Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Activation of S6 kinase in cultured vascular smooth muscle cells by submitogenic levels of thrombospondin

Purified human platelet thrombospondin was shown to activate S6 kinase in cultured vascular smooth muscle cells in a dose- (1-9 micrograms/ml) and time-dependent manner. Down regulation of epidermal growth factor and somatomedin C receptors by prior treatment of cells with their respective growth factors did not reduce this effect. Kinase activation by thrombospondin was only marginally reduced in the presence of platelet-derived growth factor specific antibody at levels that totally inhibited platelet-derived growth factor (5 ng/ml) induced activation. Additionally, thrombospondin elicits a rapid dose-dependent phosphoinositide turnover response analogous to that of platelet-derived growth factor, epidermal growth factor and somatomedin C. Prior treatment of cells with phorbol ester for 48 hrs in serum-free culture medium resulted in a small enhancement of S6 kinase activation by thrombospondin and the above mentioned growth factors but a complete loss in the ability of phorbol ester to activate this enzyme. These findings with cultured smooth muscle cells suggest a growth factor-like role for thrombospondin.     

Role of integrin-linked kinase in vascular smooth muscle cells: regulation by statins and angiotensin II

Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.     

Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells.

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- +/- 0.4-fold increase). Exogenous H(2)O(2) (50-200 microM) also stimulates Akt/PKB phosphorylation (maximal 8- +/- 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H(2)O(2) stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H(2)O(2) production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [(3)H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

The effects of thiazolidinediones on vascular smooth muscle cell activation by angiotensin II

Angiotensin II (Ang II) stimulates the activation of extracellular signal-regulated kinase (ERK), a subgroup of the mitogen-activated protein kinase (MAPK) family, in cultured vascular smooth muscle cells (VSMC). This ERK activation was recently shown to be a critical regulatory factor for Ang II-mediated migration and growth. It has been demonstrated that the thiazolidinedione troglitazone (TRO) blocked Ang II-induced DNA synthesis and migration in VSMC. Here we provide evidence for TRO to inhibit Ang II-induced ERK activation which was suggested to constitute the mechanism by which this agent blocks Ang II-induced VSMC growth and migration. We have found that pretreatment with PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase, decreased Ang II-induced AP-1 activation and that TRO is capable of inhibiting Ang II-induced AP-1 activation. On the other hand, the other thiazolidinediones pioglitazone (PIO) and rosiglitazone (ROSI) had little effect on Ang II-induced activation of ERK or AP-1, suggesting the inhibitory effects of TRO on VSMC activation by Ang II be independent of the peroxisome proliferator-activated receptor-gamma (PPARgamma) for which thiazolidinediones are ligands. Ang II-induced ERK activation was inhibited by protein kinase C (PKC)-specific inhibitor GF109203X, while TRO was also able to block PKC activator phorbol 12 myristate 13-acetate (PMA)-induced ERK activation. Accordingly, TRO may inhibit Ang II-induced MAPK activation at least partly by an inhibition of PKC. These results support the assumption that by targeting MAPK activation, TRO may inhibits the critical signaling steps leading to restenosis and atherosclerosis that may result in part from dysregulated VSMC growth and migration induced by Ang II.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Characterization of paxillin LIM domain-associated serine threonine kinases: activation by angiotensin II in vascular smooth muscle cells

Recently we reported a novel means of regulating LIM domain protein function. Paxillin LIM zinc-finger phosphorylation in response to cell adhesion regulates the subcellular localization of this cytoskeletal adaptor protein to focal adhesions, and also modulates cell adhesion to fibronectin (Brown et al. [1998] Mol. Biol. Cell 9:1803-1816). In the present study, we characterize further the protein kinases that phosphorylate paxillin LIM2 on threonine and LIM3 on serine. Analysis of the subcellular distribution of the LIM kinases demonstrated that the LIM3 protein kinase, but not the LIM2 kinase, resides within a detergent-insoluble fraction. The activities of the paxillin LIM domain kinases are differentially regulated during embryogenesis, and analysis of tissue distribution indicated a specificity in expression patterns between the LIM2 and LIM3 kinases. In addition, these protein kinases were refractory to inhibition by a panel of broad-spectrum serine/threonine kinase inhibitors, suggesting a novel derivation. The paxillin protein kinase activities were stimulated in serum-starved CHO.K1 cells by the mitogen phorbol myristate acetate (PMA), and by PMA and angiotensin II in rat aortic smooth muscle cells. In vivo labeling, phosphoamino acid analysis, and phosphopeptide mapping of paxillin immunoprecipitated from angiotensin II-stimulated smooth muscle cells confirmed an induction of paxillin serine/threonine phosphorylation and supports the contention that these newly identified paxillin kinases are dynamic components of growth factor signaling through the cytoskeleton.     

S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells

Angiotensin II (AII) increases production of reactive oxygen species from NAD(P)H oxidase, a response that contributes to vascular hypertrophy. Here we show in cultured vascular smooth muscle cells that S-glutathiolation of the redox-sensitive Cys(118) on the small GTPase, Ras, plays a critical role in AII-induced hypertrophic signaling. AII simultaneously increased the Ras activity and the S-glutathiolation of Ras (GSS-Ras) detected by biotin-labeled GSH or mass spectrometry. Both the increase in activity and GSS-Ras was labile under reducing conditions, suggesting the essential nature of this thiol modification to Ras activation. Overexpression of catalase, a dominant-negative p47(phox), or glutaredoxin-1 decreased GSS-Ras, Ras activation, p38, and Akt phosphorylation and the induction of protein synthesis by AII. Furthermore, expression of a Cys(118) mutant Ras decreased AII-mediated p38 and Akt phosphorylation as well as protein synthesis. These results show that H(2)O(2) from NAD(P)H oxidase forms GSS-Ras on Cys(118) and increases its activity leading to p38 and Akt phosphorylation, which contributes to the induction of protein synthesis. This study suggests that GSS-Ras is a redox-sensitive signaling switch that participates in the cellular response to AII.     

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells

The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.     

Age-related decrease of protein kinase G activation in vascular smooth muscle cells

The p21 (cip1/waf1) protein induces cell cycle arrest through inhibition of the activity of cdk (cyclin dependent kinase)/cyclin complexes. Expression of p21 is induced in a p53-dependent manner by DNA damage. p21 can also be induced independently of p53 by phorbol ester or okadaic acid. In this study, we have addressed the role of the PKC (protein kinase C) signaling pathway in the induction of p21 in response to PMA (phorbol myristate acetate) and okadaic acid. Levels of p21 (protein and mRNA) rapidly increased (within approximately 4 h) in U937 cells treated with PMA. The PKC-specific inhibitors RO 31-8220 and GF109203X down-regulated PMA or okadaic acid-induced p21 expression. Following persistent PKC activation, p21 mRNA levels remained elevated, indicating an enhanced stability of the mRNA. Using actinomycin D to measure mRNA stability and p21 promoter luciferase assays to measure activity, we provide evidence to support a role for the PKC signaling pathway in p21 mRNA stability. Thus, PKC regulates the amount of p21 in U937 cells at the level of mRNA accumulation and translation.     

Regulation of atrial natriuretic factor receptors by angiotensin II in rat vascular smooth muscle cells

Atrial natriuretic factor (ANF) is actively involved in the control of blood pressure and fluid homeostasis as a physiological antagonist of the renin-angiotensin system. To evaluate a possible interaction between ANF and angiotensin II (Ang-II) receptors, we investigated the effect of long term pretreatment (18 h) of rat cultured vascular smooth muscle cells with Ang-II. Binding of 125I-labeled ANF and cyclic GMP production induced by ANF were measured. After preincubation of the cells with Ang-II (1, 10, and 100 nM), the number of ANF binding sites (Bmax) was decreased by 30, 59, and 71%, respectively, with a slight decrease of the Kd values. Sar1-Ile8-Ang-II (100 nM), a specific Ang-II receptor antagonist, totally inhibited the down-regulation induced by Ang-II (10 nM). Moreover, the regulatory effect of Ang-II on ANF receptors appeared more slowly as compared to ANF homologous receptor regulation. Ang-II pretreatment did not desensitize but increased cyclic GMP production elicited by ANF, implying that only the number of non-guanylate cyclase-coupled receptors was affected. These findings, which were not observed with 100 nM of epinephrine, norepinephrine, histamine, serotonin, and Arg-vasopressin, demonstrate a specific and functional link between ANF and Ang-II receptors. This study also shows that the regulation of ANF receptors is heterogeneous, providing new evidence of multiple classes of ANF receptors.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells

We examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on Ca2+ extrusion from cultured vascular smooth muscle cells (VSMCs) incubated in the absence of added extracellular Na+ (Na+o). Previously, strong experimental evidence was presented that the Na+o-independent Ca2+ extrusion from VSMCs is effected by the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., and Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Brief (2 min) pretreatment of VSMCs with 30-300 nM PMA suppressed the intracellular Ca2+ transient induced with 1 microM ionomycin to about 60% of the control, whereas it accelerated the concomitant Na+o-independent 45Ca2+ extrusion by up to 20%. When the Ca2+ transient was induced with 0.1 microM angiotensin II, the PMA pretreatment markedly suppressed it and reduced also the rate of 45Ca2+ efflux from cells slightly. These effects of PMA were mimicked by 1-oleoyl-2-acetylglycerol, another protein kinase C activator, but were abolished by prior treatment of cells with staurosporine, an inhibitor of protein kinase C, or prior long incubation of cells with PMA. Analysis of the effect of PMA on [Ca2+]i dependence of the rate of Na+o-independent 45Ca2+ efflux revealed that PMA increased the maximum Ca2+ efflux rate without a significant change in the affinity for Ca2+. These results strongly suggest that the plasma membrane Ca2+ pump in VSMCs can be stimulated by PMA and that protein kinase C is involved in regulation of [Ca2+]i in intact VSMCs.     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

Phospholipase D in cultured rat vascular smooth muscle cells and its activation by phorbol ester

We determined the phospholipase D (PLD) activity in rat vascular smooth muscle cells by the formation of phosphatidylethanol in cells prelabeled with [3H] myristic acid. The enzyme was markedly activated by a phorbol ester (TPA). Down regulation of protein kinase C (PKC) resulted in almost complete inhibition indicating PKC-dependent mechanism of its activation. Depletion of calcium by EGTA and TMB-8 caused 53% inhibition. Chelator-stable association of PKC to membrane by TPA was observed in the absence of extracellular Ca2+. The mitogenic peptide PDGF also caused a marked stimulation of PLD. These results indicate that PLD in vascular smooth muscle cells is stimulated by TPA through the activation of PKC both by calcium-dependent and independent mechanisms.     

Angiotensin II induces expression of the c-fos gene through protein kinase C activation and calcium ion mobilization in cultured vascular smooth muscle cells

Incubation of the serum-deprived cultures of rat vascular smooth muscle cells with angiotensin II, a potent vasoconstrictor, caused a rapid and transient increase in the c-fos mRNA level. The doses of this agonist necessary for the increase in the c-fos mRNA level coincided with those for the phospholipase C-mediated hydrolysis of phosphoinositides. Moreover, protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate and Ca2+-ionophore A23187 increased the c-fos mRNA level in an additive manner. These results suggest that angiotensin II induces expression of the c-fos gene through the activation of protein kinase C and Ca2+ mobilization in cultured vascular smooth muscle cells.     

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.     

Endogenous angiotensin II enhances atherogenesis in apoprotein E-deficient mice with renovascular hypertension through activation of vascular smooth muscle cells

Renovascular hypertension is one of the most important risk factors in the development of atherosclerosis. However, very little is known about the role of angiotensin II (AII), a key regulator of blood pressure homeostasis, on renovascular hypertension-associated atherogenesis. To study a possible role of AII on atherogenesis, we generated apoE-deficient hypertensive mice with either normal or increased AII production by applying 1-kidney, 1-clip (1K1C) or 2-kidney, 1-clip (2K1C) operation, respectively. Hypertension was successfully achieved in both mice groups, and was persistent for 8 weeks. Atherosclerosis quantification showed a marked increase in lesion area in aortic sinus of 2K1C mice as compared with 1K1C mice, suggesting a potential role of endogenous AII on atherogenesis. In the immunohistochemical analysis, induction of renovascular hypertension with 2K1C for 8 weeks led to an enhanced accumulation of macrophages in the aortic sinus, which was accompanied by a parallel increase in scavenger receptor A (SRA) expression on the macrophages. In in vitro experiments, although treatment of cells with increasing concentrations of AII (0.1 to 10 microM) affects neither SRA expression nor oxLDL uptake by macrophages, conditioned media (CM) derived from AII-stimulated vascular smooth muscle cells (VSMC) increased macrophage uptake of oxLDL in association with an enhanced expression of SRA on the macrophages. These findings suggest that the increased generation of AII in renovascular hypertension may initiate and promote atherosclerosis by activation of VSMC.     

Protein kinase C inhibits insulin-induced Akt activation in vascular smooth muscle cells.

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.