. ROCK: Rho-associated kinase; FAK: focal adhesion kinase; ERK: extracellular signal-regulated kinase; MEK: MAPK/ERK kinase; MAPK: mitogen activated protein kinase; MLC: myosin light chain; MLCK: myosin light chain kinase.
      Evidence suggests that p38-mediated phosphorylation of caldesmon, a thin filament binding protein, plays a role in urokinase-stimulated VSMC migration (Fig. 3) [
      • Goncharova E.A.
      • Vorotnikov A.V.
      • Gracheva E.O.
      • et al.
      Activation of p38 MAP-kinase and caldesmon phosphorylation are essential for urokinase-induced human smooth muscle cell migration.
      ]. Importantly, urokinase-type plasminogen activator (uPA) itself can lead to the activation of RhoA, leading to VSMC migration. Interestingly, this uPA-mediated migratory pathway is unique to VSMCs. uPA stimulation of cell migration is done via binding and activation of uPA receptor (uPAR) signaling (Fig. 2). Active uPAR complexes tend to form at the leading fronts of VSMC membranes and contain the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3K) [
      • Kusch A.
      • Tkachuk S.
      • Haller H.
      • et al.
      Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2 interaction.
      ]. The active form of RhoA is recruited to VSMC leading fronts as well, where it associates with the uPAR complex [
      • Kiian I.
      • Tkachuk N.
      • Haller H.
      • et al.
      Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signaling pathway.
      ]. RhoA then activates ROCK, which phosphorylates MLC, leading to VSMC migration. However, the mechanism by which RhoA becomes recruited and activated by the uPAR complex remains poorly understood.
      p38 can also mediate VSMC migration induced by several upstream effectors. For example, pathologically elevated cyclic strain can induce VSMC migration and proliferation by suppressing Rho-GDIα (Rho GDP dissociation inhibitor alpha)- a negative regulator of several Rho family GTPases. This subsequently induces phosphorylation of Rac1 and p38, which, we hypothesize could possibly be due to increased activity of RhoA [
      • Qi Y.X.
      • Qu M.J.
      • Yan Z.Q.
      • et al.
      Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho-GDIalpha, Rac1, and p38 pathway.
      ]. Consistent with this, pharmacological inhibition of p38, RhoA, or ROCK suppressed sphingosylphosphorylcholine-induced VSMC migration [
      • Wang H.H.
      • Nakamura A.
      • Matsumoto A.
      • et al.
      Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine.
      ,
      • Li S.
      • Tanaka H.
      • Wang H.H.
      • et al.
      Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation.
      ].
      PDGF-BB is another upstream ligand that can trigger RhoA/ROCK-mediated VSMC migration. PDGF-BB is a growth factor known to regulate growth and proliferation in cells of mesenchymal origin, including glial cells, fibroblasts and VSMCs [
      • Heldin C.H.
      Structural and functional studies on platelet-derived growth factor.
      ]. It is a potent mitogen and chemoattractant of VSMCs and was shown to be secreted in detectable quantities during atherogenesis and, as a result, was suggested to mediate this pathology by virtue of its effects on VSMC migration and proliferation [
      • Raines E.W.
      PDGF and cardiovascular disease.
      ]. This is especially since PDGF‐BB activates ROCK and MLCK, both of which can then regulate MLC activity (Fig. 1) [
      • Wang J.
      • Zhang C.
      • Li C.
      • et al.
      MicroRNA-92a promotes vascular smooth muscle cell proliferation and migration through the ROCK/MLCK signalling pathway.
      ].
      Binding of PDGF-BB to its surface receptors on VSMCs can activate both ROCK isotypes, ROCK 1 and ROCK 2 [
      • Hartmann S.
      • Ridley A.J.
      • Lutz S.
      The function of rho-associated kinases ROCK1 and ROCK2 in the pathogenesis of cardiovascular disease.
      ]. However, only ROCK 1 stimulates the nuclear translocation of p42/44 MAPK, also known as ERK1/2, a key MAPK involved in cellular proliferation and migration (Fig. 2) [
      • Zhao Y.
      • Lv M.
      • Lin H.
      • et al.
      ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells.
      ]. Nucleus-localized ERK1/2 can phosphorylate a set of transcription factors that include c-fos and c-myc leading to the downregulation of the expression of numerous differentiation-specific contractile proteins. This ERK-mediated cytoskeletal remodeling eventually precipitates VSMC migration [
      • Zhao Y.
      • Lv M.
      • Lin H.
      • et al.
      ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells.
      ].
      ERK1/2 can mediate cell migration through other pathways [
      • Huang C.
      • Jacobson K.
      • Schaller M.D.
      MAP kinases and cell migration.
      ]. For instance, ERK can phosphorylate and activate MLCK, a key enzyme in the actomoysin dynamics (Fig. 3) [
      • Huang C.
      • Jacobson K.
      • Schaller M.D.
      MAP kinases and cell migration.
      ,
      • Klemke R.L.
      • Cai S.
      • Giannini A.L.
      • et al.
      Regulation of cell motility by mitogen-activated protein kinase.
      ,
      • Morrison D.L.
      • Sanghera J.S.
      • Stewart J.
      • et al.
      Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1.
      ,
      • Totsukawa G.
      • Wu Y.
      • Sasaki Y.
      • et al.
      Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts.
      ,
      • Webb D.J.
      • Donais K.
      • Whitmore L.A.
      • et al.
      FAK–Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.
      ]. It can also phosphorylate m-calpains, a family of Ca2+-activated proteolytic enzymes (Fig. 3) [
      • Dourdin N.
      • Bhatt A.K.
      • Dutt P.
      • et al.
      Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts.
      ,
      • Huttenlocher A.
      • Palecek S.P.
      • Lu Q.
      • et al.
      Regulation of cell migration by the calcium-dependent protease calpain.
      ], that are needed for focal adhesion turnover and, consequently, cell migration [
      • Cuevas B.D.
      • Abell A.N.
      • Witowsky J.A.
      • et al.
      MEKK1 regulates calpain‐dependent proteolysis of focal adhesion proteins for rear‐end detachment of migrating fibroblasts.
      ]. ERK1/2 also activates FAK and paxillin (Fig. 3), whose assembly/disassembly is involved in focal adhesion turnover and, as such, migration [
      • Huang C.
      • Jacobson K.
      • Schaller M.D.
      MAP kinases and cell migration.
      ]. These ERK-mediated cell migration pathways are well-established in many cell types. However, much remains to be understood about the extent of their contribution to ROCK-mediated VSMC migration.
      PDGF-BB also regulates VSMC migration by impacting the actin superstructure, an event critical to cell migration. Actin cytoskeleton remodeling involves a myriad of proteins like Arp2/3 (actin-related proteins 2 and 3), mammalian diaphanous proteins (mDia), cofilin, Wiscott–Aldrich syndrome protein (WASP), and profilin. These proteins regulate the polymerization/depolymerization of the actin cytoskeleton in response to several upstream signals, including PDGF-BB. PDGF-BB and other VSMC chemotactic signals can induce RhoA/ROCK activation, leading to cytoskeletal remodeling and VSMC migration by a unique mechanism [
      • Ahmed S.
      • Warren D.T.
      Vascular smooth muscle cell contractile function and mechanotransduction.
      ,
      • Yang N.
      • Higuchi O.
      • Ohashi K.
      • et al.
      Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.
      ,
      • San Martín A.
      • Lee M.Y.
      • Williams H.C.
      • et al.
      Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells.
      ,
      • Amano M.
      • Nakayama M.
      • Kaibuchi K.
      Rho-kinase/ROCK: a key regulator of the cytoskeleton and cell polarity.
      ,
      • Liao J.K.
      • Seto M.
      • Noma K.
      Rho kinase (ROCK) inhibitors.
      ,
      • Ridley A.J.
      Rho GTPases and cell migration.
      ]. The details of this process include ROCK phosphorylation and activation of LIM kinase (LIMK), which then phosphorylates and inactivates cofilin (Fig. 2). Since cofilin is needed for actin filament severing and depolymerization, the inhibition of its activity blocks lamellipodium formation and directional VSMC migration [
      • Afewerki T.
      • Sultan A.
      • Warren D.
      Emerging regulators of vascular smooth muscle cell migration.
      ,
      • Gerthoffer W.T.
      Mechanisms of vascular smooth muscle cell migration.
      ]. Notably, RhoA, independently of ROCK, can activate mDia1, an actin-nucleating protein involved in the polymerization of actin filaments, resulting in filopodia formation, an initial step in the process of cell migration (Fig. 2) [
      • Watanabe N.
      • Higashida C.
      Formins: processive cappers of growing actin filaments.
      ].
      ADMA is another upstream signal that can activate Rho/ROCK, leading to VSMC migration. In the VSMC cytoplasm, ADMA activates ROCK, which then stimulates ERK to downregulate contractile protein expression and precipitate a migratory phenotype (Fig. 2) [
      • Zhou Y.M.
      • Lan X.
      • Guo H.B.
      • et al.
      Rho/ROCK signal cascade mediates asymmetric dimethylarginine-induced vascular smooth muscle cells migration and phenotype change.
      ]. In addition, ADMA can facilitate Rho/ROCK-mediated VSMC migration via endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO). This NO can deactivate the Rho/ROCK axis in VSMCs by inhibiting the Gα(12/13) subunit of heterotrimeric G-proteins, which, in turn, suppresses Ang II-induced vascular migration [
      • Suzuki H.
      • Kimura K.
      • Shirai H.
      • et al.
      Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by the angiotensin II type-1 receptor: implication in vascular migration.
      ]. ADMA, being an endogenous inhibitor of eNOS, can relieve the repressive effect of eNOS on Rho/ROCK, allowing migration of VSMCs [
      • Zhou Y.M.
      • Lan X.
      • Guo H.B.
      • et al.
      Rho/ROCK signal cascade mediates asymmetric dimethylarginine-induced vascular smooth muscle cells migration and phenotype change.
      ].
      Rac1 is another member of the family of small GTPases that is largely implicated in VSMC migration [
      • Vicente-Manzanares M.
      • Webb D.J.
      • Horwitz A.R.
      Cell migration at a glance.
      ]. Rac1 is involved in lamellipodia formation by activating Arp 2/3, a complex required for the nucleation of actin and its polymerization [
      • Ridley A.J.
      Rho GTPases and cell migration.
      ]. Rac1 can also activate p21 activated kinase (PAK), a serine/threonine kinase that stabilizes microtubules and regulates cell polarity. Interestingly, RhoA and Rac1 exhibit an antagonistic relationship that largely coordinates the process of cytoskeletal remodeling and cell migration. This is evident through Rac1-inibhition of RhoA via multiple mechanisms, including negative feedback by p63 RhoGEF, a GEF responsible for activating RhoA [
      • Alberts A.S.
      • Qin H.
      • Carr H.S.
      • et al.
      PAK1 negatively regulates the activity of the Rho exchange factor NET1.
      ], activation of RhoGAP [
      • Nimnual A.S.
      • Taylor L.J.
      • Bar-Sagi D.
      Redox-dependent downregulation of rho by rac.
      ,
      • Wildenberg G.A.
      • Dohn M.R.
      • Carnahan R.H.
      • et al.
      p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho.
      ], or by direct RhoA degradation [
      • Sahai E.
      • Garcia-Medina R.
      • Pouysségur J.
      • et al.
      Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility.
      ,
      • Wang H.R.
      • Zhang Y.
      • Ozdamar B.
      • et al.
      Regulation of cell polarity and protrusion formation by targeting RhoA for degradation.
      ]. Reciprocal negative control of Rac1 activity by RhoA has also been described, and includes the stimulation of FilGAP [
      • Ohta Y.
      • Hartwig J.H.
      • Stossel T.P.
      FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling.
      ]. Apparently, this shared antagonism between Rac1 and RhoA is essential for achieving VSMC polarity and regulating the formation of membrane ruffles, leading edge protrusion, and cellular spreading and retraction. Furthermore, it has been reported in a human cellular model that Rac1 is inhibited by azathioprine [
      • Tiede I.
      • Fritz G.
      • Strand S.
      • et al.
      CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes.
      ]. Since RhoA and Rac1 have a mutually inhibitory relationship and usually promote opposing cellular functions [
      • Nguyen L.K.
      • Kholodenko B.N.
      • von Kriegsheim A.
      Rac1 and RhoA: networks, loops and bistability.
      ], a Rac1 inhibitor could, in some sense, be viewed or even utilized as a possible RhoA activator. This means that azathioprine can be used in scenarios where RhoA upregulation is desired. However, selectivity remains an important issue that warrants further experimentation, both from biochemical and pharmacological perspectives.
      Various non-coding RNAs, like microRNAs (miRNA), also regulate Rho/ROCK-stimulated VSMC migration. The original set of miRNA that was shown to modulate VSMC functions were called myomiRs and initially included miR-1, miR-133, miR-206 and miR-208. Later, myomiRs were expanded to include many other miRNAs [
      • Wang D.
      • Atanasov A.G.
      The microRNAs regulating vascular smooth muscle cell proliferation: a minireview.
      ,
      • Yeh Y.T.
      • Wei J.
      • Thorossian S.
      • et al.
      MiR-145 mediates cell morphology-regulated mesenchymal stem cell differentiation to smooth muscle cells.
      ,
      • Uray K.
      • Major E.
      • Lontay B.
      MicroRNA regulatory pathways in the control of the actin-myosin cytoskeleton.
      ]. miRNAs are abundantly expressed in the vascular wall, where they contribute to numerous VSMC functions. For instance, miR‐26, miR‐29a, miR31, miR133, among several others, regulate VSMC proliferation [
      • Wang D.
      • Atanasov A.G.
      The microRNAs regulating vascular smooth muscle cell proliferation: a minireview.
      ,
      • Urbich C.
      • Kuehbacher A.
      • Dimmeler S.
      Role of microRNAs in vascular diseases, inflammation, and angiogenesis.
      ]. Emerging evidence shows an important contributory role of miRNAs in the pathogenesis of several CVDs including atherosclerosis. Recently, miRNAs have been documented to be involved in VSMC phenotypic switching [
      • Zhang Y.N.
      • Xie B.D.
      • Sun L.
      • et al.
      Phenotypic switching of vascular smooth muscle cells in the 'normal region' of aorta from atherosclerosis patients is regulated by miR-145.
      ]. In addition, miRNAs have been found to regulate VSMC migration. For example, miR15b/16, miR22, miR24, miR34a, miR214, miR362-3p, miR379, miR638, and others were reported to regulate VSMC migration as well as proliferation by silencing the mRNA levels of an array of VSMC phenotype switching-related genes [
      • Wang D.
      • Atanasov A.G.
      The microRNAs regulating vascular smooth muscle cell proliferation: a minireview.
      ,
      • Yu X.
      • Li Z.
      MicroRNAs regulate vascular smooth muscle cell functions in atherosclerosis (Review).
      ]. Importantly, miRNAs are involved in pathological VSMC migration and proliferation, such as in the case of atherosclerosis [
      • Yu X.
      • Li Z.
      MicroRNAs regulate vascular smooth muscle cell functions in atherosclerosis (Review).
      ]. In regards to the signaling role of Rho/ROCK in VSMC migration, stimulation of ROCK or MLCK was found to activate Stat3 (signal transducer and activator of transcription), a transcription factor associated with cellular growth. Activated STAT3 can upregulate miR-92a, which targets Kruppel-like factor 4 (KLF4), a transcription factor that regulates cell cycle genes and contractile genes. When KLF4 is downregulated due to miR-92a action, cell migration increases (Fig. 4) [
      • Wang J.
      • Zhang C.
      • Li C.
      • et al.
      MicroRNA-92a promotes vascular smooth muscle cell proliferation and migration through the ROCK/MLCK signalling pathway.
      ]. Additionally, the pro-atherosclerotic activity of KLF4 on VSMC phenotypic transformation was found to vary according to phosphorylation, cellular state, as well as other microenvironmental factors. Compelling evidence shows that KLF4 can indeed have a marked impact on VSMC phenotype [
      • Yang C.
      • Xiao X.
      • Huang L.
      • et al.
      Role of Kruppel-like factor 4 in atherosclerosis.
      ,
      • Wei X.
      • Yang R.
      • Wang C.
      • et al.
      A novel role for the Kruppel-like factor 14 on macrophage inflammatory response and atherosclerosis development.
      ]. So much so that KLF4 was suggested as a possible therapeutic target in disease management, and as a biomarker for monitoring the progression of atherosclerosis [
      • Yang C.
      • Xiao X.
      • Huang L.
      • et al.
      Role of Kruppel-like factor 4 in atherosclerosis.
      ]. More importantly, KLF4 was shown to be a major determining factor in the switching of VSMCs between the six different phenotypes, namely osteogenic, contractile, mesenchymal, fibroblast, macrophage, and adipocyte, all based on scRNA-seq studies [

      Yap, C, Mieremet, A, de Vries, CJM, et al., Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Kruppel-Like Factor 4), Arterioscler Thromb Vasc Biol, 2021;41:2693-2707.

      ].
      Fig. 4
      Fig. 4RhoA/ROCK involvement in VSMC migration can be regulated by an array of miRNAs.
      Shp2 signaling increases the level of miR-204. miR204 then blocks RhoA/ROCK by binding to its corresponding mRNA in the cytoplasm. miR-145 can also block the activity of RhoA/ROCK via the same mechanism and can as well interfere with actin dynamics by downregulating the translation of the corresponding mRNAs. LncRNA SMILR blocks the expression of miR-141 in the cytoplasm, preventing the suppression of RhoA/ROCK. Lnc RNA430945 increases the expression of ROR2 which in turn increases the activity of RhoA/ROCK. Increased activity of RhoA/ROCK manifests as increased migration. RhoA/ROCK itself activates STAT3, a nuclear transcription factor, which then increases the level of miR-92a expression. miR-92a blocks Klf-4, a transcription factor that regulates cell cycle and contractile genes.
      Other miRNAs can also play other roles in VSMC phenotypes. For instance, preliminary evidence shows that Shp2-mediated increase in miR204 levels downregulates ROCK 1 levels in pulmonary artery VSMCs (Fig. 4) [
      • Courboulin A.
      • Paulin R.
      • Giguère N.J.
      • et al.
      Role for miR-204 in human pulmonary arterial hypertension.
      ]. Likewise, miR-145 negatively regulates RhoA/ROCK-mediated VSMC proliferation and migration, partly by decreasing mRNA levels of ROCK 1. This occurs through directly binding ROCK 1 mRNA 3′UTR (Fig. 4) [
      • Chen M.
      • Zhang Y.
      • Li W.
      • et al.
      MicroRNA-145 alleviates high glucose-induced proliferation and migration of vascular smooth muscle cells through targeting ROCK1.
      ]. In fact, miR143/miR145 are regarded as molecular switches of the phenotype of VSMCs since these miRNAs target many genes that regulate VSMC function, including different components of the Rho/ROCK cascade as well as other regulators of actin dynamics. In this way, miR143/miR145 are regarded as global regulators of VSMC phenotype switching, including the switches geared towards cellular migration and proliferation [
      • Rangrez A.Y.
      • Massy Z.A.
      • Metzinger-Le Meuth V.
      • et al.
      miR-143 and miR-145: molecular keys to switch the phenotype of vascular smooth muscle cells, Circulation.
      ].
      In a similar manner, miR-223 suppresses RhoB, leading to reduced RhoB/ROCK signaling as well as decreased migration and proliferation in VSMCs [
      • Zeng Y.
      • Zhang X.
      • Kang K.
      • et al.
      MicroRNA-223 attenuates hypoxia-induced vascular remodeling by targeting RhoB/MLC2 in pulmonary arterial smooth muscle cells.
      ]. Likewise, miR141 downregulates RhoA, causing blunted RhoA/ROCK signaling and decreased migration and proliferation [
      • Liu L.
      • Zang J.
      • Chen X.
      • et al.
      Role of miR-124 and miR-141 in the regulation of vascular reactivity and the relationship to RhoA and Rac1 after hemorrhage and hypoxia.
      ,
      • Lei S.
      • Peng F.
      • Li M.L.
      • et al.
      LncRNA-SMILR modulates RhoA/ROCK signaling by targeting miR-141 to regulate vascular remodeling in pulmonary arterial hypertension.
      ]. A reversal of this inhibition is seen with the long non-coding RNA (LncRNA)-SMILR, which directly binds to miR141 and represses its expression, thus leading to an increase in cell proliferation and migration. Concordant with this, silencing of LncRNA-SMILR robustly inhibits vascular remodeling and mitigation of pulmonary arterial hypertension in an in vivo rat model (Fig. 4) [
      • Lei S.
      • Peng F.
      • Li M.L.
      • et al.
      LncRNA-SMILR modulates RhoA/ROCK signaling by targeting miR-141 to regulate vascular remodeling in pulmonary arterial hypertension.
      ]. Furthermore, lncRNA430945 promotes the expression of receptor tyrosine kinase-like orphan receptor 2 (ROR2). ROR2 subsequently activates RhoA/ROCK, which eventually stimulates VSMC migration and proliferation (Fig. 4) [
      • Wang J.
      • Zhang C.
      • Li C.
      • et al.
      MicroRNA-92a promotes vascular smooth muscle cell proliferation and migration through the ROCK/MLCK signalling pathway.
      ].
      One important pathological manifestation of migratory dysregulation that warrants further elaboration is what occurs in atherosclerosis. Indeed, new studies focus on the role of RhoA/ROCK in trapping macrophages within atherosclerotic lesions and its contribution to atherosclerosis progression [
      • Kloc M.
      • Uosef A.
      • Kubiak J.Z.
      • et al.
      Role of macrophages and RhoA pathway in atherosclerosis.
      ]. As is well-known, atherosclerosis is a chronic inflammatory disorder that involves the progressive accumulation of lipids within arterial walls. The pathogenesis of this disease involves a multi-step process that occurs over the course of several years or even decades. The initial stage lesions start as small lipid deposits in the intimal layers. These lesions evolve into fatty streaks consisting mainly of foam cells in the subendothelial space. Further accumulation of lipids, foam cells, and immune cells leads to the disorganization and thickening of the intima, media, and adventitia and the proceeding deformation of the artery wall. More advanced atherosclerotic lesions are characterized by extensive lipid deposition, fibrous connective tissue formation and calcification. Macrophages, have been shown to play a pivotal role in the progression of atherosclerotic lesions, as they first infiltrate the damaged endothelium in the form of monocytes. Monocytes then differentiate into naïve M0 macrophages which can then be channeled into M1 (pro-inflammatory) or M2 (anti-inflammatory) or Mox (oxidate stress phenotype) [
      National Cancer Institute
      Classification & structure of blood vessels.
      ]. Macrophages phagocytize ox-LDL becoming foam cells which constitute the lipidic necrotic core of atherosclerotic plaques. M2 macrophages internalize more lipids than M1 macrophages, due to a lower expression of cholesterol efflux proteins [
      • Rj F.
      Inflammatory Atherosclerosis: Characteristics of the Injurious Agent.
      ].
      In healthy arteries, VSMCs are located in the tunica media and are well differentiated. In response to vascular injury and inflammatory triggers such as what happens in atherosclerosis, they undergo dedifferentiation and take on a proliferative, and migratory phenotype which is greatly implicated in the pathogenesis of atherosclerosis [
      • Poole J.C.
      • Florey H.W.
      Changes in the endothelium of the aorta and the behaviour of macrophages in experimental atheroma of rabbits.
      ]. Importantly, dedifferentiated VSMCs can express macrophagic markers. They also can internalize lipids and become foam cells [
      • Mestas J.
      • Ley K.
      Monocyte-endothelial cell interactions in the development of atherosclerosis.
      ].
      RhoA/ROCK is believed to be involved in the entrapment process of lipid-laden macrophages in atherosclerotic plaques. Cholesterol-laden macrophages have been shown to have lower levels of the active GTP-bound RhoA, lower level of MLC phosphorylation and decreased motility [
      • Nagao T.
      • Qin C.
      • Grosheva I.
      • et al.
      Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation.
      ]. Studies also demonstrated that constitutively active RhoA prevent cholesterol-mediated inhibition of macrophage motility. This shows that the increase in cholesterol content prevents RhoA activation and subsequently compromise macrophage migratory abilities leaving them trapped within atherosclerotic lesions as foam cells [
      • Nagao T.
      • Qin C.
      • Grosheva I.
      • et al.
      Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation.
      ]. Iterestingly, undifferentiated VSMCs under inflammatory conditions, display many macrophagic features. As such, one might envision a scenario whereby RhoA/ROCK deactivation promotes lipid-laden VSMC entrapment in atherosclerotic lesions in the form of foam cells.

      4. Mechanisms of RhoA/ROCK mediated VSMC proliferation

      VSMC phenotypic switching is heavily implicated in plaque formation and the pathogenesis of atherosclerosis. RhoA/ROCK promotes VSMC proliferation during neointima formation after balloon injury, and its inhibition suppresses neointima formation [
      • Eto Y.
      • Shimokawa H.
      • Hiroki J.
      • et al.
      Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs.
      ]. Indeed, compelling evidence shows that RhoA or ROCK inhibitors halt VSMC proliferation, leading to a blockade of neointima formation during atherosclerosis [
      • Shimokawa H.
      • Sunamura S.
      • Satoh K.
      RhoA/Rho-kinase in the cardiovascular system.
      ]. Moreover, in CADASIL, a cerebral arteriopathy caused by a mutation that amplifies the NOTCH3 (neurogenic locus notch homolog protein 3) gene, there are high levels of NOX5 leading to overproduction of reactive oxygen species (ROS). This pro-oxidant milieu precipitates endoplasmic reticulum stress, followed by enhanced RhoA/ROCK expression, resulting in pathological increase of VSMCs proliferation [
      • Shimokawa H.
      • Sunamura S.
      • Satoh K.
      RhoA/Rho-kinase in the cardiovascular system.
      ]. It is also well known that ROS play critical roles in phenotypic switch [
      • Badran A.
      • Nasser S.A.
      • Mesmar J.
      • et al.
      Reactive oxygen species: modulators of phenotypic switch of vascular smooth muscle cells.
      ].
      There is an interplay between Rho/ROCK and MEK/ERK, a major driver of proliferative phenotype. Indeed, active RhoA/ROCK stimulates the MEK/ERK signaling axis, which then triggers MEK/ERK to promote VSMC proliferation [
      • Zhou Y.M.
      • Lan X.
      • Guo H.B.
      • et al.
      Rho/ROCK signal cascade mediates asymmetric dimethylarginine-induced vascular smooth muscle cells migration and phenotype change.
      ,
      • Zhao Y.
      • Lv M.
      • Lin H.
      • et al.
      Rho-associated protein kinase isoforms stimulate proliferation of vascular smooth muscle cells through ERK and induction of cyclin D1 and PCNA.
      ,
      • Guan R.
      • Xu X.
      • Chen M.
      • et al.
      Advances in the studies of roles of Rho/Rho-kinase in diseases and the development of its inhibitors.
      ,
      • Wei C.Y.
      • Huang K.C.
      • Chou Y.H.
      • et al.
      The role of Rho-associated kinase in differential regulation by statins of interleukin-1beta- and lipopolysaccharide-mediated nuclear factor kappaB activation and inducible nitric-oxide synthase gene expression in vascular smooth muscle cells.
      ]. Contextually, ERK inhibitors or knock down of ROCK1 and ROCK2 by siRNA causes inhibition of VSMC proliferation [
      • Zhao Y.
      • Lv M.
      • Lin H.
      • et al.
      Rho-associated protein kinase isoforms stimulate proliferation of vascular smooth muscle cells through ERK and induction of cyclin D1 and PCNA.
      ]. Furthermore, inhibition of ERK1/2 is known to inhibit hyper-proliferation of VSMCs from spontaneously hypertensive rats [
      • Almajdoob S.
      • Hossain E.
      • Anand-Srivastava M.B.
      Resveratrol attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats: role of ROS and ROS-mediated cell signaling.
      ]. Likewise, RhoA/ROCK-induced ERK activation mediates mechanical stretch-induced VSMC contractility and proliferation [
      • Numaguchi K.
      • Eguchi S.
      • Yamakawa T.
      • et al.
      Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments.
      ]. Similarly, RhoA/ROCK signaling, in combination with ERK and PI3K activation, is critical for stretch-induced saphenous vein VSMC proliferation [
      • Kozai T.
      • Eto M.
      • Yang Z.
      • et al.
      Statins prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway.
      ,
      • Kozai T.
      • Eto M.
      • Yang Z.
      • et al.
      Statins prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway.
      ]. When activated, ERK can translocate to the nucleus to activate transcription factors that upregulate the expression of cyclin D1 and PCNA (proliferation cell nuclear antigen), both known to be potent cell cycle regulators (Fig. 5). Cyclin D1 and PCNA can activate VSMC cell cycle, resulting in the eventual proliferation of VSMCs [
      • Zhao Y.
      • Lv M.
      • Lin H.
      • et al.
      Rho-associated protein kinase isoforms stimulate proliferation of vascular smooth muscle cells through ERK and induction of cyclin D1 and PCNA.
      ].
      Fig. 5
      Fig. 5RhoA/ROCK signaling modulates several downstream pathways implicated in cellular proliferation.
      RhoA/ROCK activates MLCK which then phosphorylates and activates STAT3. In the nucleus, STAT3 induces the transcription of miR-92a, which in turn silences the expression of KLF4, a protein that arrests the cell cycle. This culminates in increased VSMC proliferation. Moreover, RhoA/ROCK activates MEK1/ERK leading to ERK phosphorylation and its translocation to the nucleus to activate transcription factors like c-fos or c-myc. The activated transcription factors can enhance the transcription of proliferation genes like Cyclin D and PCNA, leading to VSMC growth. RhoA/ROCK can also activate JNK which induces c-jun to transcribe cyclinD1, CDK2, and CDK4, leading to VSMC proliferation. On the other hand, ROCK itself is negatively regulated by miR-143/145 and by p63RhoGEF. miR-143/145 silences ROCK1 leading to a decrease in cell migration and proliferation. MLK 3 binds and makes p63RhoGEF less available for RhoA activation, causing a reduced RhoA/ROCK signaling and leading to a decrease in VSMC proliferation. ROCK: Rho-associated kinase; MLCK: myosin light-chain Kinase; STAT3: signal transducer and activator of transcription 3; KLF4: krüppel-like factor 4; MEK1: MAPK/ERK kinase 1; ERK: extracellular signal-regulated kinase; PCNA: proliferating cell nuclear antigen; JNK: cJun N-terminal kinase; CDK: cyclin dependent kinase.
      In addition to its role in migration, ADMA is a major contributor to Rho/ROCK/ERK-mediated VSMC proliferation (Fig. 2) [
      • Guan R.
      • Xu X.
      • Chen M.
      • et al.
      Advances in the studies of roles of Rho/Rho-kinase in diseases and the development of its inhibitors.
      ]. Similarly, JNK is involved in Rho/ROCK-induced VSMC proliferation [
      • Beier I.
      • Dusing R.
      • Vetter H.
      • et al.
      Epidermal growth factor stimulates Rac1 and p21-activated kinase in vascular smooth muscle cells.
      ]. In PDGF-BB-stimulated VSMC proliferation, JNK appears to activate the oncogenic transcription factor c-jun, which upregulates cell cycle regulators Cyclin D1, CDK2, and CDK4 leading to increased proliferation (Fig. 5). This JNK-mediated VSMC proliferation (and also migration) promotes neointima formation [
      • Tang L.
      • Dai F.
      • Liu Y.
      • et al.
      RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.
      ,
      • Tang L.
      • Dai F.
      • Liu Y.
      • et al.
      RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.
      ]. Interestingly, inhibition of RhoA suppresses PDGF-induced DNA synthesis, phosphorylation of retinoblastoma protein, and levels of p27 cell cycle inhibitor. This further confirms the role of Rho/ROCK signaling in modulating cell cycle genes and the consequent phenotypic changes [
      • Laufs U.
      • Marra D.
      • Node K.
      • et al.
      3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1).
      ].
      Neuron-derived orphan receptor (NOR-1), a transcription factor, also impacts the role of Rho/ROCK. Indeed, higher levels of NOR-1 are associated with increasing levels of CREB (cAMP response element binding protein), which then amplifies the effect of RhoA/ROCK activation, leading to enhanced proliferation of aortic VSMCs [
      • Guan R.
      • Xu X.
      • Chen M.
      • et al.
      Advances in the studies of roles of Rho/Rho-kinase in diseases and the development of its inhibitors.
      ]. In addition to its role in vascular disease [
      • Bailey S.R.
      • Eid A.H.
      • Mitra S.
      • et al.
      Rho kinase mediates cold-induced constriction of cutaneous arteries: role of alpha2C-adrenoceptor translocation.
      ,
      • Chotani M.A.
      • Mitra S.
      • Eid A.H.
      • et al.
      Distinct cAMP signaling pathways differentially regulate alpha2C-adrenoceptor expression: role in serum induction in human arteriolar smooth muscle cells.
      ,
      • Chotani M.A.
      • Mitra S.
      • Su B.Y.
      • et al.
      Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells.
      ,
      • Eid A.H.
      • Chotani M.A.
      • Mitra S.
      • et al.
      Cyclic AMP acts through Rap1 and JNK signaling to increase expression of cutaneous smooth muscle alpha2C-adrenoceptors.
      ,
      • Eid A.H.
      • Maiti K.
      • Mitra S.
      • et al.
      Estrogen increases smooth muscle expression of alpha2C-adrenoceptors and cold-induced constriction of cutaneous arteries.
      ,
      • Jeyaraj S.C.
      • Unger N.T.
      • Eid A.H.
      • et al.
      Cyclic AMP-Rap1A signaling activates RhoA to induce alpha(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.
      ,
      • Motawea H.K.
      • Jeyaraj S.C.
      • Eid A.H.
      • et al.
      Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle alpha2C-adrenoceptors through the actin-binding protein filamin-2.
      ,
      • Wehbe N.
      • Nasser S.A.
      • Al-Dhaheri Y.
      • et al.
      EPAC in vascular smooth muscle cells.
      ,
      • Anwar M.A.
      • Samaha A.A.
      • Baydoun S.
      • et al.
      Rhus coriaria L. (Sumac) evokes endothelium-dependent vasorelaxation of rat aorta: involvement of the cAMP and cGMP pathways.
      ,
      • Eid A.H.
      cAMP induces adhesion of microvascular smooth muscle cells to fibronectin via an Epac-mediated but PKA-independent mechanism.
      ], cAMP can also inhibit VSMC proliferation in a rather intriguing proposed hypothesis. Increased levels of cAMP can inhibit RhoA, leading to actin depolymerization, which in turn induces the phosphorylation and nuclear export of YAP/TAZ (Yes Associated Protein/Transcriptional Coactivator With PDZ-Binding Motif). This then diminishes the activity of TEAD (TEA domain) transcription factors, leading to reduced VSMC proliferation (Fig. 6) [
      • Kimura T.E.
      • Duggirala A.
      • Smith M.C.
      • et al.
      The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP.
      ,
      • Smith S.A.
      • Newby A.C.
      • Bond M.
      Ending restenosis: inhibition of vascular smooth muscle cell proliferation by cAMP.
      ]. Other reports show that RhoA/ROCK activation induce nuclear import of ERK1/2, resulting in phosphorylation of Elk1 (ETS domain-containing protein 1) (Fig. 6), a coactivator of the transcription factor SRF (serum response factor). In turn, phospho-ELK1/SRF mediates the transcription of proliferation-related genes, like Cyclin D1 and the transcription factor Egr1 (early growth response 1), an immediate early response gene that controls cell proliferation. RhoA/ROCK-stimulated ERK1/2 can also enhance the binding of Egr1 to its DNA binding site leading to enhanced VSMC proliferation [
      • Liu Y.
      • Suzuki Y.J.
      • Day R.M.
      • et al.
      Rho kinase-induced nuclear translocation of ERK1/ERK2 in smooth muscle cell mitogenesis caused by serotonin.
      ].
      Fig. 6
      Fig. 6Pathways of transcription factor-mediated VSMC dedifferentiation. Rho/ROCK signaling triggers the nuclear localization of myocardin/MTRF-A.
      Normally, G-actin monomers bind and sequester MTRF-A in the cytoplasm. Activated Rho/ROCK induce G-actin polymerization into F-actin fibers; MTRF-A is released from the G-actin and translocate to the nucleus. In the nucleus, MTRF-A coactivates SRF, which in turn modulates the transcription of contractile and cytoskeletal genes. In a different mechanism, RhoA/ROCK signaling induces the YAP/TAZ/TEAD transcriptional program. RhoA/ROCK stimulates YAP activation by dephosphorylation of YAP which translocates to the nucleus where it coactivates TEAD transcription factors resulting in enhanced transcription of VSMC growth-related genes. Interestingly, there is an interaction between the MTRF-A/SRF and the YAP/TAZ/TEAD pathways. RhoA/ROCK can activate MEK/ERK causing the translocation of phosphorylated ERK to the nucleus. In the nucleus, phosphorylated ERK can activate ELK1, which then competes for binding to a common site on SRF and can replace the myocardin/MRTF-A that is already bound to SRF. This leads to a downregulation of the expression of contractile proteins. KLF4 is a transcription factor that can induce VSMC dedifferentiation. KLF4 binds to G/C repressor elements that are usually close to CArG boxes, SRF DNA binding sites. KLF-4 can attenuate SRF-mediated expression of contractile proteins through three different routes. 1) KLF4 disrupts myocardin/SRF interaction, 2) KLF4 can inhibit myocardin expression, and 3) KLF4 can recruit chromatin modifying enzymes to epigenetically repress the expression of CArG/SRF-mediated expression of contractile VSMC proteins. ROCK: Rho-associated kinase; MTRF-A: myocardin related transcription factor A; SRF: serum response factor; YAP: Yes associated protein; TAZ: transcriptional coactivator with PDZ-binding motif; TEAD: TEA domain; ERK: extracellular signal-regulated kinase; MEK: MAPK/ERK kinase; ELK1: ETS domain-containing protein 1; KLF4: krüppel-like factor 4.

      5. Mechanisms of RhoA/ROCK-induced VSMC dedifferentiation

      Dedifferentiation of VSMCs during phenotypic switching involves loss or downregulation of contractility-related phenotypes and genes as well as the acquisition of an epithelioid phenotype [
      • Chang S.
      • Song S.
      • Lee J.
      • et al.
      Phenotypic modulation of primary vascular smooth muscle cells by short-term culture on micropatterned substrate.
      ]. Dedifferentiated synthetic VSMCs release matrix-digesting enzymes, migrate to injury sites, elevate their rate of proliferation, and synthesize new ECM. When the injury is resolved and a physiologic status is regained, synthetic VSMCs re-assume the contractile phenotype and resume their contractile function [
      • Milewicz D.M.
      • Kwartler C.S.
      • Papke C.L.
      • et al.
      Genetic variants promoting smooth muscle cell proliferation can result in diffuse and diverse vascular diseases: evidence for a hyperplastic vasculomyopathy, Genetics in medicine.
      ]. This phenotype shift, despite being physiological, is a critical event in vascular pathologies.
      VSMC dedifferentiation is an initial step in the process of atherosclerotic plaque formation, where it enables VSMCs to migrate into the intima to form the fibrous cap [
      • Chistiakov D.A.
      • Orekhov A.N.
      • Bobryshev Y.V.
      Vascular smooth muscle cell in atherosclerosis.
      ,
      • Owens G.K.
      • Kumar M.S.
      • Wamhoff B.R.
      Molecular regulation of vascular smooth muscle cell differentiation in development and disease.
      ]. Differentiated contractile VSMCs express transcription factors like myocardin, contractile proteins such as smooth muscle alpha actin (α-SMA) and SM myosin heavy chain, intermediate filaments like desmin and vimentin as well as matrix adhesion proteins such as α1 and β1 integrin. Synthetic dedifferentiated VSMCs, however, express calcification genes, like osteopontin or osteocalcin, gap junction proteins like connexin 43, transcription factors like KLF4, collagenase enzymes like collagenase IV, and different matrix metalloproteinases (MMPs) isoforms [
      • Rensen S.S.
      • Doevendans P.A.
      • van Eys G.J.
      Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.
      ]. The precise molecular mechanisms underlying this event are still not well understood. However, evidence suggests that this process is not only limited to downregulation of smooth muscle specific genes in contractile VSMCs, but rather that drastic cytoskeletal modifications take place due to a global modification of VSMC gene expression. Rho/ROCK signaling, being a prominent regulator of cytoskeletal dynamics in VSMCs, is positioned to play a key role during VSMC dedifferentiation.
      Rho/ROCK signaling can modulate the expression of a set of smooth muscle specific genes, leading to the modulation of VSMC differentiation status through two important pathways. First, Rho/ROCK signaling is crucial for stabilization and remodeling of the actin cytoskeleton, by both inhibiting MLCP in addition to increasing the sensitivity of MLC to Ca2+ (Fig. 1). In addition, several RhoA/ROCK direct and indirect effectors like cofilin, mDia, Arp 2/3 proteins, or WASP are regulators of actin polymerization. Contextually, inhibiting Rho/ROCK disrupts the actin cytoskeleton, indicative of the need for Rho activity in the early phase of VSMC remodeling/dedifferentiation [
      • Zheng J.P.
      • Ju D.
      • Shen J.
      • et al.
      Disruption of actin cytoskeleton mediates loss of tensile stress induced early phenotypic modulation of vascular smooth muscle cells in organ culture.
      ]. Paradoxically, Ang II-induced Rho/ROCK-mediated actin polymerization suppresses the expression of VSMC contractile proteins, an integral step for VSMC dedifferentiation, which is, in turn, a prerequisite for VSMC migration [
      • Qi Y.
      • Liang X.
      • Dai F.
      • et al.
      RhoA/ROCK pathway activation is regulated by AT1 receptor and participates in smooth muscle migration and dedifferentiation via promoting actin cytoskeleton polymerization.
      ]. This discrepancy can be explained by the fact that Ang II effects seem to be concentration-dependent. A concentration of 100 nM Ang II suppressed the expression of contractile muscle genes whereas a concentration of 1000 nM had the opposite effect [
      • Qi Y.
      • Liang X.
      • Dai F.
      • et al.
      RhoA/ROCK pathway activation is regulated by AT1 receptor and participates in smooth muscle migration and dedifferentiation via promoting actin cytoskeleton polymerization.
      ]. However, more attention is needed to better elucidate this discordance.
      Second, the remarkable effects of activated Rho/ROCK pathway on cytoskeletal dynamics not only regulate VSMC contraction, adhesion, and migration, but also include transcriptional regulation. RhoA/ROCK-induced actin polymerization can stimulate serum response factor (SRF)-mediated gene transcription via different ways. Rho/ROCK signaling, for instance, triggers the nuclear localization of the myocardin family of transcriptional coactivators, including, myocardin-related transcription factor (MTRF)-A, and MTRF-B. Indeed, G-actin monomers bind and sequester MTRF-A in the cytoplasm. However, upon Rho/ROCK activation and G-actin polymerization into F-actin fibers, MTRF-A is released from the G-actin monomers and becomes free to translocate to the nucleus. In the nucleus, MTRF-A coactivates SRF by forming a ternary complex with it at the CArG site, the SRF DNA binding site (Fig. 6) [
      • Mack C.P.
      • Somlyo A.V.
      • Hautmann M.
      • et al.
      Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization.
      ,
      • Miralles F.
      • Posern G.
      • Zaromytidou A.I.
      • et al.
      Actin dynamics control SRF activity by regulation of its coactivator MAL.
      ]. MTRFA-coactivated SRF can modulate the transcription of a repertoire of target genes most of which are muscle-specific contractile and cytoskeletal genes. For example, active Rho/ROCK induces the nuclear translocation of myocardin, leading to a new expression profile in VSMCs, which then contributes to vascular pathologies [
      • Hien T.T.
      • Turczyńska K.M.
      • Dahan D.
      • et al.
      Elevated glucose levels promote contractile and cytoskeletal gene expression in vascular smooth muscle via rho/protein kinase C and actin polymerization.
      ]. Moreover, ROCK-mediated phosphorylation of protein phosphatase 1 regulatory subunit 14A (CPI 17), a phosphorylation-dependent inhibitor of smooth muscle protein phosphatase 1α (PP1α), will activate CPI-17 15, 121. Activated CPI 17 will inhibit PP1α, which usually represses the transcription factor MEF2C (myocyte specific enhancer factor 2C). The now-active MEF2C triggers the transcription of myocardin, whose increased protein levels will increase binding and activation of SRF, leading to a gene expression pattern characteristic of differentiated VSMCs [
      • Perry R.
      • Yang C.
      • Soora N.
      • et al.
      Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1 represses MEF2-dependent gene expression.
      ,
      • Pagiatakis C.
      • Gordon J.W.
      • Ehyai S.
      • et al.
      A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression.
      ]. Interestingly, dedifferentiated VSMCs have low levels of myocardin and SRF [
      • Zheng J.P.
      • Ju D.
      • Shen J.
      • et al.
      Disruption of actin cytoskeleton mediates loss of tensile stress induced early phenotypic modulation of vascular smooth muscle cells in organ culture.
      ]. Altogether, it seems that Rho/ROCK signaling can modify the differentiated state of VSMCs through a myocardin/SRF-mediated mechanism.
      KLF4, a transcription factor that can induce VSMC dedifferentiation, is expressed by synthetic but absent in contractile VSMCs. KLF4 binds to G/C repressor elements that are usually close to CArG boxes, and can interact with SRF, leading to disruption of myocardin/MRTF-A interaction with SRF (Fig. 6). KLF4 involvement in VSMC dedifferentiation can be specifically attributed to disruption of myocardin-MRTF-A/SRF interaction, KLF4 inhibition of myocardin expression, or KLF4 recruitment of repressive epigenetic remodeling activities to VSMC genes [
      • Zhuge Y.
      • Zhang J.
      • Qian F.
      • et al.
      Role of smooth muscle cells in Cardiovascular Disease.
      ]. In addition, other transcription factors, like phospho-Elk-1, can contribute to the differentiation/dedifferentiation of VSMCs by modifying the interaction of myocardin/MRTF-A with SRF [
      • Allahverdian S.
      • Chaabane C.
      • Boukais K.
      • et al.
      Smooth muscle cell fate and plasticity in atherosclerosis.
      ,
      • Pipes G.C.
      • Creemers E.E.
      • Olson E.N.
      The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis.
      ]. Indeed, myocardin and Elk-1 compete for binding to a common site on SRF, which creates a “binary switch” through which signaling pathways can modify the VSMCs phenotypes. For example, PDGF stimulation of VSMCs leads to Elk1 phosphorylation, by ERK1/2, resulting in its binding to SRF by replacing myocardin/MRTF-A (Fig. 6) [
      • Pipes G.C.
      • Creemers E.E.
      • Olson E.N.
      The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis.
      ].
      Another transcription program elicited by RhoA/ROCK signaling is the YAP/TAZ/TEAD transcriptional program. RhoA/ROCK-transduced mechanical cues or alterations of actin dynamics can provoke YAP activation (Fig. 6) [
      • Dupont S.
      • Morsut L.
      • Aragona M.
      • et al.
      Role of YAP/TAZ in mechanotransduction.
      ,
      • Aragona M.
      • Panciera T.
      • Manfrin A.
      • et al.
      A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.
      ]. In this case, RhoA/ROCK mediates dephosphorylation of YAP, which translocates to the nucleus [
      • Dupont S.
      • Morsut L.
      • Aragona M.
      • et al.
      Role of YAP/TAZ in mechanotransduction.
      ,
      • Aragona M.
      • Panciera T.
      • Manfrin A.
      • et al.
      A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.
      ,
      • Yu F.X.
      • Zhao B.
      • Panupinthu N.
      • et al.
      Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.
      ]. Nuclear YAP binds and coactivates TEAD transcription factors, many of which are VSMC growth-related genes (Fig. 6) [
      • Kimura T.E.
      • Duggirala A.
      • Smith M.C.
      • et al.
      The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP.
      ,
      • Smith S.A.
      • Newby A.C.
      • Bond M.
      Ending restenosis: inhibition of vascular smooth muscle cell proliferation by cAMP.
      ]. Some of the Rho/ROCK/YAP/TAZ/TEAD-regulated genes are, remarkably, also regulated by RhoA/ROCK/MTRF-A/SRF, demonstrating that cytoskeletal dynamics can mediate an interaction between these pathways [
      • Smith S.A.
      • Newby A.C.
      • Bond M.
      Ending restenosis: inhibition of vascular smooth muscle cell proliferation by cAMP.
      ,
      • Yu O.M.
      • Miyamoto S.
      • Brown J.H.
      Myocardin-related transcription factor A and yes-associated protein exert dual control in G protein-coupled receptor- and RhoA-mediated transcriptional regulation and cell proliferation.
      ].
      Epigenetic regulation of the aforementioned pathways and others have also been shown to play a role in VSMC phenotype. There are many reports about epigenetic modifications taking place in the chromatin structure of several smooth muscle specific contractile proteins like SM-α-actin, SM myosin heavy chain, and SM 22α upon differentiation/dedifferentiation. For example, reduced acetylation of histone H4 was reported in PDGF-BB-stimulated VSMCs and in rat carotid artery after balloon injury [
      • Allahverdian S.
      • Chaabane C.
      • Boukais K.
      • et al.
      Smooth muscle cell fate and plasticity in atherosclerosis.
      ]. Similarly, disruption of telomeric silencing 1-like (DOT1L), the only known methylation writer at histone 3 lysine 79 (H3K79), has been shown to contribute to VSMC proliferation and consequently intimal hyperplasia [
      • Huang Y.
      • Urabe G.
      • Zhang M.
      • et al.
      Nullifying epigenetic writer DOT1L attenuates neointimal hyperplasia.
      ]. Evidence supporting this notion is provided by a study that looked into genome-wide DNA methylation changes in atherosclerotic aorta [
      • Lacey M.
      • Baribault C.
      • Ehrlich K.C.
      • et al.
      Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.
      ]. This study showed that hypermethylation of differentially methylated regions at aorta enhancer chromatin were noted in several genes that play critical roles in VSMC phenotype and in atherosclerosis [
      • Lacey M.
      • Baribault C.
      • Ehrlich K.C.
      • et al.
      Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.
      ]. These proteins include elastin, myocardin, α2 smooth muscle actin as well as smooth muscle myosin, among others [
      • Lacey M.
      • Baribault C.
      • Ehrlich K.C.
      • et al.
      Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.
      ].
      Post transcriptionally, VSMC differentiation/dedifferentiation can be regulated by miRNAs. In VSMCs, miR-1, miR-21, miR −23, miR −125b, miR-143/145 and miR-155 were found to be involved in the different aspects of VSMC phenotype switching [
      • Yeh Y.T.
      • Wei J.
      • Thorossian S.
      • et al.
      MiR-145 mediates cell morphology-regulated mesenchymal stem cell differentiation to smooth muscle cells.
      ,
      • Uray K.
      • Major E.
      • Lontay B.
      MicroRNA regulatory pathways in the control of the actin-myosin cytoskeleton.
      ]. The miR143/145 cluster is a particularly potent promoter of the VSMC contractile phenotype, while other miRNAs like miR21, miR221, or miR222 favor a dedifferentiated and proliferative VSMC phenotype. Many of these miRNA can target the transcription factors that mediate differentiation/dedifferentiation of VSMC. For example, miR143/145 can target KLF4, Elk1 and myocardin [
      • Wang D.
      • Atanasov A.G.
      The microRNAs regulating vascular smooth muscle cell proliferation: a minireview.
      ]. A recent study found that RhoA/ROCK can induce miR-145 expression to silence KLF4 and induce the differentiation of mesenchymal stem cells into functional VSMCs [
      • Yeh Y.T.
      • Wei J.
      • Thorossian S.
      • et al.
      MiR-145 mediates cell morphology-regulated mesenchymal stem cell differentiation to smooth muscle cells.
      ].
      While the early stage of dedifferentiation involves destabilization of actin cytoskeleton, later stages encompass an intricate network of signaling events, most notable of which are the MAPKs, ERK1/2 and p38 signaling [
      • Guo H.
      • Makarova N.
      • Cheng Y.
      • et al.
      The early- and late stages in phenotypic modulation of vascular smooth muscle cells: differential roles for lysophosphatidic acid.
      ]. For example, MAPK activation, in a Rho/ROCK dependent manner, contributes to the late stage of dedifferentiation [
      • Muniyappa R.
      • Xu R.
      • Ram J.L.
      • et al.
      Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells.
      ,
      • Guo H.
      • Makarova N.
      • Cheng Y.
      • et al.
      The early- and late stages in phenotypic modulation of vascular smooth muscle cells: differential roles for lysophosphatidic acid.
      ]. Likewise, ADMA contributes to VSMC dedifferentiation in a process that involves the nuclear translocation of ERK2 [
      • Zhou Y.M.
      • Lan X.
      • Guo H.B.
      • et al.
      Rho/ROCK signal cascade mediates asymmetric dimethylarginine-induced vascular smooth muscle cells migration and phenotype change.
      ]. Nevertheless, the mechanism of late stage VSMC dedifferentiation downstream of MAPKs is still not fully understood and warrants future investigation.

      6. Effect of RhoA on VSMC apoptosis

      Apoptosis is an intricately regulated but only recently studied component of VSMC behavior. It is integral to multiple critical events, such as vascular remodeling, thrombosis and atherosclerosis, among others [
      • Aravani D.
      • Foote K.
      • Figg N.
      • et al.
      Cytokine regulation of apoptosis-induced apoptosis and apoptosis-induced cell proliferation in vascular smooth muscle cells.
      ]. During these processes, apoptotic VSMCs usually undergo extensive cytoskeletal rearrangements. Since Rho/ROCK signaling contributes to the actin cytoskeleton stability and remodeling, its involvement in VSMC apoptosis warrants discussion. For instance, Ang II–induced abdominal aortic aneurysm (AAA) in apoE-knockout mice is associated with a significant increase in apoptosis in aortic wall VSMCs. Inhibition of ROCK by fasudil blunted Ang II–induced apoptosis of cells of the aortic wall and reduced AAA [
      • Wang Y.X.
      • Martin-McNulty B.
      • da Cunha V.
      • et al.
      Fasudil, a Rho-kinase inhibitor, attenuates angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice by inhibiting apoptosis and proteolysis.
      ]. Contextually, Rho/ROCK inactivation can partly account for the elaborate alterations that occur at the level of the actin cytoskeleton during VSMC apoptosis. Inhibition of RhoA/Rac-1 disrupts the integrity of the actin cytoskeleton of rat VSMCs in vivo and in vitro [
      • Kang S.
      • Kim K.
      • Noh J.Y.
      • et al.
      Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/Rac-1 activity.
      ]. Concurrently, bcl-2-modifying factor (Bmf), a pro-apoptotic factor that binds to actin filaments, dissociated and moved into the mitochondria. Concomitantly, dissipation of mitochondrial membrane potential, caspase-3 activation and eventually apoptosis ensue (Fig. 7) [
      • Kang S.
      • Kim K.
      • Noh J.Y.
      • et al.
      Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/Rac-1 activity.
      ]. As such, administration of actin stabilizing agents can rescue these cells from contractile dysfunction as well as apoptosis, which both confirms the integrity of the previous findings and also suggests that disruption of RhoA/Rac-1 signaling leads to disruption of cytoskeletal integrity, which in turn can induce apoptosis [
      • Kang S.
      • Kim K.
      • Noh J.Y.
      • et al.
      Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/Rac-1 activity.
      ].
      Fig. 7
      Fig. 7Numerous stressors inhibit the RhoA/ROCK pathway leading to alteration of cytoskeletal stability and other phenomena like apoptosis.
      Concomitant with disruption of the actin cytoskeleton integrity, Bmf, a pro-apoptotic factor attached to actin filaments, dissociates and moves into the mitochondria, where dissipation of mitochondrial membrane potential, caspase-3 activation and ultimately apoptosis ensue. Separately, Rho/ROCK can independently activate JNK in many cell types resulting in cell apoptosis. Finally, RhoA/ROCK activation can disrupt the vimentin cytoskeleton resulting in eventual VSMC apoptosis. ROCK: Rho-associated kinase; Bmf: Bcl-2-modifying factor; JNK: c-Jun N-terminal kinase.
      Apart from its direct effect on the actin cytoskeleton, Rho/ROCK can mediate apoptosis through a variety of pathways in different cell types [
      • Lacal J.C.
      Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid-dependent signaling.
      ]. For example, in rat cardiomyocytes, the Rho/ROCK pathway induces apoptosis by activation of MAPKs p38 and JNK [
      • Zhou H.
      • Sun Y.
      • Zhang L.
      • et al.
      The RhoA/ROCK pathway mediates high glucose-induced cardiomyocyte apoptosis via oxidative stress, JNK, and p38MAPK pathways.
      ]. Activation of JNK was documented to induce apoptosis in VSMCs (Fig. 7) [
      • Guo D.
      • Wang Y.W.
      • Ma J.
      • et al.
      Study on the role of Cathepsin B and JNK signaling pathway in the development of cerebral aneurysm.
      ,
      • Han Y.
      • Wu G.
      • Deng J.
      • et al.
      Cellular repressor of E1A-stimulated genes inhibits human vascular smooth muscle cell apoptosis via blocking P38/JNK MAP kinase activation.
      ]. It is noteworthy to mention that Rho/ROCK signaling was found to elicit JNK activation in VSMCs [
      • Yang H.
      • Villani R.M.
      • Wang H.
      • et al.
      The role of cellular reactive oxygen species in cancer chemotherapy.
      ]. As such, one could envisage a mechanism whereby Rho/ROCK activates JNK in VSMCs in order to induce apoptosis; however, this requires further investigation.
      Rho/ROCK can negatively regulate apoptosis in VSMCs. For example, inhibition of ROCK led to inhibition of VSMC migration and an enhancement of apoptosis of VSMCs, causing a reduction of neointima formation after vascular injury [
      • Shibata R.
      • Kai H.
      • Seki Y.
      • et al.
      Role of Rho-associated kinase in neointima formation after vascular injury.
      ]. In another study, the same group confirmed that ROCK inhibition enhances apoptosis by increasing Bax expression and thereby reducing neointima formation [
      • Shibata R.
      • Kai H.
      • Seki Y.
      • et al.
      Rho-kinase inhibition reduces neointima formation after vascular injury by enhancing Bax expression and apoptosis.
      ]. Others also reported that shear stress downregulates the expression of Rho-GDIα and Rac1. Since Rho-GDIα downregulation promotes VSMC migration and apoptosis, this suggests that RhoA or Rac1 act to enhance apoptosis of VSMCs during shear stress [
      • Qi Y.X.
      • Qu M.J.
      • Long D.K.
      • et al.
      Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis: a proteomic analysis.
      ]. Nonetheless, the evidence for the involvement of Rho/ROCK signaling in VSMC apoptosis is evolving and awaits further validation.

      7. Mechanisms of RhoA/ROCK-mediated modulation of VSMC extracellular matrix deposition

      Within the vascular system, the ECM has structural and regulatory roles. ECM is involved in relaying mechanical signals to cells and providing enough elasticity for blood flow, all while maintaining the tensile strength needed to handle changes in pressure [
      • George S.
      • Johnson J.
      Extracellular Matrix and Smooth Muscle Cells.
      ]. Major producers of ECM within the vessel wall are VSMCs. As a result, pathological states, most notable of which were atherosclerotic plaque formation and arterial hypertension, were reported to emerge when synthesis of ECM proteins by VSMCs was dysregulated [
      • Ponticos M.
      • Smith B.D.
      Extracellular matrix synthesis in vascular disease: hypertension, and atherosclerosis.
      ]. Similar to other aspects of VSMC phenotypic switching, dysregulated ECM deposition seems to engage extensive cytoskeletal modifications. Rho/ROCK signaling, being a chief modifier of actin filaments stability, is suggested to contribute to regulation of the process of VSMC ECM deposition [
      • Lim S.M.
      • Trzeciakowski J.P.
      • Sreenivasappa H.
      • et al.
      RhoA-induced cytoskeletal tension controls adaptive cellular remodeling to mechanical signaling.
      ].
      RhoA/ROCK can mediate the process of ECM deposition through multiple downstream signaling pathways that mostly emanate from TGF-β as an upstream signal [
      • Cicha I.
      • Yilmaz A.
      • Klein M.
      • et al.
      Connective tissue growth factor is overexpressed in complicated atherosclerotic plaques and induces mononuclear cell chemotaxis in vitro.
      ,
      • Ruiz-Ortega M.
      • Rodriguez-Vita J.
      • Sanchez-Lopez E.
      • et al.
      TGF-beta signaling in vascular fibrosis.
      ,
      • Cajot J.F.
      • Bamat J.
      • Bergonzelli G.E.
      • et al.
      Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.
      ]. TGF-β signaling via the Smad pathway has prominent roles in VSMCs stiffness and fibrosis. It is involved in the regulation of expression of most VSMC ECM-related proteins, including the pro-fibrotic mediator CTGF (connective tissue growth factor), the main ECM component Type I Collagen (Col-1), and the plasminogen activator inhibitor-type 1 (PAI-1; SERPINE1) [
      • Cicha I.
      • Yilmaz A.
      • Klein M.
      • et al.
      Connective tissue growth factor is overexpressed in complicated atherosclerotic plaques and induces mononuclear cell chemotaxis in vitro.
      ,
      • Ruiz-Ortega M.
      • Rodriguez-Vita J.
      • Sanchez-Lopez E.
      • et al.
      TGF-beta signaling in vascular fibrosis.
      ,
      • Cajot J.F.
      • Bamat J.
      • Bergonzelli G.E.
      • et al.
      Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.
      ].
      Initially, links between RhoA/ROCK and ECM deposition were reported while studying the effect of statins on TGF-β/SMAD pathway in vascular cells and also during atherosclerosis [
      • Rodriguez-Vita J.
      • Sanchez-Galan E.
      • Santamaria B.
      • et al.
      Essential role of TGF-beta/Smad pathway on statin dependent vascular smooth muscle cell regulation.
      ]. It was found that blockade of RhoA with either statins or RhoA specific inhibitors will exacerbate TGF-β-induced increase of Smad3 phosphorylation and ECM deposition [
      • Rodriguez-Vita J.
      • Sanchez-Galan E.
      • Santamaria B.
      • et al.
      Essential role of TGF-beta/Smad pathway on statin dependent vascular smooth muscle cell regulation.
      ]. Murine transcriptome studies have yielded an independent significant connection between Smad3 and aortic dilatation pathology in the setting of hereditary thoracic aortic aneurysm; loss of contact between VSMC and ECM, which is implicated in aneurysm pathogenesis, was linked to Smad3 activity and VSMC response to microenvironmental factors [
      • Bramel E.E.
      • Creamer T.J.
      • Saqib M.
      • et al.
      Postnatal Smad3 inactivation in murine smooth muscle cells elicits a temporally and regionally distinct transcriptional response.
      ]. Moreover, inhibition of RhoA/ROCK pathway can increase the expression of TGF-β and its receptor TGF-β receptor type II (TRII). Overall, RhoA/ROCK signaling serves to reduce TGF-β levels. These signaling activities suggest that hyperactivation of RhoA may impinge on this pathway during pathological conditions, leading to unstable plaque formation or a modulation of elastic ECM deposition by VSMCs [
      • Rodriguez-Vita J.
      • Sanchez-Galan E.
      • Santamaria B.
      • et al.
      Essential role of TGF-beta/Smad pathway on statin dependent vascular smooth muscle cell regulation.
      ]. Aneurysmal formation, due to its link with abnormal ECM deposition, may also be influenced by RhoA, as its correlation with Smad3 activity has already been established by transcriptome analysis, albeit in animal models [
      • Bramel E.E.
      • Creamer T.J.
      • Saqib M.
      • et al.
      Postnatal Smad3 inactivation in murine smooth muscle cells elicits a temporally and regionally distinct transcriptional response.
      ].
      PAI-1 is involved in the modulation of VSMC stiffness [
      • Cajot J.F.
      • Bamat J.
      • Bergonzelli G.E.
      • et al.
      Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.
      ]. PAI-1 inhibits tissue plasminogen activator, preventing the cleavage of plasminogen into active plasmin, which in turn can activate MMPs. PAI-1 blocks plasmin-mediated degradation of fibrin and hence ECM degradation [
      • Cajot J.F.
      • Bamat J.
      • Bergonzelli G.E.
      • et al.
      Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.
      ]. Activated ROCK is an activator of PAI-1 expression, since inhibition of RhoA/ROCK inhibited Ang II-induced PAI-1 activation [
      • Samarakoon R.
      • Higgins S.P.
      • Higgins C.E.
      • et al.
      TGF-beta1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60(c-src)/EGFR(Y845) and Rho/ROCK signaling.
      ]. In addition, ERK1/2 was implicated in RhoA/ROCK-mediated activation of PAI-1 expression [
      • Samarakoon R.
      • Higgins S.P.
      • Higgins C.E.
      • et al.
      TGF-beta1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60(c-src)/EGFR(Y845) and Rho/ROCK signaling.
      ].
      MMPs mediate remodeling of the ECM under physiological and pathological states. MMPs degrade the ECM to allow for cell migration and their proteolytic activity is also needed to control the availability of growth factors in close proximity with cells. During phenotypic switching, synthetic VSMCs elevate the expression of several MMPs like MMP-2, MMP-9, and other matrix digesting enzymes, possibly as a part of the dedifferentiation process [
      • Newby A.C.
      Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates.
      ]. As such, MMPs contribute to formation and remodeling of the vasculature. In addition, remodeling of the ECM is a requirement for VSMC migration, and upregulated MMP activation induces VSMC migration and proliferation [
      • Newby A.C.
      Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates.
      ,
      • Belo V.A.
      • Guimarães D.A.
      • Castro M.M.
      Matrix metalloproteinase 2 as a potential mediator of vascular smooth muscle cell migration and chronic vascular remodeling in hypertension.
      ]. Consistent with this assumption is the failure of in vitro migration of VSMCs from MMP-2 knock out mice [
      • Deatrick K.B.
      • Luke C.E.
      • Elfline M.A.
      • et al.
      The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling.
      ]. Likewise, MMP-9 is necessary for VSMC migration and proliferation [
      • Cho A.
      • Reidy M.A.
      Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury.
      ]. Moreover, dysregulated MMP activity can lead to vascular pathologies like hypertension-induced arterial changes and persistent hypertension [
      • Belo V.A.
      • Guimarães D.A.
      • Castro M.M.
      Matrix metalloproteinase 2 as a potential mediator of vascular smooth muscle cell migration and chronic vascular remodeling in hypertension.
      ].
      The interplay between Rho and MMPs is becoming more lucid. MMPs, which can destabilize the atherosclerotic plaque, are themselves regulated by RhoA/ROCK signaling [
      • Newby A.C.
      Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates.
      ]. For instance, PDGF-BB-induced MMP-2 expression in rat VSMCs is mediated via ROCK, ERK and p38 MAPKs [
      • Cui Y.
      • Sun Y.W.
      • Lin H.S.
      • et al.
      Platelet-derived growth factor-BB induces matrix metalloproteinase-2 expression and rat vascular smooth muscle cell migration via ROCK and ERK/p38 MAPK pathways.
      ]. Also, Rho/ROCK mediates PDGF-induced secretion of MMP-9 in saphenous vein VSMCs via ERK, p38, and NFκB [
      • Turner N.A.
      • O'Regan D.J.
      • Ball S.G.
      • et al.
      Simvastatin inhibits MMP-9 secretion from human saphenous vein smooth muscle cells by inhibiting the RhoA/ROCK pathway and reducing MMP-9 mRNA levels.
      ]. Taken together, regulation of MMP expression by RhoA/ROCK appears to be integral to VSMC-mediated ECM deposition during phenotypic switching.
      Src homology region 2 domain-containing phosphatase-2 (SHP2) is a protein tyrosine phosphatase that acts upstream of TGFβ1-SMAD2 pathway and contributes to VSMC ECM stiffness [
      • Gong H.
      • Ni J.
      • Xu Z.
      • et al.
      Shp2 in myocytes is essential for cardiovascular and neointima development.
      ]. It was found that Ang II activates SHP2, which deactivates RhoGAP p190A, causing RhoA to be mainly present as the active RhoA-GTP form. This activates ROCK, which then inhibits the TGFβ/Smad pathway [
      • Bregeon J.
      • Loirand G.
      • Pacaud P.
      • et al.
      Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells.
      ]. This Ang II-mediated signaling via SHP2/RhoGAP p190A/RhoA/TGFβ/SMAD appears to be also an important player in phenotypic regulation. Remarkably, an opposing observation was reported in the case of Marfan Syndrome aortic aneurysms, where high levels of active RhoA-GTP paradoxically favor ECM deposition [
      • Crosas-Molist E.
      • Meirelles T.
      • López-Luque J.
      • et al.
      Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.
      ]. It was proposed that this phenomenon may be indirectly related to chronic activation of TGF-β and overexpression of myocardin in Marfan syndrome vascular cells [
      • Crosas-Molist E.
      • Meirelles T.
      • López-Luque J.
      • et al.
      Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.
      ]. Yet, the underlying mechanism remains to be elucidated.
      There are other important roles that VSMCs play in pathological states. For example, during atherosclerosis and when phosphate concentration is high, VSMCs actively drive the deposition of calcified matrix within the vessel wall [
      • Shanahan C.M.
      Mechanisms of vascular calcification in CKD-evidence for premature ageing?.
      ,
      • Lee S.J.
      • Lee I.K.
      • Jeon J.H.
      Vascular calcification-new insights into its mechanism.
      ]. Deposition of mineralized ECM is a characteristic of dedifferentiated synthetic VSMCs during pathological phenotype switching [
      • Shanahan C.M.
      Mechanisms of vascular calcification in CKD-evidence for premature ageing?.
      ]. Pathological dedifferentiation of VSMCs can cause them to follow osteogenic, chondrogenic, or even macrophage cell lineages that can form foam cells. Differentiation into osteoblast-like cells entails secretion of mineralized ECM components like osteocalcin, leading to vascular calcification [
      • Lee S.J.
      • Lee I.K.
      • Jeon J.H.
      Vascular calcification-new insights into its mechanism.
      ,
      • Pustlauk W.
      • Westhoff T.H.
      • Claeys L.
      • et al.
      Induced osteogenic differentiation of human smooth muscle cells as a model of vascular calcification.
      ]. Despite this understanding, the mechanisms underlying the osteogenic differentiation of VSMCs are still poorly understood [
      • Lee S.J.
      • Lee I.K.
      • Jeon J.H.
      Vascular calcification-new insights into its mechanism.
      ]. A recent study reported that inhibition of ROCK causes a reduction in VSMC calcification [
      • Tsuda T.
      • Imanishi M.
      • Oogoshi M.
      • et al.
      Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells.
      ]. Another recent study reported that RhoA/ROCK positively regulates VSMC calcification, where Wnt3a, a pleiotropic cellular function signal transducer protein, mediates calcification of VSMCs via the Rho/ROCK/JNK pathway [
      • Oh Y.J.
      • Kim H.
      • Kim A.J.
      • et al.
      Reduction of secreted frizzled-related protein 5 drives vascular calcification through wnt3a-mediated rho/ROCK/JNK signaling in chronic kidney disease.
      ]. Conversely, Rho/ROCK has also been shown to be a negative regulator of bovine VSMC deposition of calcified matrix [
      • Chen N.X.
      • Chen X.
      • O'Neill K.D.
      • et al.
      RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC), American journal of physiology.
      ]. Collectively, more research is needed to unravel the role of RhoA/ROCK in VSMC deposition of calcified matrix.

      8. Mechanisms of RhoA/ROCK-induced vascular stiffness

      Vascular stiffness is correlated with the rigidity of the vessel wall. An increase in vascular stiffness is implicated in a number of cardiovascular disorders since it can lead to increased vascular impedance and impairment of the elastic component of vessel structure [
      • Cheung Y-f
      Chapter 6 - systemic circulation.
      ]. In addition, increased arterial stiffness is associated with end-organ remodeling, such as arterial intima-media thickening in adults [
      • Valisno J.A.C.
      • May J.
      • Singh K.
      • et al.
      BCL11B regulates arterial stiffness and related target organ damage.
      ]. Vascular stiffness is modified by an alteration of the composition of the main components of the vessel wall viz: elastin, collagen, and smooth muscle cells. Stiffness of VSMCs per se has a major contribution to arterial stiffness [
      • Qiu H.
      • Zhu Y.
      • Sun Z.
      • et al.
      Short communication: vascular smooth muscle cell stiffness as a mechanism for increased aortic stiffness with aging.
      ,
      • Zhu Y.
      • Qiu H.
      • Trzeciakowski J.P.
      • et al.
      Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging.
      ]. In this context, Rho/ROCK signaling contributes to both VSMC and arterial stiffness.
      The transcriptional program that takes place due to the action of myocardin/SRF transcriptional program is the main driver behind VSMC stiffness that manifests in response to hypertension, among other cues [
      • Lacolley P.
      • Li Z.
      • Challande P.
      • et al.
      SRF/myocardin: a novel molecular axis regulating vascular smooth muscle cell stiffening in hypertension.
      ]. Rho/ROCK signaling can directly induce myocardin nuclear translocation, the subsequent activation of myocardin/SRF transcriptional program, and eventually the switch to a stiffer VSMC phenotype (Fig. 8). This can culminate in the stiffness of the whole vascular wall and the alteration of the hemodynamic properties of the blood vessel [
      • Zhou N.
      • Lee J.J.
      • Stoll S.
      • et al.
      Rho kinase regulates aortic vascular smooth muscle cell stiffness via actin/SRF/myocardin in hypertension.
      ].
      Fig. 8
      Fig. 8Mechanisms of RhoA/ROCK-induced VSMC stiffness.
      TGF-β, PLK1, or oxidized low-density lipoprotein (oxLDL), can induce ROCK to activate RhoA, which then triggers myocardin family transcription factors like MFTR-A to co-activate serum response factor (SRF). This then initiates expression of target genes like Myosin-related transcription factor, SMA, smoothelin, SM22, and calponin-1, proteins that heighten VSMC stiffness. In addition, Cullin-3 can inhibit ROCK and modulate the expression of these genes. In addition, stress signals can induce PECAM-1 to activate PI3K, which then inhibits ROCK via PKA. TGF-β: transforming growth factor β; PLK1: polo like kinase 1; oxLDL: oxidized low-density lipoprotein; ROCK: Rho-associated kinase; MRTF-A: myocardin-related transcription factor; SRF: serum response factor; SMA: smooth muscle actin; MYH: smooth muscle myosin heavy chain; SMTN: smoothelin, α-SMA: α-smooth muscle actin, SM22: smooth muscle protein 22; CUL3: cullin-3; PECAM-1: platelet endothelial cell adhesion molecule 1; PI3K: phosphoinositide 3-kinase; PKA: protein kinase A.
      Transforming growth factor beta (TGF-β), is a pleiotropic cytokine involved in many cellular processes including cell proliferation, differentiation, apoptosis, and migration. VSMCs obtained from Marfan Syndrome patients exhibit overactive RhoA/ROCK pathway owing to an overactive TGF-β signaling. Such cells, as discussed above, exhibit increased expression of contractile protein markers, indicative of increased cellular stiffness. In addition, higher levels of nuclear-translocated myosin-related transcription factor A were observed. These findings propose a pathway that involves increased TGF-β signaling as the trigger of eventual RhoA/ROCK activation, leading to increased stiffness. This pathway may increase nuclear localization of myosin related transcription factor A, which can start a new transcription program leading to phenotypic switching of VSMCs [
      • Crosas-Molist E.
      • Meirelles T.
      • López-Luque J.
      • et al.
      Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.
      ]. However, due to the paradoxical findings for RhoA/ROCK in Marfan syndrome patient VSMCs, this particular finding warrants verification.
      RhoA/ROCK signaling can contribute to VSMC stiffness by other mechanisms involving cullin-3, polo-like kinase (Plk1), or neurolipin-2 (NLP-2). Cullin-3 is a component of the machinery responsible for RhoA degradation [
      • Agbor L.N.
      • Ibeawuchi S.C.
      • Hu C.
      • et al.
      Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism.
      ]. A cullin-3 loss-of-function mutation (CUL3Δ9) leads to accumulation of RhoA, and as a result increased arterial stiffness (Fig. 8) [
      • Agbor L.N.
      • Ibeawuchi S.C.
      • Hu C.
      • et al.
      Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism.
      ]. Polo-like kinase 1 (Plk1), a serine/threonine kinase normally expressed during cellular division, regulates Ang II-mediated activation of RhoA/ROCK in VSMCs, causing stress fiber formation which effectively increases cellular stiffness (Fig. 8). Specific deletion of Plk1 in VSMCs causes aberrant arrangement of cellular stress fibers and decreased arterial elasticity [
      • de Cárcer G.
      • Wachowicz P.
      • Martínez-Martínez S.
      • et al.
      Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis.
      ]. In addition, Plk1 modulates Ang II–dependent activation of RhoA, placing Plk1 upstream of RhoA in the regulation of VSMC stiffness [
      • de Cárcer G.
      • Wachowicz P.
      • Martínez-Martínez S.
      • et al.
      Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis.
      ]. In bladder smooth muscle cells, activation of NRP2, a cellular receptor protein, downregulates RhoA/ROCK [
      • Bielenberg D.R.
      • Seth A.
      • Shimizu A.
      • et al.
      Increased smooth muscle contractility in mice deficient for neuropilin 2.
      ]. This leads to a reduction of cellular stiffness due to reduced phosphorylation of MLC [
      • Bielenberg D.R.
      • Seth A.
      • Shimizu A.
      • et al.
      Increased smooth muscle contractility in mice deficient for neuropilin 2.
      ]. While this pathway is active in bladder smooth muscle, testing the potential effect of NRP2 signaling on VSMC phenotype switching and cellular stiffness in particular is worthy of investigation [
      • Bielenberg D.R.
      • Seth A.
      • Shimizu A.
      • et al.
      Increased smooth muscle contractility in mice deficient for neuropilin 2.
      ].

      9. Rho/ROCK-mediated phenotypic switching in “venous” SMCs

      While this review focuses mostly on Rho/ROCK-related pathways that govern phenotypic behavior in arterial territories, it is important to note that phenotypic switching is not limited to arterial tree. Indeed, it was shown that venous SMCs can also undergo phenotypic changes that are implicated in multiple physiological and pathological processes [
      • Zhao J.
      • Jourd'heuil F.L.
      • Xue M.
      • et al.
      ,
      • Bezhaeva T.
      • de Vries M.R.
      • Geelhoed W.J.
      • et al.
      Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.
      ,
      • Bezhaeva T.
      • Wong C.
      • de Vries M.R.
      • et al.
      Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.
      ]. For instance, arteriovenous fistula failure as well as coronary artery venous graft disease are well-known vascular pathological entities where dysfunctional SMC phenotypes lead to maladaptive remodeling at the venous side culminating in thrombosis, stenosis or obstruction [
      • Zhao J.
      • Jourd'heuil F.L.
      • Xue M.
      • et al.
      ,
      • Bezhaeva T.
      • de Vries M.R.
      • Geelhoed W.J.
      • et al.
      Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.
      ,
      • Bezhaeva T.
      • Wong C.
      • de Vries M.R.
      • et al.
      Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.
      ,
      • Shiu Y.T.
      • Rotmans J.I.
      • Geelhoed W.J.
      • et al.
      Arteriovenous conduits for hemodialysis: how to better modulate the pathophysiological vascular response to optimize vascular access durability.
      ,
      • Wadey K.
      • Lopes J.
      • Bendeck M.
      • et al.
      Role of smooth muscle cells in coronary artery bypass grafting failure.
      ]. It is worth mentioning here that in AV fistulas, Rho/ROCK alters the eNOS pathway, a major regulator of endothelial line properties and a known contributor to the arterialization of veins [
      • Bai H.
      • Wei S.
      • Xie B.
      • et al.
      Endothelial nitric oxide synthase (eNOS) mediates neointimal thickness in arteriovenous fistulae with different anastomotic angles in rats.
      ,
      • Satish M.
      • Gunasekar P.
      • Agrawal D.K.
      Pro-inflammatory and pro-resolving mechanisms in the immunopathology of arteriovenous fistula maturation.
      ].
      Despite some similarities in the endpoint of phenotypic switching in arteries and veins, there are many differences at the cellular aspect in the behavior of venous versus arterial VSMCs. Indeed, the molecular pathways dictating these phenotypic behaviors are different and their alteration account for the arterial adaption of veins in the case of arteriovenous fistulas as well as venous grafts. In vein grafts, Rho/ROCK is induced under pulsatile stretch conditions in venous tissues and is shown to trigger venous SMCs phenotypic changes, inducing a transition from the quiescent, or contractile, phenotype, into a synthetic, migratory, and proliferative phenotype [
      • Muto A.
      • Yi T.
      • Harrison K.D.
      • et al.
      Eph-B4 prevents venous adaptive remodeling in the adult arterial environment.
      ]. One translational aspect of this difference is noticeable when statins are part of a therapeutic regimen, as these drugs are known to prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway [
      • Kozai T.
      • Eto M.
      • Yang Z.
      • et al.
      Statins prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway.
      ,
      • Muto A.
      • Yi T.
      • Harrison K.D.
      • et al.
      Eph-B4 prevents venous adaptive remodeling in the adult arterial environment.
      ]. In particular, simvastatin suppresses the RhoA/ROCK pathway leading to inhibition of MMP-9, a matrix metalloproteinase associated with intimal hyperplasia in saphenous vein (SV) bypass grafts [
      • Foo S.S.
      • Turner C.J.
      • Adams S.
      • et al.
      Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly.
      ].
      Likewise, SMCs were found to show distinct metabolic patterns when subjected to venous versus arterial mechanical stretches [
      • Tang Y.
      • Jia Y.
      • Fan L.
      • et al.
      MFN2 prevents neointimal hyperplasia in vein grafts via destabilizing PFK1.
      ]. Indeed, mechanical stretch activates the Rho/ROCK signaling in venous SMCs, which is then followed by phosphorylation and activation of JNK. JNK then phosphorylates and inactivates specific protein 1 (SP1), a transcription factor involved in mitofusin-2 (MFN2) expression. MFN2 is a mechanoresponsive protein that interacts with Phosphofructokinase 1 (PFK1) to mediate PFK1 degradation and therefore suppresses glycolysis in VSMCs. As such, when SP1 is suppressed by active JNK, venous SMCs switch to the glycolytic pathway favoring cellular proliferation and migration [
      • Tang Y.
      • Jia Y.
      • Fan L.
      • et al.
      MFN2 prevents neointimal hyperplasia in vein grafts via destabilizing PFK1.
      ].
      One factor believed to play a prominent role in defining venous identity is Ephrin type-B receptor 4 (Eph-B4). This receptor, a member of the Eph receptor tyrosine kinase (RTK) family, has been described not only as a marker of venous endothelial cells but also as player in the behavior of venous smooth muscle cells [
      • Muto A.
      • Yi T.
      • Harrison K.D.
      • et al.
      Eph-B4 prevents venous adaptive remodeling in the adult arterial environment.
      ]. Importantly, Eph-B4 signaling in venous graft SMCs is believed to limit proper venous adaptation to the arterial environment. In this context, vein graft adaptation is associated with intimal thickening, largely comprised of mature SMCs that spatially localize with decreased Eph-B4 expression [
      • Kudo F.A.
      • Muto A.
      • Maloney S.P.
      • et al.
      Venous identity is lost but arterial identity is not gained during vein graft adaptation.
      ]. Apparently, Rho/ROCK can act as a downstream effector of EphB4 in multiple cell lines. Activation of EphB4 receptor, by its ligand ephrin-B2-Fc, stimulates RhoA and induces cell migration via actin cytoskeleton reorganization [
      • Yang N.Y.
      • Pasquale E.B.
      • Owen L.B.
      • et al.
      The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization.
      ]. In other cells, EphrinB2/EphB4 signaling was implicated in junctional stability of the endothelial line via Rac1/Rho-mediated regulation of cytoskeletal contractility [
      • Frye M.
      • Stritt S.
      • Ortsäter H.
      • et al.
      EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity.
      ]. Given this interaction between ephrinB2/EphB4 and Rho/ROCK, and since both elements are expressed in venous SMCs, it would be tempting to speculate that this interaction modulates some phenotypic behavior of such cells. Nonetheless, this requires further investigation to elucidate the major components of this newly proposed pathway.

      10. Current and potential therapeutic uses

      Fasudil hydrochloride is a RhoA/ROCK inhibitor that was approved for cerebrovascular vasospasm since 1994 [
      • Montagnoli T.L.
      • da Silva J.S.
      • Sudo S.Z.
      • et al.
      ROCK inhibition as potential target for treatment of pulmonary hypertension.
      ]. This may suggest some level of clinical safety for RhoA/ROCK inhibitors despite the theoretical complication raised by the pleiotropic action of Rho/ROCK signaling on a multitude of cell types. Fasudil is also used in the treatment of glaucoma [
      • Testa V.
      • Ferro Desideri L.
      • Della Giustina P.
      • et al.
      An update on ripasudil for the treatment of glaucoma and ocular hypertension.
      ], pulmonary hypertension [
      • Montagnoli T.L.
      • da Silva J.S.
      • Sudo S.Z.
      • et al.
      ROCK inhibition as potential target for treatment of pulmonary hypertension.
      ], and coronary vasospam [
      • Montagnoli T.L.
      • da Silva J.S.
      • Sudo S.Z.
      • et al.
      ROCK inhibition as potential target for treatment of pulmonary hypertension.
      ]. Recently, other RhoA/ROCK inhibitors have been proposed as treatments for sepsis cardiovascular complications [
      • Hahmeyer M.
      • da Silva-Santos J.E.
      Rho-proteins and downstream pathways as potential targets in sepsis and septic shock: what have we learned from basic research.
      ] and diabetic macular edema [
      • Mateos-Olivares M.
      • García-Onrubia L.
      • Valentín-Bravo F.J.
      • et al.
      Rho-kinase inhibitors for the treatment of refractory diabetic macular oedema.
      ]. It has also been suggested to be beneficial in the management of Raynaud's disease [
      • Bailey S.R.
      • Eid A.H.
      • Mitra S.
      • et al.
      Rho kinase mediates cold-induced constriction of cutaneous arteries: role of alpha2C-adrenoceptor translocation.
      ,
      • Jeyaraj S.C.
      • Unger N.T.
      • Eid A.H.
      • et al.
      Cyclic AMP-Rap1A signaling activates RhoA to induce alpha(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.
      ,
      • Motawea H.K.
      • Jeyaraj S.C.
      • Eid A.H.
      • et al.
      Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle alpha2C-adrenoceptors through the actin-binding protein filamin-2.
      ,
      • Fardoun M.M.
      • Nassif J.
      • Issa K.
      • et al.
      Raynaud's phenomenon: a brief review of the underlying mechanisms.
      ] and multiple sclerosis [
      • Aktas O.
      • Küry P.
      • Kieseier B.
      • et al.
      Fingolimod is a potential novel therapy for multiple sclerosis.
      ]. Besides the possibly extensive side effect profile imposed by the activity of RhoA/ROCK in several tissues, limitations for the use of RhoA/ROCK inhibitors include their teratogenicity, as it has been shown to participate in fetal cardiac development [
      • Shimokawa H.
      • Sunamura S.
      • Satoh K.
      RhoA/Rho-kinase in the cardiovascular system.
      ]. The selectivity of the inhibition exerted by the inhibitors also affects the possible clinical use, especially that RhoA/ROCK inhibitors might reduce the activity of other kinases, increasing the risk of undesirable side effects [
      • Cai A.
      • Li L.
      • Zhou Y.
      Pathophysiological effects of RhoA and Rho-associated kinase on cardiovascular system.
      ] with long-term or high dose use. In addition to that, the selectivity of RhoA/ROCK inhibitors to ROCK1 versus ROCK2 isoforms might have some pharmacological implications.
      Over the past 15 years, three dimensional molecular design technologies have become robust approaches for discovering new chemical entities against multiple molecular targets and, among them, structure-based virtual screening is the most prominent. Briefly, thousands of structures from different chemical databases are screened in silico, and a defined number of candidates are examined for biological potency, sensitivity and specificity through experimental trials. In the context of Rho/ROCKs inhibitors, multiple novel drugs are the manifestation of that computational technology [
      • Pan P.
      • Shen M.
      • Yu H.
      • et al.
      Advances in the development of Rho-associated protein kinase (ROCK) inhibitors.
      ]. Such advancement has allowed the modeling of new Rho/ROCK inhibitory agents with high specificity and sensitivity compared to old drugs such as fasudil. For instance, many of these drugs showed superior potency and selectivity against ROCK kinases than towards other kinases like protein kinase A and C [
      • Chen Y.T.
      • Bannister T.D.
      • Weiser A.
      • et al.
      Chroman-3-amides as potent Rho kinase inhibitors.
      ].They also showed high selectivity for ROCK 2 isoforms [
      • Chen Y.T.
      • Bannister T.D.
      • Weiser A.
      • et al.
      Chroman-3-amides as potent Rho kinase inhibitors.
      ,
      • Feng Y.
      • Yin Y.
      • Weiser A.
      • et al.
      Discovery of substituted 4-(pyrazol-4-yl)-phenylbenzodioxane-2-carboxamides as potent and highly selective Rho kinase (ROCK-II) inhibitors.
      ]. These include drugs of the 2,3-diaminopyrazines derivatives, benzodioxane derivatives and benzyl pyridinethiazole-based amide derivatives among many others. Recently, a long-acting hypoxia -activated prodrug of fasudil was designed for the purpose of minimizing the systemic side effects of the parent drug. This prodrug has shown exceptional selectivity against cancer cells as well as hypoxic regions of the lungs in patients with pulmonary hypertension. Consequently, the clinical implications of this novel agent might be promising [
      • Al-Hilal T.A.
      • Hossain M.A.
      • Alobaida A.
      • et al.
      Design, synthesis and biological evaluations of a long-acting, hypoxia-activated prodrug of fasudil, a ROCK inhibitor, to reduce its systemic side-effects.
      ].
      To utilize these advances for mitigating vein graft failure, stenosis and neointimal hyperplasia, a certain degree a vasospecificity is needed in order to load Rho/ROCK inhibitors in the desired location while minimizing the systemic side effects of these agents. To achieve such purpose, multiple mechanistic strategies for drug delivery have been designed. Early testing with a rhosin (RhoA inhibitor)-eluting stent in a leporine carotid model yielded attenuation of neointima formation as compared to a bare metal stent [
      • Huang C.
      • Zhou M.
      • Zheng X.
      RhoA inhibitor-eluting stent attenuates restenosis by inhibiting YAP signaling.
      ]. Additionally, intimal hyperplasia after balloon injury was reduced upon administration of anagliptin in a murine model, through the action of the SOD-1/RhoA/JNK pathway [
      • Li Q.
      • Zhang M.
      • Xuan L.
      • et al.
      Anagliptin inhibits neointimal hyperplasia after balloon injury via endothelial cell-specific modulation of SOD-1/RhoA/JNK signaling in the arterial wall.
      ]. These preliminary results suggested, at the very least, a potential use for RhoA/ROCK inhibitors for preventing predictable, procedural, VSMC pathology mediated vascular injury events.
      One recent strategy for dealing with venous graft failure involves a novel complex electro-spun external sheath which can slowly/gradually and time-dependently release loaded fasudil dihydrochloride, everolimus and simvastatin at the level of a vein graft [
      • Zhang Y.
      • Fang Q.
      • Niu K.
      • et al.
      Time-dependently slow-released multiple-drug eluting external sheath for efficient long-term inhibition of saphenous vein graft failure.
      ]. The combined effect of mechanical restriction exerted by the sheath and loaded fasudil can alleviate the damage on the endothelial line and trigger endothelial repair through Rho/ROCK activation to prevent early phase graft failure [
      • Bafford R.
      • Sui X.X.
      • Wang G.
      • et al.
      Angiotensin II and tumor necrosis factor-alpha upregulate survivin and Kruppel-like factor 5 in smooth muscle cells: potential relevance to vein graft hyperplasia.
      ,
      • Meisel S.R.
      • Ouyang Y.
      • Fishbein M.C.
      • et al.
      Prolonged hypercholesterolemia-induced tissue factor expression in rabbit vein grafts: a potential mechanism for graft failure.
      ]. Middle phase graft failure due to SMC proliferation is efficiently inhibited by the loaded everolimus. Late stage graft failure principally caused by atherosclerosis is prevented by slow-released simvastatin [
      • Conte M.S.
      Molecular engineering of vein bypass grafts.
      ,
      • Kobayashi M.
      • Ishida F.
      • Takahashi T.
      • et al.
      Preventive effect of MK-733 (simvastatin), an inhibitor of HMG-CoA reductase, on hypercholesterolemia and atherosclerosis induced by cholesterol feeding in rabbits.
      ]. It is expected that, in the near future, these and other targeted delivery approaches take the front seat in the management of several cardiovascular diseases.

      11. Conclusion

      While important in various physiological processes, VSMC phenotypic switching, when dysregulated, can contribute to multiple vascular pathologies like atherosclerosis, intimal hyperplasia and hypertension. The relationship between dysregulated VSMC phenotypes and Rho/ROCK signaling demands extensive future investigation, and is a requisite for novel therapeutic approaches as well as for understanding of vascular function. Despite possible challenges relating to non-selectivity, teratogenicity, and pleiotropy, the development of “vasospecific” RhoA/ROCK inhibitors offers a valuable prospective tool for the management of vascular disease.

      CRediT authorship contribution statement

      Tedy Sawma: Writing – original draft. Abdullah Shaito: Writing – original draft. Nicolas Najm: Writing – original draft. Munir Sidani: Writing – original draft. Alexander Orekhov: Writing – review & editing. Ahmed F. El-Yazbi: Writing – review & editing. Rabah Iratni: Writing – review & editing. Ali H. Eid: Conceptualization, Writing – review & editing, Resources, Supervision, Project administration.

      Declaration of interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      References

        • Chen Y.
        • Su X.
        • Qin Q.
        • et al.
        New insights into phenotypic switching of VSMCs induced by hyperhomocysteinemia: role of endothelin-1 signaling.
        Biomed. Pharmacother. 2020; 123109758
        • Gomez D.
        • Owens G.K.
        Smooth muscle cell phenotypic switching in atherosclerosis.
        Cardiovasc. Res. 2012; 95: 156-164
        • Rensen S.S.
        • Doevendans P.A.
        • van Eys G.J.
        Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.
        Neth. Heart J.: monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation. 2007; 15: 100-108
        • Rzucidlo E.M.
        • Martin K.A.
        • Powell R.J.
        Regulation of vascular smooth muscle cell differentiation.
        J. Vasc. Surg. 2007; 45: A25-A32
        • Petsophonsakul P.
        • Furmanik M.
        • Forsythe R.
        • et al.
        Role of vascular smooth muscle cell phenotypic switching and calcification in aortic aneurysm formation.
        Arterioscler. Thromb. Vasc. Biol. 2019; 39: 1351-1368
        • Zhuge Y.
        • Zhang J.
        • Qian F.
        • et al.
        Role of smooth muscle cells in Cardiovascular Disease.
        Int. J. Biol. Sci. 2020; 16: 2741-2751
        • Conklin A.C.
        • Nishi H.
        • Schlamp F.
        • et al.
        Meta-analysis of smooth muscle lineage transcriptomes in atherosclerosis and their relationships to in vitro models.
        Immunometabolism. 2021; 3
        • Badran A.
        • Nasser S.A.
        • Mesmar J.
        • et al.
        Reactive oxygen species: modulators of phenotypic switch of vascular smooth muscle cells.
        Int. J. Mol. Sci. 2020; 21: 8764
        • Fardon M.M.
        • Dehaini H.
        • Baydoun E.
        • et al.
        Estrogen attenuates phenotypic switch of human arteriolar smooth muscle cells: role of p53 and pRb.
        Faseb. J. 2020; 34 (1-1)
        • Rensen S.S.M.
        • Doevendans P.A.F.M.
        • van Eys
        • Gjjm
        Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.
        Neth. Heart J. 2007; 15: 100-108
        • Gui Y.
        • Zheng H.
        • Cao R.Y.
        Foam cells in atherosclerosis: novel insights into its origins, consequences, and molecular mechanisms.
        Front. Cardiovasc Med. 2022; 9845942
        • Matsui T.
        • Amano M.
        • Yamamoto T.
        • et al.
        Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.
        EMBO J. 1996; 15: 2208-2216
        • Kureishi Y.
        • Kobayashi S.
        • Amano M.
        • et al.
        Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.
        J. Biol. Chem. 1997; 272: 12257-12260
        • Crestani S.
        • Webb R.C.
        • da Silva-Santos J.E.
        High-salt intake augments the activity of the RhoA/ROCK pathway and reduces intracellular calcium in arteries from rats.
        Am. J. Hypertens. 2017; 30: 389-399
        • Eto M.
        • Kitazawa T.
        • Matsuzawa F.
        • et al.
        Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor.
        Structure. 2007; 15: 1591-1602
        • Kitazawa T.
        • Eto M.
        • Woodsome T.P.
        • et al.
        Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility.
        J. Biol. Chem. 2000; 275: 9897-9900
        • Wang Y.
        • Zheng X.R.
        • Riddick N.
        • et al.
        ROCK isoform regulation of myosin phosphatase and contractility in vascular smooth muscle cells.
        Circ. Res. 2009; 104: 531-540
        • Calò L.A.
        • Davis P.A.
        • Pagnin E.
        • et al.
        Increased level of p63RhoGEF and RhoA/Rho kinase activity in hypertensive patients.
        J. Hypertens. 2014; 32: 331-338
        • Gerthoffer W.T.
        Mechanisms of vascular smooth muscle cell migration.
        Circ. Res. 2007; 100: 607-621
        • Chava K.R.
        • Karpurapu M.
        • Wang D.
        • et al.
        CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.
        Arterioscler. Thromb. Vasc. Biol. 2009; 29: 809-815
        • Qi Y.
        • Liang X.
        • Dai F.
        • et al.
        RhoA/ROCK pathway activation is regulated by AT1 receptor and participates in smooth muscle migration and dedifferentiation via promoting actin cytoskeleton polymerization.
        Int. J. Mol. Sci. 2020; 21: 5398
        • Muniyappa R.
        • Xu R.
        • Ram J.L.
        • et al.
        Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells.
        Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1762-H1768
        • Mugabe B.E.
        • Yaghini F.A.
        • Song C.Y.
        • et al.
        Angiotensin II-induced migration of vascular smooth muscle cells is mediated by p38 mitogen-activated protein kinase-activated c-Src through spleen tyrosine kinase and epidermal growth factor receptor transactivation.
        J. Pharmacol. Exp. Therapeut. 2010; 332: 116-124
        • Huang J.
        • Xie L.D.
        • Luo L.
        • et al.
        Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro.
        Mol. Cell. Biochem. 2014; 390: 115-121
        • Huang C.
        • Jacobson K.
        • Schaller M.D.
        MAP kinases and cell migration.
        J. Cell Sci. 2004; 117: 4619-4628
        • Chava K.R.
        • Karpurapu M.
        • Wang D.
        • et al.
        CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.
        Arterioscler. Thromb. Vasc. Biol. 2009; 29: 809-815
        • Goncharova E.A.
        • Vorotnikov A.V.
        • Gracheva E.O.
        • et al.
        Activation of p38 MAP-kinase and caldesmon phosphorylation are essential for urokinase-induced human smooth muscle cell migration.
        Biol. Chem. 2002; 383: 115-126
        • Kusch A.
        • Tkachuk S.
        • Haller H.
        • et al.
        Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2 interaction.
        J. Biol. Chem. 2000; 275: 39466-39473
        • Kiian I.
        • Tkachuk N.
        • Haller H.
        • et al.
        Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signaling pathway.
        Thromb. Haemostasis. 2003; 89: 904-914
        • Qi Y.X.
        • Qu M.J.
        • Yan Z.Q.
        • et al.
        Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho-GDIalpha, Rac1, and p38 pathway.
        J. Cell. Biochem. 2010; 109: 906-914
        • Wang H.H.
        • Nakamura A.
        • Matsumoto A.
        • et al.
        Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine.
        Am. J. Physiol. Heart Circ. Physiol. 2009; 296: H1683-H1693
        • Li S.
        • Tanaka H.
        • Wang H.H.
        • et al.
        Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation.
        Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H1262-H1272
        • Heldin C.H.
        Structural and functional studies on platelet-derived growth factor.
        EMBO J. 1992; 11: 4251-4259
        • Raines E.W.
        PDGF and cardiovascular disease.
        Cytokine Growth Factor Rev. 2004; 15: 237-254
        • Wang J.
        • Zhang C.
        • Li C.
        • et al.
        MicroRNA-92a promotes vascular smooth muscle cell proliferation and migration through the ROCK/MLCK signalling pathway.
        J. Cell Mol. Med. 2019; 23: 3696-3710
        • Hartmann S.
        • Ridley A.J.
        • Lutz S.
        The function of rho-associated kinases ROCK1 and ROCK2 in the pathogenesis of cardiovascular disease.
        Front. Pharmacol. 2015; 6: 276
        • Zhao Y.
        • Lv M.
        • Lin H.
        • et al.
        ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells.
        IUBMB Life. 2012; 64: 194-202
        • Klemke R.L.
        • Cai S.
        • Giannini A.L.
        • et al.
        Regulation of cell motility by mitogen-activated protein kinase.
        J. Cell Biol. 1997; 137: 481-492
        • Morrison D.L.
        • Sanghera J.S.
        • Stewart J.
        • et al.
        Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1.
        Biochem. Cell Biol. = Biochimie et biologie cellulaire. 1996; 74: 549-557
        • Totsukawa G.
        • Wu Y.
        • Sasaki Y.
        • et al.
        Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts.
        J. Cell Biol. 2004; 164: 427-439
        • Webb D.J.
        • Donais K.
        • Whitmore L.A.
        • et al.
        FAK–Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.
        Nat. Cell Biol. 2004; 6: 154-161
        • Dourdin N.
        • Bhatt A.K.
        • Dutt P.
        • et al.
        Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts.
        J. Biol. Chem. 2001; 276: 48382-48388
        • Huttenlocher A.
        • Palecek S.P.
        • Lu Q.
        • et al.
        Regulation of cell migration by the calcium-dependent protease calpain.
        J. Biol. Chem. 1997; 272: 32719-32722
        • Cuevas B.D.
        • Abell A.N.
        • Witowsky J.A.
        • et al.
        MEKK1 regulates calpain‐dependent proteolysis of focal adhesion proteins for rear‐end detachment of migrating fibroblasts.
        EMBO J. 2003; 22: 3346-3355
        • Ahmed S.
        • Warren D.T.
        Vascular smooth muscle cell contractile function and mechanotransduction.
        Vessel Plus. 2018; 2: 36
        • Yang N.
        • Higuchi O.
        • Ohashi K.
        • et al.
        Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.
        Nature. 1998; 393: 809-812
        • San Martín A.
        • Lee M.Y.
        • Williams H.C.
        • et al.
        Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells.
        Circ. Res. 2008; 102: 432-438