PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.     

miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

Proteasome-dependent inactivation of Akt is essential for 12-O-tetradecanoylphorbol 13-acetate-induced apoptosis in vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. Here we investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on SMC apoptosis. We found that TPA treatment induced SMC apoptosis through the rapid downregulation of Akt phosphorylation. The inhibition of Akt activation by TPA was markedly reduced by inhibitors of protein phosphatase 2A and proteasome. Moreover, TPA promoted the ubiquitination of p-Akt, whereas inhibition of TPA-induced PKC activation suppressed the downregulation and ubiquitination of p-Akt. Taken together, these results demonstrate that TPA triggers inactivation of Akt, at least in part, through PKC and Ubiquitin–proteasome degradation, thereby contributing to SMC apoptosis.     

Vascular smooth muscle G(q) signaling is involved in high blood pressure in both induced renal and genetic vascular smooth muscle-derived models of hypertension

More than 30% of the US population has high blood pressure (BP), and less than a third of people treated for hypertension have it controlled. In addition, the etiology of most high BP is not known. Having a better understanding of the mechanisms underlying hypertension could potentially increase the effectiveness of treatment. Because G(q) signaling mediates vasoconstriction and vascular function can cause BP abnormalities, we were interested in determining the role of vascular smooth muscle (VSM) G(q) signaling in two divergent models of hypertension: a renovascular model of hypertension through renal artery stenosis and a genetic model of hypertension using mice with VSM-derived high BP. Inhibition of VSM G(q) signaling attenuated BP increases induced by renal artery stenosis to a similar extent as losartan, an ANG II receptor blocker and current antihypertensive therapy. Inhibition of G(q) signaling also attenuated high BP in our genetic VSM-derived hypertensive model. In contrast, BP remained elevated 25% following treatment with losartan, and prazosin, an alpha(1)-adrenergic receptor antagonist, only decreased BP by 35%. Inhibition of G(q) signaling attenuated VSM reactivity to ANG II and resulted in a 2.4-fold rightward shift in EC(50). We also determined that inhibition of G(q) signaling was able to reverse VSM hypertrophy in the genetic VSM-derived hypertensive model. These results suggest that G(q) signaling is an important signaling pathway in two divergent models of hypertension and, perhaps, optimization of antihypertensive therapy could occur with the identification of particular G(q)-coupled receptors involved.     

Oxygen-dependent signaling in pulmonary vascular smooth muscle

The pulmonary circulation constricts in response to acute hypoxia, which is reversible on reexposure to oxygen. On exposure to chronic hypoxia, in addition to vasoconstriction, the pulmonary vasculature undergoes remodeling, resulting in a sustained increase in pulmonary vascular resistance that is not immediately reversible. Hypoxic pulmonary vasoconstriction is physiological in the fetus, and there are many mechanisms by which the pulmonary vasculature relaxes at birth, principal among which is the acute increase in oxygen. Oxygen-induced signaling mechanisms, which result in pulmonary vascular relaxation at birth, and the mechanisms by which chronic hypoxia results in pulmonary vascular remodeling in the fetus and adult, are being investigated. Here, the roles of cGMP-dependent protein kinase in oxygen-mediated signaling in fetal pulmonary vascular smooth muscle and the effects of chronic hypoxia on ion channel activity and smooth muscle function such as contraction, growth, and gene expression were discussed.     

Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification

Osteopontin (OPN) is a non-collagenous, glycosylated phosphoprotein associated with biomineralization in osseous tissues, as well as ectopic calcification. We previously reported that osteopontin was co-localized with calcified deposits in atherosclerotic lesions, and that osteopontin potently inhibits calcium deposition in a human smooth muscle cell (HSMC) culture model of vascular calcification. In this report, the role of phosphorylation in osteopontin's mineralization inhibitory function was examined. The ability of OPN to inhibit calcification completely depended on post-translational modifications, since bacteria-derived recombinant OPN did not inhibit HSMC mineralization. Following casein kinase II treatment, phosphorylated OPN (P-OPN) dose-dependently inhibited calcification of HSMC cultured in vitro about as effectively as native OPN. The inhibitory effect of osteopontin depended on the extent of phosphorylation. To determine the specific structural domains of OPN important for inhibition of calcification, we compared OPN fragments (N-terminal, C-terminal, and full-length), and compared the inhibitory effect of both phosphorylated and non-phosphorylated fragments. While none of the non-phosphorylated OPN fragments effected calcification, P-OPN caused dose dependent inhibition of HSMC calcification. P-OPN was treated with alkaline phosphatase to create dephosphorylated OPN. Dephosphorylated OPN did not have an inhibitory effect on calcification. The expression of OPN mRNA and P-OPN secretion by HSMC were decreased in a time-dependent manner during culture calcification. These results indicate that phosphorylation is required for the inhibitory effect of OPN on HSMC calcification, and that regulation of OPN phosphorylation represents one way in which mineralization may be controlled by cells.     

Cardioprotective prostacyclin signaling in vascular smooth muscle

Prostacyclin plays an important cardioprotective role, which has been increasingly appreciated in recent years in light of adverse effects of COX-2 inhibitors in clinical trials. This cardioprotection is thought to be mediated, in part, by prostacyclin inhibition of platelet aggregation. Multiple lines of evidence suggest that prostacyclin additionally protects from cardiovascular disease by pleiotropic effects on vascular smooth muscle. Genetic deletion of the prostacyclin receptor in mice revealed an important role for prostacyclin in preventing the development of atherosclerosis, intimal hyperplasia, and restenosis. In vitro studies have shown these effects may be due to prostacyclin inhibition of vascular smooth muscle cell proliferation and migration. Prostacyclin has also been shown to promote vascular smooth muscle cell differentiation at the level of gene expression through the Gs/cAMP/PKA pathway. Recently identified single nucleotide polymorphisms in the prostacyclin receptor that compromise receptor function suggest that some genetic variations may predispose individuals to increased cardiovascular disease. Herein, we review the literature on the cardioprotective effects of prostacyclin on vascular smooth muscle, and the underlying molecular signaling mechanisms. Understanding the role of prostacyclin and other eicosanoid mediators in the vasculature may lead to improved therapeutic and preventative options for cardiovascular disease.     

Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous HDGF only in the nucleus and target a green fluorescent protein (GFP)-HDGF fusion to the nucleus. To define the features of the HDGF molecule that are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus demonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys --> Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34(cdc2) kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.     

Urokinase-induced signaling in human vascular smooth muscle cells is mediated by PDGFR-beta

Urokinase (uPA)-induced signaling in human vascular smooth muscle cells (VSMC) elicits important cellular functional responses, such as cell migration and proliferation. However, how intracellular signaling is linked to glycolipid-anchored uPA receptor (uPAR) is unknown. We provide evidence that uPAR activation by uPA induces its association with platelet-derived growth factor receptor (PDGFR)-beta. The interaction results in PDGF-independent PDGFR-beta activation by phosphorylation of cytoplasmic tyrosine kinase domains and receptor dimerization. Association of the receptors as well as the tyrosine kinase activity of PDGFR-beta are decisive in mediating uPA-induced downstream signaling that regulates VSMC migration and proliferation. These findings provide a molecular basis for mechanisms VSMC use to induce uPAR- and PDGFR-directed signaling. The processes may be relevant to VSMC function and vascular remodeling.     

Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.     

Autocrine VEGF signaling is required for vascular homeostasis

Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.     

Jagged-1/Notch3 signaling transduction pathway is involved in apelin-13-induced vascular smooth muscle cells proliferation

The apelin/apelin receptor （APJ, apelin-angiotensin receptor-like 1） system is a newly deorphanized G protein- coupled receptor system. Both apelin and APJ that are important regulatory factors are expressed in the cardio- vascular system. Our previous studies demonstrated that apelin-13 significantly stimulated vascular smooth muscle cell （VSMC） proliferation. In this paper, our data sug- gested that the Jagged-l/Notch3 signaling transduction pathway is involved in apelin-13-induced VSMC prolifer- ation by promoting the expression of Cyclin D1. Results indicated that apelin-13 stimulates the proliferation of VSMC and the expression of Jagged-1 and Notch3 in con- centration- and time-dependent manners. The increased expression of Jagged-1 and Notch3 induced by apelin-13 could be abolished by extracellular signal-regulated protein kinase （ERK） blockade. PD98059 （ERK inhibitor） can inhibit the activation of Jagged-I/Notch3 induced by apelin- 13. Down-regulation of Notch3 using small interfering RNA inhibits the expression of Cyclin DI and prevents apelin- 13-induced VSMC proliferation. In conclusion, Jagged-I/ Notch3 signaling transduction pathway is involved in VSMC proliferation induced by apelin-13.     

Fibrinogen is chemotactic for vascular smooth muscle cells

We studied the effect of fibrinogen on the migration of bovine aortic smooth muscle cells in culture, using a Neuro Probe 48-well micro chemotaxis chamber. Fibrinogen stimulated the migration of the cells dose-dependently at concentrations from 30 to 1000 micrograms/ml. A modified checkerboard analysis of the response demonstrated that the effect was largely chemotactic in nature. The present results suggest that fibrinogen may play an important role in the pathogenesis of arterial intimal thickening and atherosclerosis.     

Ouabain-induced signaling and vascular smooth muscle cell proliferation

The hypothesis of this study is that the sodium pump complex acts as an intracellular signal-transducing molecule in canine vascular smooth muscle cells through its interaction with other membrane and cytoskeletal proteins. We have demonstrated that 1 nm ouabain induced transactivation of the epidermal growth factor receptor (EGFR), resulting in increased proliferation and bromodeoxyuridine (BrdUrd) uptake. Immunoprecipitation and Western blotting showed that the EGFR and Src were phosphorylated within 5 min of 10(-9) m ouabain stimulation. Both ouabain-induced DNA synthesis (BrdUrd uptake) and MAPK42/44 phosphorylation were inhibited by the Src inhibitor PP2, the EGFR kinase inhibitor AG1478, the tyrosine kinase inhibitor genistein, and the MEK1 inhibitor PD98059. Ouabain concentrations higher than 1 nm had little or no stimulating effect on proliferation or BrdUrd uptake but did minimally activate ERK1/2. Thus, low concentrations of ouabain, which do not inhibit the sodium pump sufficiently to perturb the resting cellular ionic milieu, initiate a transactivational signaling cascade leading to vascular smooth muscle cell proliferation.     

Phorbol 12-myristate 13-acetate prevents isoproterenol-induced morphological change in cultured vascular smooth muscle cells

The effect of phorbol 12-myristate 13-acetate (PMA) on isoproterenol (ISO)- and dibutyryl cAMP (dBcAMP)-induced morphological change and cytoskeletal reorganization was studied in cultured vascular smooth muscle cells (VSMC) using the fluorescence staining of actin and microtubules. The treatment of VSMC with 1.0 μM of ISO or with 1.0 mM of dBcAMP for 90 min induced the disruption of actin-containing stress fibers followed by cytoplasmic arborization. The addition of 100 or 10 nM of PMA prevented both the destruction of actin fibers and cell arborization induced either by ISO or by dBcAMP. However, PMA rather enhanced cAMP production stimulated by ISO. I-Oleoyl-2-acetylsn-glycerol (100 μg/ml) mimicked this inhibitory effect of PMA whereas 4a-phorbol 12,13-didecanoate (100 nM) failed to block the arborization. These results indicated that the inhibition of arborization by PMA was mediated through the activation of protein kinase C. Colchicine at 5.0 μM also had an inhibitory effect on ISO- and dBcAMP-induced cell arborization. However, immunofluorescence studies revealed that colchicine but not PMA elicited the reorganization of microtubules, suggesting that the effect of PMA was mediated through a mechanism different from that of colchicine. These observations indicated that the morphology of VSMC was regulated through the alteration of cytoskeletal organization induced by cAMP-mediated and by protein kinase C-dependent systems.     

PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; chi(2) analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.     

NF-kappaB is required for TNF-alpha-directed smooth muscle cell migration.

Migration of vascular smooth muscle cells (VSMC) is a crucial event in the formation of vascular stenotic lesions. Tumor necrosis factor-alpha (TNF-alpha) is elaborated by VSMC in atherosclerosis and following angioplasty. We investigated the role of nuclear factor-kappaB (NF-kappaB) in human VSMC migration induced by TNF-alpha. Adenoviral expression of a mutant form of the inhibitor of NF-kappaB, IkappaB-alphaM, suppressed TNF-alpha-triggered degradation of cellular IkappaB-alpha, inhibited activation of NF-kappaB, and attenuated TNF-alpha-induced migration. Further, IkappaB-alphaM suppressed TNF-alpha-stimulated release of interleukin-6 and -8 (IL-6 and IL-8). Neutralization of IL-6 and IL-8 with appropriate antibodies reduced TNF-alpha-induced VSMC migration. Addition of recombinant IL-6 and IL-8 stimulated migration. Collectively, our data provide initial evidence that TNF-alpha-mediated VSMC migration requires NF-kappaB activation and is associated with induction of IL-6 and IL-8 which act in an autocrine manner.