Advertisement

Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemia-induced neovascularization in hypercholesterolemic conditions

      Highlights

      • Neovascularization is impaired in animal models and patients presenting hypercholesterolemia.
      • MicroRNA-146b is reduced by hypercholesterolemia, which leads to TRAF6-dependent inflammation and impaired angiogenesis.
      • Forced expression of miR-146b could reduce inflammation and improve neovascularization in atherosclerotic conditions.

      Abstract

      Background and aims

      MicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions.

      Methods

      Next generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE−/−) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro.

      Results

      We found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE−/− mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE−/− mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs).

      Conclusions

      Hypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

      Graphical abstract

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Steinberg D.
        Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime.
        Nat. Med. 2002; 8: 1211-1217
        • Hansson G.K.
        Inflammation, atherosclerosis, and coronary artery disease.
        N. Engl. J. Med. 2005; 352: 1685-1695
        • Libby P.
        • Ridker P.M.
        • Hansson G.K.
        • et al.
        Inflammation in atherosclerosis: from pathophysiology to practice.
        J. Am. Coll. Cardiol. 2009; 54: 2129-2138
        • Ridker P.M.
        • Everett B.M.
        • Thuren T.
        • et al.
        Antiinflammatory therapy with canakinumab for atherosclerotic disease.
        N. Engl. J. Med. 2017; 377: 1119-1131
        • Gimbrone Jr., M.A.
        • Garcia-Cardena G.
        Endothelial cell dysfunction and the pathobiology of atherosclerosis.
        Circ. Res. 2016; 118: 620-636
        • Gareus R.
        • Kotsaki E.
        • Xanthoulea S.
        • et al.
        Endothelial cell-specific NF-kappaB inhibition protects mice from atherosclerosis.
        Cell Metabol. 2008; 8: 372-383
        • Cheng H.S.
        • Sivachandran N.
        • Lau A.
        • et al.
        MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways.
        EMBO Mol. Med. 2013; 5: 1017-1034
        • Taganov K.D.
        • Boldin M.P.
        • Chang K.J.
        • et al.
        NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.
        Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12481-12486
        • Chen L.J.
        • Chuang L.
        • Huang Y.H.
        • et al.
        MicroRNA mediation of endothelial inflammatory response to smooth muscle cells and its inhibition by atheroprotective shear stress.
        Circ. Res. 2015; 116: 1157-1169
        • Li K.
        • Ching D.
        • Luk F.S.
        • et al.
        Apolipoprotein E enhances microRNA-146a in monocytes and macrophages to suppress nuclear factor-kappaB-driven inflammation and atherosclerosis.
        Circ. Res. 2015; 117: e1-e11
        • Echavarria R.
        • Mayaki D.
        • Neel J.C.
        • et al.
        Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: role of miR-146b-5p.
        Cardiovasc. Res. 2015; 106: 465-477
        • Losordo D.W.
        • Dimmeler S.
        Therapeutic angiogenesis and vasculogenesis for ischemic disease: part 1: angiogenic cytokines.
        Circulation. 2004; 109: 2487-2491
        • D'Amore P.A.
        • Thompson R.W.
        Mechanisms of angiogenesis.
        Annu. Rev. Physiol. 1987; 49: 453-464
        • Asahara T.
        • Murohara T.
        • Sullivan A.
        • et al.
        Isolation of putative progenitor endothelial cells for angiogenesis.
        Science. 1997; 275: 964-967
        • Asahara T.
        • Masuda H.
        • Takahashi T.
        • et al.
        Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.
        Circ. Res. 1999; 85: 221-228
        • Urbich C.
        • Dimmeler S.
        Endothelial progenitor cells: characterization and role in vascular biology.
        Circ. Res. 2004; 95: 343-353
        • Van Belle E.
        • Rivard A.
        • Chen D.
        • et al.
        Hypercholesterolemia attenuates angiogenesis but does not preclude augmentation by angiogenic cytokines.
        Circulation. 1997; 96: 2667-2674
        • Couffinhal T.
        • Silver M.
        • Kearney M.
        • et al.
        Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice.
        Circulation. 1999; 99: 3188-3198
        • Tirziu D.
        • Moodie K.L.
        • Zhuang Z.W.
        • et al.
        Delayed arteriogenesis in hypercholesterolemic mice.
        Circulation. 2005; 112: 2501-2509
        • Chen J.Z.
        • Zhang F.R.
        • Tao Q.M.
        • et al.
        Number and activity of endothelial progenitor cells from peripheral blood in patients with hypercholesterolaemia.
        Clin. Sci. (Lond.). 2004; 107: 273-280
        • Desjarlais M.
        • Dussault S.
        • Dhahri W.
        • et al.
        MicroRNA-150 modulates ischemia-induced neovascularization in atherosclerotic conditions.
        Arterioscler. Thromb. Vasc. Biol. 2017; 37: 900-908
        • Maingrette F.
        • Dussault S.
        • Dhahri W.
        • et al.
        Psychological stress impairs ischemia-induced neovascularization: protective effect of fluoxetine.
        Atherosclerosis. 2015; 241: 569-578
        • Annex B.H.
        Therapeutic angiogenesis for critical limb ischaemia.
        Nat. Rev. Cardiol. 2013; 10: 387-396
        • Paterson M.R.
        • Kriegel A.J.
        MiR-146a/b: a family with shared seeds and different roots.
        Physiol. Genom. 2017; 49: 243-252
        • Zhu K.
        • Pan Q.
        • Zhang X.
        • et al.
        MiR-146a enhances angiogenic activity of endothelial cells in hepatocellular carcinoma by promoting PDGFRA expression.
        Carcinogenesis. 2013; 34: 2071-2079
        • Zhu H.Y.
        • Bai W.D.
        • Liu J.Q.
        • et al.
        Up-regulation of FGFBP1 signaling contributes to miR-146a-induced angiogenesis in human umbilical vein endothelial cells.
        Sci. Rep. 2016; 6: 25272
        • Li Y.
        • Zhu H.
        • Wei X.
        • et al.
        LPS induces HUVEC angiogenesis in vitro through miR-146a-mediated TGF-beta1 inhibition.
        Am J Transl Res. 2017; 9: 591-600
        • Stickel N.
        • Prinz G.
        • Pfeifer D.
        • et al.
        MiR-146a regulates the TRAF6/TNF-axis in donor T cells during GVHD.
        Blood. 2014; 124: 2586-2595
        • Bruneau S.
        • Datta D.
        • Flaxenburg J.A.
        • et al.
        TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor.
        Biochem. Biophys. Res. Commun. 2012; 419: 66-71
        • Silvestre J.S.
        • Mallat Z.
        • Tedgui A.
        • et al.
        Post-ischaemic neovascularization and inflammation.
        Cardiovasc. Res. 2008; 78: 242-249
        • Coughlin C.M.
        • Salhany K.E.
        • Wysocka M.
        • et al.
        Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.
        J. Clin. Investig. 1998; 101: 1441-1452
        • Kopp H.G.
        • Hooper A.T.
        • Broekman M.J.
        • et al.
        Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization.
        J. Clin. Investig. 2006; 116: 3277-3291
        • Fajardo L.F.
        • Kwan H.H.
        • Kowalski J.
        • et al.
        Dual role of tumor necrosis factor-alpha in angiogenesis.
        Am. J. Pathol. 1992; 140: 539-544
        • Gardiner T.A.
        • Gibson D.S.
        • de Gooyer T.E.
        • et al.
        Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy.
        Am. J. Pathol. 2005; 166: 637-644
        • Vasa M.
        • Fichtlscherer S.
        • Aicher A.
        • et al.
        Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.
        Circ. Res. 2001; 89: E1-7
        • Heinrich E.M.
        • Dimmeler S.
        MicroRNAs and stem cells: control of pluripotency, reprogramming, and lineage commitment.
        Circ. Res. 2012; 110: 1014-1022
        • Zhao J.L.
        • Rao D.S.
        • O'Connell R.M.
        • et al.
        MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells in mice.
        Elife. 2013; 2e00537
        • Su Z.F.
        • Sun Z.W.
        • Zhang Y.
        • et al.
        Regulatory effects of miR-146a/b on the function of endothelial progenitor cells in acute ischemic stroke in mice.
        Kaohsiung J. Med. Sci. 2017; 33: 369-378
        • Pronk C.J.
        • Veiby O.P.
        • Bryder D.
        • et al.
        Tumor necrosis factor restricts hematopoietic stem cell activity in mice: involvement of two distinct receptors.
        J. Exp. Med. 2011; 208: 1563-1570
        • Fang J.
        • Bolanos L.C.
        • Choi K.
        • et al.
        Ubiquitination of hnRNPA1 by TRAF6 links chronic innate immune signaling with myelodysplasia.
        Nat. Immunol. 2017; 18: 236-245