Mechanical stretch increases MMP-2 production in vascular smooth muscle cells via activation of PDGFR-?/Akt signaling pathway.
ABSTRACT: Increased blood pressure, leading to mechanical stress on vascular smooth muscle cells (VSMC), is a known risk factor for vascular remodeling via increased activity of matrix metalloproteinase (MMP) within the vascular wall. This study aimed to identify cell surface mechanoreceptors and intracellular signaling pathways that influence VSMC to produce MMP in response to mechanical stretch (MS). When VSMC was stimulated with MS (0-10% strain, 60 cycles/min), both production and gelatinolytic activity of MMP-2, but not MMP-9, were increased in a force-dependent manner. MS-enhanced MMP-2 expression and activity were inhibited by molecular inhibition of Akt using Akt siRNA as well as by PI3K/Akt inhibitors, LY293002 and AI, but not by MAPK inhibitors such as PD98059, SP600125 and SB203580. MS also increased Akt phosphorylation in VSMC, which was attenuated by AG1295, a PDGF receptor (PDGFR) inhibitor, but not by inhibitors for other receptor tyrosine kinase including EGF, IGF, and FGF receptors. Although MS activated PDGFR-? as well as PDGFR-? in VSMC, MS-induced Akt phosphorylation was inhibited by molecular deletion of PDGFR-? using siRNA, but not by inhibition of PDGFR-?. Collectively, our data indicate that MS induces MMP-2 production in VSMC via activation of Akt pathway, that is mediated by activation of PDGFR-? signaling pathways.
Project description:Hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), caused by loss-of-function mutations in either the TSC1 or TSC2 gene, leads to the development of tuberous sclerosis complex (TSC), a benign tumor syndrome with multiple affected organs. mTORC1-mediated inhibition of AKT constrains the tumor progression of TSC, but the exact mechanisms remain unclear. Herein we showed that loss of TSC1 or TSC2 downregulation of platelet-derived growth factor receptor ? (PDGFR?) expression was mediated by mTORC1. Moreover, mTORC1 inhibited PDGFR? expression via suppression of forkhead box O3a (FOXO3a)-mediated PDGFR? gene transcription. In addition, ectopic expression of PDGFR? promoted AKT activation and enhanced proliferation and tumorigenic capacity of Tsc1- or Tsc2-null mouse embryonic fibroblasts (MEFs), and vice versa. Most importantly, rapamycin in combination with AG1295, a PDGFR inhibitor, significantly inhibited growth of TSC1/TSC2 complex-deficient cells <i>in vitro</i> and <i>in vivo</i>. Therefore, downregulated FOXO3a/PDGFR?/AKT pathway exerts a protective effect against hyperactivated mTORC1-induced tumorigenesis caused by loss of TSC1/TSC2 complex, and the combination of rapamycin and AG1295 may be a new effective strategy for TSC-associated tumors treatment.
Project description:Testosterone deficiency is associated with a higher incidence of cardiovascular diseases in men. However, its effect on cell senescence, which plays a causal role in vascular aging, remains unclear. Here, we tested the hypothesis that testosterone alleviated vascular smooth muscle cell (VSMC) senescence and collagen synthesis via growth arrest-specific protein 6 (Gas6)/Axl- and Akt/FoxO1a-dependent pathways. Testosterone significantly ameliorated angiotensin II-induced VSMC senescence and collagen overexpression. In addition, testosterone inhibited angiotensin II-induced matrix metalloproteinase-2 (MMP-2) activity, which played a pivotal role in facilitating age-related collagen deposition. Testosterone increased the expression of tissue inhibitor of metalloproteinase-2 but decreased the expression of MMP-2 and membrane type-1 metalloproteinase which contributed to increase MMP-2 activity. The effects on VSMCs senescence and collagen synthesis were mediated by restoration of angiotensin II-induced downregulation of Gas6 and Axl expression and a subsequent reduction of Akt and FoxO1a phosphorylation. The effects of testosterone were reversed by a Gas6 blocker, Axl-Fc, and a specific inhibitor of Axl, R428. Treatment of VSMCs with PI3K inhibitor LY294002 abrogated the downregulating effect of testosterone on MMP-2 activity. Furthermore, when FoxO1a expression was silenced by using a specific siRNA, the inhibitory effect of testosterone on MMP-2 activity was revered as well, that indicated this process was Akt/FoxO1a dependence. Taken together, Gas6/Axl and Akt/FoxO1a were involved in protective effects of testosterone on VSMCs senescence and collagen synthesis. Our results provide a novel mechanism underlying the protective effect of testosterone on vascular aging and may serve as a theoretical basis for testosterone replacement therapy.
Project description:<h4>Objective</h4>It has been suggested that autoantibodies in systemic sclerosis (SSc) may induce the differentiation of cultured fibroblasts into myofibroblasts through platelet-derived growth factor receptor (PDGFR) activation. The present study aims to characterize the effects of SSc IgG on vascular smooth muscle cells (VSMCs) and to determine if stimulatory autoantibodies directed to the PDGFR can be detected, and whether they induce a profibrotic response in primary cultured VSMCs.<h4>Methods</h4>Cultured VSMCs were exposed to IgG fractions purified from SSc-patient or control sera. VSMC responses were then analyzed for ERK1/2 and Akt phosphorylation, PDGFR immunoprecipitation, cellular proliferation, protein synthesis, and pro-fibrotic changes in mRNA expression.<h4>Results</h4>Stimulatory activity in IgG fractions was more prevalent and intense in the SSc samples. SSc IgG immunoprecipitated the PDGFR with greater avidity than control IgG. Interestingly, activation of downstream signaling events (e.g. Akt, ERK1/2) was independent of PDGFR activity, but required functional EGFR. We also detected increased protein synthesis in response to SSc IgG (p<0.001) and pro-fibrotic changes in gene expression (Tgfb1 +200%; Tgfb2 -23%; p<0.001)) in VSMCs treated with SSc IgG.<h4>Conclusion</h4>When compared to control IgG, SSc IgG have a higher stimulation index in VSMCs. Although SSc IgG interact with the PDGFR, the observed remodeling signaling events occur through the EGFR in VSMC. Our data thus favour a model of transactivation of the EGFR by SSc-derived PDGFR autoantibodies and suggest the use of EGFR inhibitors in future target identification studies in the field of SSc.
Project description:Peroxynitrite is a potent oxidizing and nitrating species formed in a diffusion-limited reaction between nitrogen monoxide and superoxide. It induces apoptosis through unknown mechanisms and is believed to interfere with receptor tyrosine kinase signalling through nitration of tyrosine residues. One pathway emanating from receptor tyrosine kinases is that leading to activation of the anti-apoptotic kinase Akt. In the present study we provide evidence that peroxynitrite, administered to cells using two different delivery systems, results in the dose- and time-dependent activation of Akt. Akt activation is rapid and followed by phosphorylation of glycogen synthase kinase-3, an established substrate of Akt. Akt activation is inhibited in the presence of the phosphoinositide 3-kinase (PI-3K) inhibitors wortmannin and LY294002, and by treatment with the platelet-derived growth factor (PDGF) receptor (PDGFR) inhibitor AG1295, indicating a requirement for PDGFR and PI-3K in mediating peroxynitrite-induced Akt activation. Accordingly, the PDGFR-A and PDGFR-B isoforms were shown to undergo rapid tyrosine phosphorylation on treatment with peroxynitrite. Prior exposure of cells to peroxynitrite interferes with PDGF-induced Akt phosphorylation. Our findings suggest that Akt activation occurs as an acute response to peroxynitrite treatment and could play an important role in influencing cell survival and/or alter the cellular response to other growth regulatory signals.
Project description:The phenotype of smooth muscle cells (SMCs) plays an important role in vascular function in health and disease. We investigated the mechanism of modulation of SMC phenotype (from contractile to synthetic) induced by the synergistic action of a growth factor (platelet-derived growth factor, PDGF-BB) and a cytokine (interleukin, IL-1beta). Human aortic SMCs grown on polymerized collagen showed high expression levels of contractile markers (smooth muscle alpha-actin, myosin heavy chain, and calponin). These levels were not significantly affected by PDGF-BB and IL-1beta individually, but decreased markedly after the combined usage of PDGF-BB and IL-1beta. PDGF/IL-1beta costimulation also induced a sustained phosphorylation of Akt and p70 ribosomal S6 kinase (p70S6K). The effects of PDGF/IL-1beta costimulation on contractile marker expression and Akt and p70S6K phosphorylation were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 and by adenovirus expressing a dominant-negative Akt, and they were mimicked by constitutively active Akt. PDGF-BB/IL-1beta induced a sustained phosphorylation of PDGF receptor (PDGFR)-beta and its association with IL-1 receptor (IL-1R1). Such activation and association of receptors were blocked by a PDGFR-beta neutralizing antibody (AF385), an IL-1R1 antagonist (IL-1ra), as well as a specific inhibitor of PDGFR-beta phosphorylation (AG1295); these agents also eliminated the PDGF-BB/IL-1beta-induced signaling and phenotypic modulation. PDGF-BB/IL-1beta inhibited the polymerized collagen-induced serum response factor DNA binding activity in the nucleus, and this effect was mediated by the PDGFR-beta/IL-1R1 association and phosphatidylinositol 3-kinase/Akt/p70S6K pathway. Our findings provide insights into the mechanism of SMC phenotypic modulation from contractile to synthetic, e.g., in atherosclerosis.
Project description:<h4>Background</h4>The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN's effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury in vivo and in vitro.<h4>Methods and results</h4>Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H2O2 robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; P<0.001) as determined by TUNEL assay. In contrast, siRNA-mediated knock-down of PTEN attenuated the basal as well as H2O2-induced apoptosis of VSMC. Mechanistically, overexpression of PTEN prevented serum-induced Akt-phosphorylation, whereas siRNA-mediated knock down of PTEN augmented Akt-activation. Moreover, co-transfection of PTEN and a constitutive active Akt mutant prevented PTEN-dependent augmentation of VSMC apoptosis, indicating, that PTEN regulates VSMC apoptosis by inhibition of Akt phosphorylation/activation.<h4>Conclusion</h4>By interfering with the PI3-K/Akt-dependent survival signalling, the oxidative stress-induced up regulation of PTEN in VSMC of injured arteries augments the sensitivity of VSMC to apoptotic stimuli in the early phase following vascular injury, augmenting the initial injury and cell loss of the injured vessel wall. Thus, these data add to our understanding of PTEN's role during vascular remodelling.
Project description:Pathologic proliferation and migration of vascular smooth muscle cells (VSMCs) exacerbate cardiovascular disease. MicroRNAs (miRNAs), as endogenous inhibitors of protein synthesis, are expected to modulate pathologic proliferation of VSMCs. Here we report that both platelet-derived growth factor receptor (PDGFR) targeting miR-9 and a small molecule that increases miR-9 can inhibit the serum-induced proliferation of VSMCs. First, based on miRNA-target prediction databases and empirical data, we have selected miR-9 as a potent anti-proliferative miRNA in VSMCs. Further examination indicated that miR-9 directly targets PDGFR disrupting downstream signaling cascades, and this resulted in inhibition of VSMC proliferation and migration. Exogenous delivery of miR-9 inhibited VSMC proliferation in vitro, and a small molecule that increased miR-9 expression also inhibited neointima formation following balloon injury in vivo. We provide evidence of miRNA-mediated modulation of VSMC proliferation and further demonstrate that small molecule-mediated regulation of miRNA targeting a key regulator of VSMC proliferation is a viable therapeutic strategy for treating vascular disease involving pathologic VSMC proliferation such as restenosis.
Project description:BACKGROUND: Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. METHODS: In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. RESULTS: Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. CONCLUSION: From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases.
Project description:There is an unmet need for treatments to reduce abdominal aortic aneurysm (AAA) progression. Vascular smooth muscle cell (VSMC) apoptosis precipitates AAA formation, whereas VSMC proliferation repairs the vessel wall. We previously demonstrated that over-expression of EC4-Fc (truncated N-cadherin), or deletion of matrix-metalloproteinase-7 (Mmp-7) reduced VSMC apoptosis in mouse atherosclerotic plaques. Additionally, MMP-7 promotes VSMC apoptosis by cleavage of N-cadherin. We investigated their combined effect on AAA formation. Increased apoptosis and proliferation were observed in human AAA (HAAA) sections compared to normal aortae (HA). This coincided with increased MMP-7 activity and reduced N-cadherin protein levels in HAAA sections compared to HA. Using a mouse model of aneurysm formation, we showed that the combination of Mmp-7 deletion and EC4-Fc overexpression significantly increased AAA severity. Medial apoptosis and proliferation were both significantly reduced in these mice compared to control mice. In vitro, MMP-7 inhibition and EC4-Fc administration significantly supressed human aortic VSMC apoptosis (via activation of PI-3 kinase/Akt signalling) and proliferation. In conclusion, combined Mmp-7 deletion and systemic over-expression of EC4-Fc reduced both proliferation and apoptosis. Reduced proliferation-mediated repair over-rides any benefit of reduced apoptosis, increasing aneurysm severity. Future studies should therefore focus on retarding VSMC apoptosis whilst promoting VSMC proliferation.
Project description:Neuroinflammation is a landmark of neuroinflammatory and neurodegenerative diseases. Matrix metalloproteinase (MMP)-9, one member of MMPs, has been shown to contribute to the pathology of these brain diseases. Several experimental models have demonstrated that lipopolysaccharide (LPS) exerts a pathological role through Toll-like receptors (TLRs) in neuroinflammation and neurodegeneration. However, the mechanisms underlying LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1) are not completely understood. Here, we applied pharmacological inhibitors and siRNA transfection to assess the levels of MMP-9 protein, mRNA, and promoter activity, as well as protein kinase phosphorylation in RBA-1 cells triggered by LPS. We found that LPS-induced expression of pro-form MMP-9 and cell migration were mediated through TLR4, proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and Jun amino-terminal kinase (JNK)1/2 signaling molecules in RBA-1 cells. In addition, LPS-stimulated binding of c-Jun to the MMP-9 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, which was blocked by pretreatment with c-Src inhibitor II, PF431396, AG1296, LY294002, Akt inhibitor VIII, p38 MAP kinase inhibitor VIII, SP600125, and tanshinone IIA. These results suggest that in RBA-1 cells, LPS activates a TLR4/c-Src/Pyk2/PDGFR/PI3K/Akt/p38 MAPK and JNK1/2 pathway, which in turn triggers activator protein 1 (AP-1) activation and ultimately induces MMP-9 expression and cell migration.