MAO-A-induced mitogenic signaling is mediated by reactive oxygen species, MMP-2, and the sphingolipid pathway

The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.     

Urotensin-II and cardiovascular remodeling

Urotensin-II (U-II), a cyclic undecapeptide, and its receptor, UT, have been linked to vascular and cardiac remodeling. In patients with coronary artery disease (CAD), it has been shown that U-II plasma levels are significantly greater than in normal patients and the severity of the disease is increased proportionally to the U-II plasma levels. We showed that U-II protein and mRNA levels were significantly elevated in the arteries of patients with coronary atherosclerosis in comparison to healthy arteries. We observed U-II expression in endothelial cells, foam cells, and myointimal and medial vSMCs of atherosclerotic human coronary arteries. Recent studies have demonstrated that U-II acts in synergy with mildly oxidized LDL inducing vascular smooth muscle cell (vSMC) proliferation. Additionally, U-II has been shown to induce cardiac fibrosis and cardiomyocyte hypertrophy leading to cardiac remodeling. When using a selective U-II antagonist, SB-611812, we demonstrated a decrease in cardiac dysfunction including a reduction in cardiomyocyte hypertrophy and cardiac fibrosis. These findings suggest that U-II is undoubtedly a potential therapeutic target in treating cardiovascular remodeling.     

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.     

Upregulation of Intermediate-Conductance Ca2+-Activated K+ Channels (KCNN4) in Porcine Coronary Smooth Muscle Requires NADPH Oxidase 5 (NOX5)

Aims

NADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca²⁺-activated K⁺ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors.

Methods and Results

Dihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration.

Conclusions

Our findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.     

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1–100 μM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.     

Insulin-like growth factor I and protein kinase C activation stimulate pulmonary artery smooth muscle cell proliferation through separate but synergistic pathways

Smooth muscle cell (SMC) hyperplasia is an important component of vascular remodeling in chronic hypoxic pulmonary hypertension. The mechanisms underlying SMC proliferation in the remodeling process are poorly understood, but may involve insulin-like growth factor I (IGF-I). This study investigates the potential proliferative effects of IGF-I on SMC cultured from the pulmonary arteries (PA) of neonatal calves. We hypothesized that IGF-I stimulates PA SMC proliferation through a protein kinase C (PKC)-independent pathway, but that PKC activation would augment this proliferative response. Incorporation of 3H-thymidine was used as an index of cellular proliferation, and was correlated with subsequent changes in cell counts. Under serum-free conditions, IGF-I (100 ng/ml) induced a 6-fold increase in thymidine incorporation by quiescent PA SMC. This stimulation was not blocked by dihydrosphingosine, an inhibitor of PKC activation. Phorbol myristate acetate (PMA) (1 nM), a membrane-permeable PKC activator, induced a 12-fold increase in thymidine incorporation which was 70% inhibited by dihydrosphingosine. Co-incubation with IGF-I and PMA caused a 60-fold increase in thymidine incorporation, which was 30% inhibited by dihydrosphingosine. This synergistic increase in thymidine incorporation was associated with a subsequent significant increase in cell number. PKC-downregulated cells (1,000 nM PMA x 30 hr) proliferated in response to IGF-I but not PMA, and did not demonstrate synergism with the combination of IGF-I and PMA. The threshold concentrations of IGF-I and PMA for synergism were approximately 1 ng/ml and 1 pM, respectively. We conclude that IGF-I stimulates neonatal PA SMC proliferation via a PKC-independent pathway, and that trace amounts of PKC activators are capable of synergistically augmenting this response. We speculate that the synergistic stimulation of SMC proliferation by IGF-I and PKC activators may play an important role in hypertensive pulmonary vascular remodeling.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

Shedding of dipeptidyl peptidase 4 is mediated by metalloproteases and up-regulated by hypoxia in human adipocytes and smooth muscle cells

Dipeptidyl peptidase 4 is an important drug target for diabetes and a novel adipokine. However, it is unknown how soluble DPP4 (sDPP4) is cleaved from the cell membrane and released into the circulation. We show here that MMP1, MMP2 and MMP14 are involved in DPP4 shedding from human vascular smooth muscle cells (SMC) and MMP9 from adipocytes. Hypoxia increased DPP4 shedding from SMC which is associated with increased mRNA expression of MMP1. Our data suggest that constitutive as well as hypoxia-induced DPP4 shedding occurs due to a complex interplay between different MMPs in cell type-specific manner.     

Activation of cytosolic phospholipase A2 and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation

Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PLs), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study, we investigated the role of key enzymes involved in PL metabolism, the PL-hydrolyzing enzyme phospholipase A2 (PLA2) and the PL-synthesizing enzyme lysophosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic SMCs (BASMCs). Concomitantly with the induction of BASMC proliferation, thrombin activated cytosolic PLA2 (cPLA2-alpha), expressed by selective release of arachidonic acid and mRNA expression, as well as LPAAT, expressed by nonselective incorporation of fatty acid and mRNA expression. Specific inhibitors of these enzymes, arachidonyl-trifluoromethyl-ketone for cPLA2 and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA2 and LPAAT activation in thrombin-induced SMC proliferation. Thrombin acts through the protease-activated receptor (PAR-1), and, accordingly, we found that thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent thrombin-induced mRNA expression of cPLA2 and LPAAT, implying that the activation of cPLA2 and LPAAT is essential but not sufficient for thrombin-induced proliferation of BASMCs.