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Hydroxytyrosol suppresses MMP-9 and COX-2 activity and expression in activated human monocytes via PKCα and PKCβ1 inhibition

      Highlights

      • Hydroxytyrosol blunts MMP-9 release and extracellular matrix invasive potential.
      • Hydroxytyrosol reduces COX-2 expression and activity in human activated monocytes.
      • Hydroxytyrosol down-regulates phorbol ester-induced NF-κB activation.
      • Conventional PKCs are involved in MMP-9 and COX-2 induction by phorbol ester.
      • Hydroxytyrosol reduces PKCα and PKCβ1 activation.

      Abstract

      Objective

      Hydroxytyrosol (HT), the major olive oil antioxidant polyphenol in cardioprotective Mediterranean diets, is endowed with anti-inflammatory and anti-atherosclerotic activity. The production of cyclooxygenase (COX)-2-dependent inflammatory eicosanoids and the functionally linked release of matrix metalloproteinase (MMP)-9 by macrophages likely contribute to plaque instability leading to acute coronary events. Objective of the study was to examine the HT effects on inflammatory markers in human activated monocytes, including MMP-9 and COX-2 activity and expression and explore HT underlying mechanisms.

      Methods and results

      Human peripheral blood mononuclear cells (PBMC) and U937 monocytes were treated with 1–10 μmol/L HT before activation with phorbol myristate acetate (PMA). HT blunted monocyte matrix invasive potential and reduced MMP-9 release and expression at zymography, ELISA and RT-PCR, with an IC50 = 10 μmol/L ( P< 0.05), without affecting tissue inhibitor of metalloproteinase (TIMP)-1. Moreover, HT inhibited prostaglandin (PG)E2 production and COX-2 expression, without affecting COX-1. These effects were mediated by inhibition of transcription factor nuclear factor (NF)-κB and protein kinase C (PKC)α and PKCβ1 activation.

      Conclusion

      HT, at nutritionally relevant concentrations, reduces MMP-9 and COX-2 induction in activated human monocytes via PKCα and PKCβ1 inhibition, thus featuring novel anti-inflammatory properties. Overall, such results contribute to explaining the vascular protective effects by olive oil polyphenols in Mediterranean diets.

      Graphical abstract

      Keywords

      1. Introduction

      Monocytes/macrophages play a crucial role in the inflammatory processes associated with atherosclerotic plaque development and progression toward instability [
      • Libby P.
      Inflammation and cardiovascular disease mechanisms.
      ]. Macrophages resident in human and experimental atherosclerosis co-localize with and release active matrix metalloproteinases (MMPs), including the gelatinase MMP-9, which is specialized in the digestion of basement membrane collagens and elastin, and is implicated in atherogenesis, unstable coronary syndromes, and in aortic aneurysms [
      • Dollery C.M.
      • Libby P.
      Atherosclerosis and proteinase activation.
      ]. Reactive oxygen species (ROS) have been implicated in MMP-9 zymogen activation and, accordingly, the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited both macrophage gelatinolytic activity and MMP-9 expression in vitro and in experimental atheroma [
      • Galis Z.S.
      • Asanuma K.
      • Godin D.
      • Meng X.
      N-acetyl-cysteine decreases the matrix-degrading capacity of macrophage-derived foam cells: new target for antioxidant therapy?.
      ]. Therefore, MMP-9 expression and activity represent an attractive therapeutic target susceptible to antioxidant modulation.
      As a part of the inflammatory responses within atheroma, macrophage COX-2-dependent eicosanoids, mainly PGE2, participate in the pathogenesis of plaque growth and instability, through a variety of processes, including inflammation, enhanced leukocyte–endothelial interactions, increased vascular tone, platelet aggregation and proteolysis [
      • Linton M.F.
      • Fazio S.
      Cyclooxygenase-2 and inflammation in atherosclerosis.
      ]. PGE2 is capable of inducing the expression and activity of MMP-9 and MMP-2 in macrophages through a PGE2–cAMP pathway [
      • Pavlovic S.
      • Du B.
      • Sakamoto K.
      • et al.
      Targeting prostaglandin E2 receptors as an alternative strategy to block cyclooxygenase-2-dependent extracellular matrix-induced matrix metalloproteinase-9 expression by macrophages.
      ].
      Virgin olive oil, the most representative food of the traditional Mediterranean diet, is associated with protection from cardiovascular morbidity and mortality [
      • Trichopoulou A.
      • Costacou T.
      • Bamia C.
      • Trichopoulos D.
      Adherence to a Mediterranean diet and survival in a Greek population.
      ,
      • Estruch R.
      • Ros E.
      • Salas-Salvado J.
      • et al.
      Primary prevention of cardiovascular disease with a Mediterranean diet.
      ]. Many of the beneficial effects can be attributed to virgin olive oil minor components [
      • Covas M.I.
      Olive oil and the cardiovascular system.
      ,
      • Ruano J.
      • Lopez-Miranda J.
      • de la Torre R.
      • et al.
      Intake of phenol-rich virgin olive oil improves the postprandial prothrombotic profile in hypercholesterolemic patients.
      ], including the antioxidant polyphenol hydroxytyrosol (HT), endowed with potent anti-platelet, anti-inflammatory and anti-atherogenic properties [
      • Granados-Principal S.
      • Quiles J.L.
      • Ramirez-Tortosa C.L.
      • Sanchez-Rovira P.
      • Ramirez-Tortosa M.C.
      Hydroxytyrosol: from laboratory investigations to future clinical trials.
      ]. High intake of phenol-rich olive oils repressed the expression of several pro-inflammatory and pro-atherosclerotic genes, thereby promoting a less inflammatory profile in peripheral blood mononuclear cells [
      • Camargo A.
      • Ruano J.
      • Fernandez J.M.
      • et al.
      Gene expression changes in mononuclear cells in patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil.
      ]. However, HT effects on MMP-9 expression in human monocytes and molecular mechanisms underlying HT anti-inflammatory properties are not well understood. Therefore, we sought to evaluate the anti-inflammatory potential of HT in human monocytes primed by phorbol myristate acetate (PMA) by evaluating its effects on MMP-9 expression and its relation with COX-2/PGE2 pathway, and we investigated underlying mechanisms.

      2. Materials and methods

      A more detailed description of methods and chemicals used in this study is provided in the online Supporting Methods.

      2.1 Materials

      Hydroxytyrosol (3,4-dihydroxyphenylethanol) and prostaglandin (PG)E2 were obtained from Cayman (Cayman Chemical, Ann Arbor, MI). The non-specific PKC inhibitor Ro-318220 and the selective PKCδ inhibitor rottlerin were obtained from Alexis Biochemicals. The COX-2 selective inhibitor NS-398, the selective PKCα and β inhibitor Gö 6976, and the selective PKCβ2 inhibitor CGP53353 were from Calbiochem (La Jolla, CA). All other chemicals were purchased from Sigma–Aldrich, unless otherwise indicated.

      2.2 Cell culture and treatment

      Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood of healthy donors in accordance with the principles outlined in the Declaration of Helsinki. Human cells were obtained from discarded materials and treated anonymously. The human monocytic cell line U937 was purchased from the American Type Culture Collection (Rockville, MD). To evaluate HT effects on monocyte activation, PBMC and U937 cells were cultured in the absence or presence of 1–10 μmol/L HT for 1 h before stimulation with 30 nmol/L phorbol myristate acetate (PMA) for 0–24 h. Alternatively, U937 monocytes were fully differentiated into macrophages by 100 nmol/L PMA stimulation for 48 h, and treated with 0.1–10 μmol/L HT for additional 24 h. In some studies, COX or PKC inhibitors were added 30 min before PMA stimulation. At the end of treatment, conditioned media were collected for zymography and ELISA, and cells were rinsed with ice-cold PBS and collected for Western and RT-PCR analysis. Detailed protocols are provided in the online Supporting Methods.

      2.3 MMP zymography

      MMP-9 release in conditioned media was evaluated by gelatin zymography, which is detailed in the online Supporting Methods. Briefly, conditioned media were mixed with non-reducing Laemmli sample buffer and subjected to electrophoresis on SDS-8% polyacrylamide gel containing 1 mg/mL gelatin. After electrophoresis, gels were stained with Coomassie brilliant blue and analyzed by Scion Image software.

      2.4 MMP-9 and TIMP-1 protein release

      The levels of secreted MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 protein in conditioned media were quantified using the highly specific Biotrack ELISA system (GE Healthcare, Freiburg, Germany), according to the manufacturer's instructions.

      2.5 Measurement of cyclooxygenase activity

      The concentration of PGE2 in the conditioned media was determined with a competitive enzyme immunoassay from Cayman, according to the manufacturer's instructions.

      2.6 Cell lysis and immunoblotting

      Total and fractionated cellular proteins were isolated and immunoblotted as previously described [
      • Massaro M.
      • Habib A.
      • Lubrano L.
      • et al.
      The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKC epsilon inhibition.
      ], using antibodies against COX-2 (Cayman), β-actin (Sigma), COX-1, PKCα, PKCβ1, Na+/K+-ATPase α1 subunit, p65 subunit of NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA), and H1 histone (Active Motif, La Hulpe, Belgium).

      2.7 RNA isolation and analysis

      Monocytic cells were treated with 1–10 μmol/L HT for 1 h before stimulation with 30 nmol/L PMA for 4 h. Total RNA was isolated using the acid guanidinium thiocyanate method as detailed in online Supporting Methods. Briefly, RNA (1 μg) was used as a template for cDNA synthesis and then semiquantitative and quantitative PCR was carried out using specific primers for MMP-9, COX-2, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and β-actin or 18 S as endogenous controls.

      2.8 Preparation of nuclear protein extracts and electrophoretic mobility shift assay (EMSA)

      U937 cells (5 × 106) were treated with 0–10 μmol/L HT for 1 h and then stimulated with 30 nmol/L PMA for additional 1 h, after which nuclear proteins were purified as detailed in online Supporting Methods. Usually 5 μg of nuclear proteins were used for EMSA assay after binding to 32P-labeled NF-κB consensus oligonucleotides. Shifted bands were electrophoresed and autoradiographed.

      2.9 NF-κB activation

      NF-κB activation was assayed using the Active Motif's ELISA-based “TransAM kit” (Active Motive, La Hulpe, Belgium), following the manufacturer's protocol. For further details, see the online Supporting Methods.

      2.10 Assessment of PKC translocation

      After exposure to HT and subsequent stimulation with PMA for 20 min, monocytes were harvested and fractionated into cytosolic and membrane (particulate) fractions as previously described [
      • Massaro M.
      • Habib A.
      • Lubrano L.
      • et al.
      The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKC epsilon inhibition.
      ]. The membrane and cytosolic fractions were separated by SDS/PAGE and immunoblotted to detect specific isoform translocation using antibodies against PKCα and β1. Na+/K+ ATPase and β-actin served as internal controls for the separation of membrane and cytosolic fractions, respectively.

      2.11 Statistical analysis

      Results are expressed as means ± SD of at least three independent experiments performed in triplicate. The Student's t test was used for comparing means between control group and compound-treated group. Multiple comparisons were performed by one-way ANOVA. A P level <0.05 was considered statistically significant.

      3. Results

      3.1 HT inhibits PMA-induced MMP-9 and COX-2 activity and expression

      To evaluate HT effect on monocytes activation, human monocytes from PBMC and U937 cells were pretreated with HT and activated by PMA, an exogenous protein kinase C (PKC) activator, to express the inflammatory mediators MMP-9 and COX-2. Firstly, we found that HT inhibited PMA-induced MMP-9 protein release at gelatin zymography (Fig. 1A and B), and at ELISA (Fig. 1C), in a concentration-dependent manner with a significant inhibitory effect already evident at 1 μmol/L (P < 0.05). These effects occurred without any influence on cell viability as assessed by cell count, morphology, amido-black, and MTT assays (not shown). As a functional correlate of MMP-9 inhibition, we found that HT concentration-dependently reduced the extracellular matrix invasive potential of activated monocytes, measured as fluorescent gelatin degradation (Supplementary Fig. 1). Since MMP-9 activity is tightly regulated at multiple levels [
      • Dollery C.M.
      • Libby P.
      Atherosclerosis and proteinase activation.
      ], we assessed whether HT influenced the levels of tissue inhibitor of metalloproteinase (TIMP)-1, the MMP-9 specific inhibitor. HT did not modify PMA-induced TIMP-1 levels (Supplementary Fig. 2). In agreement with the modulation of MMP-9 protein release, HT significantly reduced PMA-induced MMP-9 mRNA levels (Fig. 1D).
      Figure thumbnail gr1
      Fig. 1Inhibition of PMA-induced MMP-9 activity and expression by HT. PBMC (A and C) and U937 cells (B–D) were treated for 1 h with HT (0–10 μmol/L) and then stimulated with 30 nmol/L PMA for 24 h (A–C) or 4 h (D). MMP-9 release was assessed by gelatin zymography (A and B), and by ELISA (C), and mRNA levels by semi-quantitative RT-PCR (D). In A and B, representative zymographies are shown. Densitometric values of MMP-9 gelatinolytic bands are expressed as percent vs PMA (n = 4). (D) MMP-9 mRNA levels were normalized to β-actin and expressed as fold induction vs PMA, made = 1 (n = 3). *P < 0.05 vs PMA alone.
      Secondly, we analyzed HT effect on the pro-inflammatory COX-2/PGE2 axis, and found that HT reduced PMA-induced PGE2 production by both PBMC and U937 cells with a significant effect already evident at 1 μmol/L (P< 0.05) (Fig. 2A). Such effect was associated with a reduction in COX-2, but not COX-1, protein expression (Fig. 2B and C). HT exerted a pre-translational effect since it reduced stimulated COX-2 mRNA levels at RT-PCR (Fig. 2D). Worthy, HT did not influence both MMP-9 and COX-2 in unstimulated condition (not shown). Similar inhibitory effect exerted by HT on both PGE2 and MMP-9 release was obtained in human monocyte-derived macrophages (Supplementary Fig. 3).
      Figure thumbnail gr2
      Fig. 2Suppression of PMA-induced activity and expression of COX-2 by HT. PBMC and U937 cells were treated for 1 h with HT (0–10 μmol/L) and then stimulated with 30 nmol/L PMA for 16 h (A–C) or 4 h (D). After treatments, the conditioned media were analyzed for PGE2 levels by ELISA (A). Protein extracts of U937 cells (B) and PBMC (C) were analyzed by Western blotting for COX-1 and COX-2 and normalized to β-actin. (D) mRNA levels for COX-2 in U937 cells were analyzed by semi-quantitative RT-PCR and COX-2/β-actin mRNA ratio is reported as percent vs PMA. Bars represent mean ± SD (n = 3). *P < 0.05 vs PMA alone.
      Having previously verified the similarity of behavior of PBMC and U937 cells in response to stimuli and HT, further experiments were restricted to U937 cells.

      3.2 Involvement of COX-2 activity in MMP-9 expression and role of HT

      Since COX-2 activity has been involved in the regulation of MMP expression [
      • Pavlovic S.
      • Du B.
      • Sakamoto K.
      • et al.
      Targeting prostaglandin E2 receptors as an alternative strategy to block cyclooxygenase-2-dependent extracellular matrix-induced matrix metalloproteinase-9 expression by macrophages.
      ], we verified such involvement in our experimental conditions and HT interference with this pathway. We analyzed the time-dependence of both COX-2 and MMP-9 induction upon PMA treatment, showing that COX-2 protein expression, already evident 2 h after PMA, preceded MMP-9 release (Supplementary Fig. 4). U937 cells exposure to exogenous PGE2 for 24 h increased MMP-9 release (Supplementary Fig. 5A), confirming the involvement of a PGE2-dependent pathway in MMP-9 induction. The specific inhibition of COX-1 activity by a low concentration of indomethacin (0.5 μmol/L) did not significantly affect MMP-9 production, whereas concentrations able to inhibit also COX-2 activity (5 μmol/L) resulted in a significant reduction in MMP-9 activity, thus suggesting the selective – albeit not exclusive – involvement of COX-2-derived prostaglandins in MMP-9 induction (Supplementary Fig. 5B). This was also confirmed by the use of the selective COX-2 inhibitor NS-398 (Supplementary Fig. 5B).
      To establish if HT effects on MMP-9 production were mediated by the reduction in COX-2 activity and PGE2 production, U937 cells were co-treated with both HT and NS-398 prior to PMA stimulation. Although 5 μmol/L NS-398 abolished PMA-induced PGE2 production (Supplementary Fig. 5C), a residual MMP-9 activity was evident (Supplementary Fig. 5D), thus indicating that MMP-9 induction by PMA was only in part dependent on COX-2 activity in our experimental conditions. Moreover, co-treatment with HT and NS-398 determined an additional reduction in MMP-9 activity compared with NS-398 alone (Supplementary Fig. 5D), suggesting that HT-mediated inhibition of MMP-9 also involved a direct COX-2-independent mechanism.

      3.3 HT suppresses NF-κB activation in human monocytoid cells

      Since both COX-2 and MMP-9 gene expression are regulated by the redox-sensitive transcription factor NF-κB [
      • Sato H.
      • Seiki M.
      Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells.
      ,
      • Tanabe T.
      • Tohnai N.
      Cyclooxygenase isozymes and their gene structures and expression.
      ], we verified HT effect on PMA-stimulated NF-κB activation. We found that 10 μmol/L HT caused a 60% reduction of NF-κB activation, as showed by a decrease in the amount of NF-κB shifted complex induced by PMA at EMSA as well as in the p65 NF-κB subunit DNA binding activity, as assessed by an ELISA-based method (P < 0.05) (Fig. 3A and B). In accordance, HT reduced p65 NF-κB subunit nuclear translocation and IκBα cytosolic levels at Western analysis (Fig. 3C and D), suggesting an interference by HT with the signaling pathways upstream of NF-κB activation.
      Figure thumbnail gr3
      Fig. 3HT suppression of PMA-mediated NF-κB activation, p65 NF-κB nuclear translocation and IκB levels. U937 cells were treated with HT for 1 h and then stimulated with PMA for additional 1 h, after which nuclear proteins were assessed by EMSA (A), ELISA (B) or Western analysis (C). (A) The autoradiography shows shifted bands of 32P-labeled NF-κB consensus oligonucleotide upon binding to nuclear protein extracts. Compared with the unstimulated condition, PMA induced the appearance of a shifted band of labeled oligonucleotides, indicating the formation of DNA–protein complexes. HT reduces the intensity of the shifted bands. The specificity of the NF-κB shifted band was determined by competition with unlabeled (cold) wild type and mutant NF-κB oligonucleotides. (B) NF-κB activity was measured in nuclear extracts using an ELISA-based TransAM kit (n = 3). (C) Nuclear proteins were assayed by Western analysis for p65 NF-κB subunit and data were normalized to H1 histone. (D) U937 cells were treated with HT for 1 h and then stimulated with PMA for 20 min, after which cytosolic proteins were assessed by Western analysis for IκBα. *P < 0.05 vs PMA alone.
      In the same experimental conditions, HT (1–10 μmol/L) in a concentration-dependent manner significantly reduced the mRNA levels of other NF-κB-regulated pro-inflammatory genes, such as MCP-1, ICAM-1, IL-1β, and TNF-α (Supplementary Fig. 6).

      3.4 HT decreases PKCα and PKCβ1 activation

      To dissect HT effects on the signaling events leading to NF-κB activation, we analyzed HT effects on PKC activation as induced by PMA. By using several PKC inhibitors, we found that PMA-induced COX-2 expression and MMP-9 release as well as p65 NF-κB subunit nuclear translocation are mediated by PKCα and PKCβ1 activation (Fig. 4). This suggests that conventional PKCα and PKCβ1 are implicated in the signaling events upstream of MMP-9 and COX-2 activation, and represent potential molecular targets for HT effects. Moreover, we also verified that MCP-1, ICAM-1, IL-1β, and TNF-α gene expression in response to PMA involved PKCα and PKCβ1 activation (data not shown). We therefore evaluated the HT effect on PKCα and PKCβ1 membrane translocation, a marker of PKC activation. PMA activated both PKCα and PKCβ1, but pre-treatment with HT before PMA stimulation significantly reduced PKCβ1 and, to a lesser extent, PKCα membrane translocation, without significantly affecting two internal controls, Na+/K+ ATPase and β-actin (Fig. 5A and B).
      Figure thumbnail gr4
      Fig. 4Role of conventional PKCs in MMP-9 and COX-2 expression and p65 NF-κB nuclear translocation. U937 cells were either left untreated or treated with the general PKC inhibitor Ro-318220 (Ro), the conventional PKC α/β1-selective inhibitor Gö 6976 (Gö), the PKCδ-specific inhibitor rottlerin (Rot), or the PKCβ2-specific inhibitor CGP53353 (CGP) for 30 min before PMA stimulation for 24 h (A and B) or 1 h (C). The conditioned media were analyzed for MMP-9 activity by zymography (A), total protein extracts were analyzed for COX-2 and β-actin (B) and nuclear proteins for p65 NF-κB by Western analysis (C). Representative immunoblots and quantifications are shown. Data are expressed as percent vs PMA alone and represent mean ± SD (n = 3). *P < 0.05 vs PMA alone.
      Figure thumbnail gr5
      Fig. 5The effect of HT on PMA-mediated membrane translocation of selected PKC isoforms. U937 cells were treated for 1 h with 10 μmol/L HT before 30 nmol/L PMA stimulation for 20 min. Equal amounts of particulate (membrane) and cytosolic fractions were assayed by Western analysis, using an antibody toward the PKCα or PKCβ1 isoform. Representative immunoblots (A) and quantifications (B) are shown. Densitometric values of PKC isoforms are expressed as percent vs PMA. Bars represent mean ± SD (n = 3). *P < 0.05 vs PMA alone.

      4. Discussion

      This study shows that the olive oil polyphenol HT, at nutritionally relevant concentrations, inhibits COX-2 and MMP-9 activity and expression in human activated monocytes via inhibition of PKCα and PKCβ1.
      We have here found that HT suppressed MMP-9 protein and mRNA levels in PMA-activated human monocytes and reduced extracellular matrix degradation without affecting TIMP-1 expression. To the best of our knowledge, this is the first study reporting a significant HT inhibition of MMP-9 expression in human activated monocytes, and is therefore in contrast with the report by Dell'Agli et al., who failed to show any modulatory effect by HT on TNFα-induced MMP-9 in THP-1 cells [
      • Dell'Agli M.
      • Fagnani R.
      • Galli G.V.
      • et al.
      Olive oil phenols modulate the expression of metalloproteinase 9 in THP-1 cells by acting on nuclear factor-kappaB signaling.
      ]. Such discrepancy may be due to differences in the stimuli used (PMA vs TNFα), which recruit and activate different signaling cascades. The ability of HT to inhibit MMP-9 also in activated primary human monocytes, i.e. PBMC, as well as in human monocyte-derived macrophages, besides the U937 cell line, provides further evidence for the HT efficacy here demonstrated.
      Being MMP-9 induction partially regulated by COX-2/PGE2 axis, we confirmed the ability of exogenous PGE2 to induce MMP-9 release and inferred a significant contribution of PMA-induced COX-2 in the PGE2-dependent MMP-9 secretion. We found that HT reduced COX-2 protein and mRNA expression, as well as PGE2 production, by PMA-activated monocytes. Nevertheless, the observed HT suppression of MMP-9 occurred through both COX-2-dependent and -independent pathways, given that the ability of HT to decrease MMP-9 activity persisted after complete block of COX-2-mediated PGE2 production. Our results on COX-2 modulation by HT are in line with other in vitro reports in human monocytes demonstrating an attenuation by HT of LPS-induced ROS-mediated COX-2 transcription [
      • Zhang X.
      • Cao J.
      • Jiang L.
      • Zhong L.
      Suppressive effects of hydroxytyrosol on oxidative stress and nuclear factor-kappaB activation in THP-1 cells.
      ,
      • Rosignoli P.
      • Fuccelli R.
      • Fabiani R.
      • Servili M.
      • Morozzi G.
      Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes.
      ]. However, at variance from previous studies where high HT concentrations (50–200 μmol/L) were used, we here demonstrate for the first time significant HT modulating effects on COX-2 as well as on MMP-9 in human monocytes at low micromolar concentrations (1–10 μmol/L), as achievable in human biological fluids after daily intake of extra-virgin olive oil (25–50 mL) [
      • Ruano J.
      • Lopez-Miranda J.
      • de la Torre R.
      • et al.
      Intake of phenol-rich virgin olive oil improves the postprandial prothrombotic profile in hypercholesterolemic patients.
      ,
      • Vissers M.N.
      • Zock P.L.
      • Katan M.B.
      Bioavailability and antioxidant effects of olive oil phenols in humans: a review.
      ]. These effects are consistent with the ability of polyphenol-rich olive oils to lower eicosanoid production and COX-2 expression in humans [
      • Camargo A.
      • Ruano J.
      • Fernandez J.M.
      • et al.
      Gene expression changes in mononuclear cells in patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil.
      ,
      • Visioli F.
      • Caruso D.
      • Grande S.
      • et al.
      Virgin olive oil study (VOLOS): vasoprotective potential of extra virgin olive oil in mildly dyslipidemic patients.
      ,
      • Llorente-Cortes V.
      • Estruch R.
      • Mena M.P.
      • et al.
      Effect of Mediterranean diet on the expression of pro-atherogenic genes in a population at high cardiovascular risk.
      ].
      Proposed molecular mechanisms for HT inhibition of MMP-9 and COX-2 involve its potent antioxidant properties [
      • Zhang X.
      • Cao J.
      • Zhong L.
      Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells.
      ,
      • Scoditti E.
      • Calabriso N.
      • Massaro M.
      • et al.
      Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer.
      ]. Oxidative stress-associated inflammation is thought to provoke vascular events in atherogenesis. HT reduction of intracellular oxidant stress by direct ROS scavenging and/or by regulating antioxidant/prooxidant enzymes [
      • Scoditti E.
      • Calabriso N.
      • Massaro M.
      • et al.
      Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer.
      ,
      • Martin M.A.
      • Ramos S.
      • Granado-Serrano A.B.
      • et al.
      Hydroxytyrosol induces antioxidant/detoxificant enzymes and Nrf2 translocation via extracellular regulated kinases and phosphatidylinositol-3-kinase/protein kinase B pathways in HepG2 cells.
      ,
      • Zrelli H.
      • Matsuoka M.
      • Kitazaki S.
      • Zarrouk M.
      • Miyazaki H.
      Hydroxytyrosol reduces intracellular reactive oxygen species levels in vascular endothelial cells by upregulating catalase expression through the AMPK-FOXO3a pathway.
      ] may prevent or diminish intracellular activation of redox-sensitive genes, including MMP-9 [
      • Yoo H.G.
      • Shin B.A.
      • Park J.S.
      • et al.
      IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells.
      ] and COX-2 [
      • Barbieri S.S.
      • Eligini S.
      • Brambilla M.
      • Tremoli E.
      • Colli S.
      Reactive oxygen species mediate cyclooxygenase-2 induction during monocyte to macrophage differentiation: critical role of NADPH oxidase.
      ], through the inhibition of redox-sensitive transcription factors, such as NF-κB. NF-κB transcriptionally regulates genes implicated in inflammation and atherogenesis [
      • de Winther M.P.
      • Kanters E.
      • Kraal G.
      • Hofker M.H.
      Nuclear factor kappaB signaling in atherogenesis.
      ]. In resting conditions, NF-κB comprises a p50/p65 heterodimer bound to the inhibitory subunit IκB in the cytosol, and can be activated by phosphorylation and subsequent degradation of IκB, allowing NF-κB to translocate into the nucleus. Since NF-κB is involved in both COX-2 and MMP-9 gene expression [
      • Sato H.
      • Seiki M.
      Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells.
      ,
      • Tanabe T.
      • Tohnai N.
      Cyclooxygenase isozymes and their gene structures and expression.
      ], we investigated HT effect directly on NF-κB activation in PMA-activated monocytes. The reduced nuclear translocation and activation of the p65 NF-κB subunit suggest an interference by HT with the upstream events leading to NF-κB activation. HT was also able of reducing the expression of other NF-κB-dependent pro-inflammatory genes, such as cytokines (TNF-α, IL-1β), immune receptor (ICAM-1), and chemokine (MCP-1), thus featuring a generalized NF-κB-mediated anti-inflammatory action. Therefore, our results are consistent with and extend previous studies demonstrating an attenuation by HT of NF-κB activation as a unifying mechanism for its anti-inflammatory and anti-atherogenic actions [
      • Camargo A.
      • Ruano J.
      • Fernandez J.M.
      • et al.
      Gene expression changes in mononuclear cells in patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil.
      ,
      • Zhang X.
      • Cao J.
      • Jiang L.
      • Zhong L.
      Suppressive effects of hydroxytyrosol on oxidative stress and nuclear factor-kappaB activation in THP-1 cells.
      ,
      • Zhang X.
      • Cao J.
      • Zhong L.
      Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells.
      ,
      • Carluccio M.A.
      • Siculella L.
      • Ancora M.A.
      • et al.
      Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals.
      ].
      One potential downstream target of ROS is PKC activity, which can be inhibited by antioxidants and has also been implicated in mediating the expression of COX-2 [
      • Tzeng J.I.
      • Chen B.C.
      • Chang H.M.
      • et al.
      Involvement of phosphatidylcholine-phospholipase C and protein kinase C in peptidoglycan-induced nuclear factor-kappaB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages.
      ] and MMP-9 [
      • Park M.J.
      • Park I.C.
      • Lee H.C.
      • et al.
      Protein kinase C-alpha activation by phorbol ester induces secretion of gelatinase B/MMP-9 through ERK 1/2 pathway in capillary endothelial cells.
      ], as well as NF-κB activation [
      • Rahman A.
      • Bando M.
      • Kefer J.
      • Anwar K.N.
      • Malik A.B.
      Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription.
      ] in response to different stimuli, including phorbol esters. In addition, PKC activity itself has been reported to induce ROS generation via NADPH oxidase activation [
      • Fontayne A.
      • Dang P.M.
      • Gougerot-Pocidalo M.A.
      • El-Benna J.
      Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation.
      ]. Accordingly, antioxidants, such as polyphenols [
      • Pignatelli P.
      • Di Santo S.
      • Buchetti B.
      • Sanguigni V.
      • Brunelli A.
      • Violi F.
      Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment.
      ,
      • Woo J.H.
      • Lim J.H.
      • Kim Y.H.
      • et al.
      Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta signal transduction.
      ] and vitamin E [
      • Cachia O.
      • Benna J.E.
      • Pedruzzi E.
      • Descomps B.
      • Gougerot-Pocidalo M.A.
      • Leger C.L.
      Alpha-tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47(phox) membrane translocation and phosphorylation.
      ], decrease superoxide production via inhibition of PKC. Here we found that conventional PKCα and PKCβ1 are involved in the signal transduction upstream of MMP-9 and COX-2. Moreover, for the first time we here found that HT decreased the activation of both PKCα and PKCβ1. Therefore, PKC activity represents a potential proximal node in the HT-mediated suppression of MMP-9 and COX-2, as well as of other pro-inflammatory genes. HT inhibitory effect on PKC activation may represent a potential mechanism underlying the reduction of NADPH oxidase activity, as recently reported [
      • Rosignoli P.
      • Fuccelli R.
      • Fabiani R.
      • Servili M.
      • Morozzi G.
      Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes.
      ]. Potential mechanisms for PKC inhibition include some enhancement of phosphatase activity responsible for PKC inactivation, as demonstrated in human colon carcinoma cells [
      • Guichard C.
      • Pedruzzi E.
      • Fay M.
      • et al.
      Dihydroxyphenylethanol induces apoptosis by activating serine/threonine protein phosphatase PP2A and promotes the endoplasmic reticulum stress response in human colon carcinoma cells.
      ]. Further studies are however warranted to precisely establish the level of HT interference in the signaling upstream of PKC activation in our experimental conditions. The described PKCα and PKCβ1 inhibition by HT may open up avenues for dietary bioactive molecules to favorably impact a number of vascular alterations involving PKC, such as increased oxidative stress, permeability, contractility, extracellular matrix synthesis, foam cell formation, angiogenesis, leukocyte adhesion, and cytokine activation, all linked to cardiovascular diseases [
      • Rask-Madsen C.
      • King G.L.
      Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance.
      ]. Although we here demonstrated new anti-inflammatory properties of HT, our study is limited by the in vitro design and therefore its physiological relevance should be confirmed in appropriate animal models and in humans.

      5. Conclusion

      In conclusion, we have here demonstrated for the first time that HT, at nutritionally relevant concentrations, inhibits MMP-9 gene and protein expression in human activated monocytes and macrophages. The HT effect on MMP-9 is multifaceted because MMP-9 inhibition by HT occurs through a direct effect on the signaling pathway leading to MMP-9 up-regulation, and only in part depends on the inhibition of the COX-2/PGE2 axis, which also contributes to MMP-9 induction. This is at partial variance from what previously shown by us in endothelial cells [
      • Scoditti E.
      • Calabriso N.
      • Massaro M.
      • et al.
      Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer.
      ]. The multiple inhibitory effects of HT on MMP-9, COX-2 as well as on other pro-inflammatory genes are mediated by the interference with an early step in the signaling cascade consisting in the inhibition of classical PKC activation. This is the first demonstration of PKC inhibition by a typical Mediterranean diet polyphenol in human inflammatory cells, thus contributing to explain the attenuation of redox-sensitive NF-κB-mediated activation of vascular inflammation by Mediterranean diets.

      Conflict of interest

      None to declare.

      Acknowledgments

      We thank “A. Di Summa” Hospital, Laboratory of Clinical Pathology and Toxicology, Brindisi (Italy), for providing technical assistance in cell culturing. This work was supported by grants from Apulian Region (Italy), POR Strategic Projects [grant numbers CIP PS_101 , CIP PS_008 ].

      Appendix A. Supplementary data

      The following is the supplementary data related to this article:

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