Signaling Pathways In Smooth Muscle Cells Research Articles

Inhibition of the Akt1-mTORC1 Axis Alters Venous Remodeling to Improve Arteriovenous Fistula Patency

Arteriovenous fistulas (AVFs) are the most common access created for hemodialysis, but approximately 40% fail to mature, suggesting a clinical need. In a mouse model, reduced Akt1 expression reduces AVF wall thickness and diameter. We hypothesized that inhibition of Akt1-mTORC1 axis alters venous remodeling associated with failure of AVF. A C57BL6/J mouse aortocaval fistula model was used (male, 9-12 weeks). Mice were injected with 100 μg of vehicle or rapamycin (intraperitoneal) daily. The AVFs were harvested at days 3, 7, and 21 for comparison of wall thickness, macrophage markers, and expression level of mTOR signaling proteins using Western blot and immunofluorescence intensity. AVF patency and diameter were assessed with ultrasound. Rapamycin reduced AVF wall thickness at days 7 and 21 (P < .05; n = 6) with no change in AVF diameter (P = .5). Rapamycin decreased the proliferation rate of smooth muscle cells (SMCs) and macrophages (P < .05), with reduced M1 (inducible nitric oxide synthase, tumor necrosis factor α) and M2 (interleukin 10, CD206) macrophages at days 3 and 7 (P < .05). Rapamycin treatment was associated with diminished phosphorylation of Akt1 and the mTORC1 pathway members mTOR (Ser2481), 4EBP1, and p70S6K (P < .05; n =6) but not of the mTORC2 pathway members mTOR (Ser2448) and serum and glucocorticoid-regulated kinase 1 (P > .4). With rapamycin treatment, there was decreased p-Akt1 and p-mTORC1 intensity in SMCs and macrophages at days 3, 7, and 21 (P < .05). There was also reduced p-endothelial nitric oxide synthase intensity in SMCs but not in endothelial cells. After depletion of macrophages with clodronate liposomes, there was reduced wall thickness (P < .01, day 21), SMC proliferation, and p-mTORC1 intensity in SMCs (P = .001); however, AVF patency was reduced (P < .05). Rapamycin improved AVF patency by day 42 (P = .04; n = 14); these AVFs showed less thickening (P < .05) and had similar p-Akt1 and p-mTORC1 intensity in macrophages compared with control (P > .5). However, rapamycin treatment initiated after day 21 did not improve patency (P = .62). Rapamycin improves AVF patency by reducing early inflammation and wall thickening while attenuating the Akt1-mTORC1 signaling pathway in SMCs and macrophages. Macrophages are associated with AVF wall thickening, and M2-type macrophages may play a mechanistic role in AVF maturation. Rapamycin is a potential translational strategy to improve AVF patency.

MicroRNA-129-5p inhibits vascular smooth muscle cell proliferation by targeting Wnt5a.

Aberrant smooth muscle cells (SMCs) play important roles in the formation of abdominal aortic aneurysm (AAA). Although the molecular mechanism of AAA formation has been investigated, there is a lack of understanding concerning the role of microRNAs (miRNAs) in AAA, which the current study aimed to address. Firstly, miRNA array analysis was performed in order to compare the miRNA profiles in a mouse model of AAA with those in normal control mice, and differentially expressed miRNAs were identified. miR-129-5p was selected for further analysis, and was used to transfect human SMCs. The results of an MTT assay revealed that miR-129-5p inhibited the proliferation of SMCs, and flow cytometry indicated that apoptosis was induced. Bioinformatic analysis predicted that Wnt5a was the potential target gene of miR-129-5p, and this was verified by luciferase assay. In summary, miR-129-5p inhibits cellular proliferation, induces apoptosis and modulates the Wnt5a signaling pathway in SMCs.

Abstract 511: Emilin1 Controls Myogenic Response of Resistance Arteries by Modulation of TRPC6 Through the TGFß/ALK5/ADAM17/EGFR Pathway

Emilin1 (E1), a protein of the extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, that releases the growth factor. E1 null mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 null phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER T2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. TAM induced Cre activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1 fl/fl mice carrying the Smmhc-CreER T2 (Smmhc-CreER T2 E1 fl/fl ) were given TAM, E1 protein completely disappeared and blood pressure significantly increased, as compared to E1 fl/fl . While no structural changes of arteries were detected, we found that mesenteric arteries of Smmhc-CreER T2 E1 fl/fl displayed an increased myogenic response to pressure as compared to E1 fl/fl . Since the hypertensive phenotype of Smmhc-CreER T2 E1 fl/fl mice was rescued by a treatment with a TGFβ neutralizing antibody, we investigated the molecular mechanism in SMCs that, downstream to this pathway, could lead to an increased Ca 2+ -mediated contractility. In particular, also the increased myogenic response was rescued by exposure of resistance arteries to a TGFβ neutralizing antibody and, more important, by the inhibition of TGFβ receptor ALK5, obtained by in vivo vessels transfection with an adenovirus carrying a dominant negative ALK5 mutant. Moreover, we found that the increased TGFβ signaling, by phosphorylating the ADAM17/EGFR cascade, converged on overactivation of the Transient receptor potential cation channel C6 (TRPC6), rescued by in vivo vessels transfection with an adenovirus carrying a dominant negative TRPC6 mutant. Our results demonstrate that E1 exerts a homeostatic control of myogenic response by regulating the TRPC6 Ca 2+ channel, through a modulation of the TGFβ/ALK5/ADAM17/EGFR signaling pathway in SMCs.

Abstract 9566: LRP1 Deficiency Promotes Smooth Muscle Cell Apoptosis in Atherosclerotic Plaques via PDGF Receptor Mediated Downregulation of Bcl-2

Apoptosis of smooth muscle cells (SMCs) promotes instability of atherosclerotic plaques. Apoptosis is believed to be suppressed by growth factor dependent activation of PI 3-kinase (PI3K). We investigated the effect of LRP1 knockout driven elevated platelet-derived growth factor beta receptor (PDGFR) signaling on SMC apoptosis in atherosclerotic plaques. We quantified apoptotic TUNEL-positive SMCs in lesions from 12 months old LDLR-/-/smLRP1-/- double knockout mice. LDLR (low-density lipoprotein receptor) deficiency accelerates atherosclerosis in mice. LDLR-/-/smLRP1-/- increased the percentage of TUNEL-positive cells compared to LDLR-/-/smLRP1+/+ controls (3.6±0.7% vs. 0.7±0.3%,respectively, P &lt; 0.05, n = 6). Mutation of the PI3K binding site in the PDGFR in LDLR-/-/smLRP1-/--F2/F2 mice suppressed SMC apoptosis (2.4%±0.3%, P &lt; 0.05, n = 6) suggesting that the PDGFR exerts proapoptotic effects via PI3K. In vitro studies (Cell Death Detection ELISA) revealed that chronic stimulation of SMCs with PDGF-BB (96 h) led to 3.1±0.4-fold increased apoptosis (n=3, P&lt;0.05). To characterize proapoptotic PDGFR driven signaling pathways we analysed PDGFR mutants in which the binding sites for different signaling molecules were deleted. Lack of PI3K binding completely abolished the proapoptotic effect, whereas a mutant which only binds PI3K was able to induce apoptosis like the WT receptor. Further analysis of PDGFR mutants revealed that activation of PI3K led to induction of c-Myc and decreased expression of antiapoptotic Bcl-2 as assessed by RT-PCR and Westernblotting. siRNA mediated knockdown of c-Myc prevented both, Bcl-2 downregulation and PDGF induced apoptosis. Consistently, immunohistochemical analysis of atherosclerotic plaques from LDLR-/-/smLRP1-/- and LDLR-/-/smLRP1-/--F2/F2 mice revealed that PDGFR dependent activation of PI3K impairs the expression of antiapoptotic Bcl-2. In conclusion, our study identified a new PDGFR driven proapoptotic signaling pathway in SMCs. Chronically elevated PDGFR/PI3K signaling downregulates Bcl-2 via induction of c-Myc. This has direct implications in atherogenesis, as PDGF promotes cell death in atherosclerotic plaques, which can ultimately lead to plaque instability and rupture.

Elevated phosphorus modulates vitamin D receptor-mediated gene expression in human vascular smooth muscle cells

Clinical observations show that an increase in serum inorganic phosphorus (Pi) is linked to higher cardiovascular (CV) mortality, while vitamin D receptor (VDR) agonist therapy is associated with survival benefit in stage 5 chronic kidney disease. Smooth muscle cells (SMCs) play an important role in CV pathophysiology, but the interaction between Pi and the VDR signaling pathway in SMCs is not known. Real-time RT-PCR studies revealed that elevated Pi (2.06 mM) modulated VDR-mediated regulation of a panel of genes including thrombomodulin and osteopontin in SMCs. DNA microarray results demonstrated that increasing Pi from 0.9 to 2.06 mM exerted a widespread modulating effect on VDR-mediated gene expression. A total of 325 target genes were affected by paricalcitol at 0.9 mM Pi, with 195 up- and 130 downregulated. The number of target genes affected by paricalcitol at 2.06 mM Pi decreased to 86, with 55 up- and 31 downregulated. VDR-mediated gene expression in As4.1 cells (a juxtaglomerular cell-like cell line derived from kidney tumors in SV40 T-antigen transgenic mice) and peroxisome proliferator-activated receptor (PPAR)gamma-mediated gene expression in SMCs were also altered by elevated Pi, suggesting that the observation is not unique to VDR in SMCs. Mechanism analysis showed that elevated Pi had no significant effect on VDR or PPARgamma protein level but altered the cytosolic vs. nuclear distribution of NF-kappaB or nuclear receptor corepressor 1 (NCoR1). Our results demonstrate for the first time that elevated Pi affects VDR-mediated gene expression in human coronary artery SMCs and the effect is not limited to VDR in SMCs.

MEK hyperphosphorylation coincides with cell cycle shut down of cultured smooth muscle cells

Smooth muscle cells (SMCs) form the backbone of arteries and their proliferation hallmarks collateral vessel growth, a process termed arteriogenesis, as well as pathogenic responses such as restenosis. Since signaling pathways in SMCs are the main targets for therapeutic interventions, we aimed to determine how and to what extent the activation of the ubiquitous MEK-ERK signaling pathway correlates with important in vivo phenomena such as dedifferentiation, nuclear activation and proliferation of SMCs. Specificity of this pathway was monitored using MEK inhibitors UO126 and PD98059 in platelet derived growth factor-AB (PDGF-AB)- and fibroblast growth factor-2 (FGF-2)-stimulated SMCs. PDGF-AB induced a rapid MEK activation followed by phosphorylation of the MEK substrates ERK1/2 while FGF-2 showed a less pronounced and delayed activation. Both growth factors triggered a marked phosphorylation of c-Myc and expression of Egr1. Pretreatment with MEK inhibitors suppressed the activation of the ERK cascade, abolished the down-regulation of desmin and led to cell cycle arrest. However, the reversibility of p27Kip1 down-regulation by UO126 was mainly observed after PDGF-AB stimulation, indicating MEK independent p27Kip1 down-regulation by FGF-2. Surprisingly, treatment of SMCs with UO126 or PD98059 increased the level of MEK phosphorylation in a dose dependent manner at serine residues 217/221 in the presence as well as in the absence of both growth factors. Our results strongly imply that depending on the environmental context phosphorylation of serines 217/221 serves as an "on" as well as an "off " switch.

Signaling pathways for arsenic-stimulated vascular endothelial growth factor-a expression in primary vascular smooth muscle cells.

Chronic arsenic exposure is associated with an increased risk for cancer, cardiovascular disease (including ischemic heart disease and hypertension), peripheral vascular disease, and diabetes. Arsenic causes blood vessel growth and remodeling in vivo and cell specific, dose-dependent induction vascular endothelial growth factor-A (VEGF), which is essential for both processes. The current study examined the hypothesis that low, environmentally relevant levels of trivalent arsenic (AsIII) activate discrete signaling pathways in vascular smooth muscle cells (SMC) to induce expression of VEGF. AsIII caused a progressive increase in VEGF mRNA levels over a 48 h period in primary porcine SMC with a threshold of 1-2.5 microM. VEGF protein levels increased with a similar concentration dependence and time course. Hypoxia inducible factor-1alpha (HIF-1alpha) protein and mRNA levels also increased in response to AsIII. However, unlike the response to an iron chelator, AsIII-induced VEGF was not inhibited by siRNA directed toward HIF-1alpha. Instead, a novel protein kinase C, PKCdelta, was activated by AsIII to induce VEGF and stabilize HIF-1alpha. Consistent with this activation, AsIII caused coordinate increases in the levels of the intracellular second messenger diacyglycerol (DAG). These data suggest that AsIII induced divergent signaling pathways in SMCs that lead to independent increases in VEGF expression and HIF-1alpha signaling. However, these pathways both require initial increases in DAG levels and PKC activity.

Troglitazone enhances glucose uptake and inhibits mitogen-activated protein kinase in human aortic smooth muscle cells

The thiazolidinedione analogue troglitazone is an antidiabetic agent that improves insulin resistance in rodents and humans. Although coronary artery disease is common in patients with the insulin resistance syndrome, the effects of troglitazone on smooth muscle cells (SMC) have not been fully elucidated. We therefore examined the effects of troglitazone on cell growth and glucose uptake in human aortic SMC. Mitogen-activated protein (MAP) kinase activity and glucose transporter (Glut) 1 mRNA levels were also studied. In the absence of troglitazone, insulin (10 −7 M) caused a 2-fold increase of DNA synthesis in SMC and troglitazone suppressed the increase of DNA synthesis in a dose-dependent manner. This growth suppression was accompanied by inhibition of MAP kinase activity. On the other hand, troglitazone significantly increased Glut 1 mRNA and enhanced glucose uptake in SMC. These results suggest that troglitazone affects the insulin signaling pathways in SMC and suppresses growth while promoting glucose uptake. Our findings support the application of troglitazone as an inhibitor of SMC proliferation in patients with insulin resistance.