Vascular Smooth Muscle Cell Hypertrophy Research Articles

The mitochondria mediate the induction of NOX1 gene expression by aldosterone in an ATF-1-dependent manner

High aldosterone (Ald) levels can induce hypertrophy of vascular smooth muscle cells (VSMCs), which carries high risks of heart failure. A previous study showed that Ald induces hypertrophy of VSMCs by up-regulating NOX1, a catalytic subunit of NADPH oxidase that produces superoxides. However, the precise mechanism remains unknown. Diphenylene iodonium (DPI) is known as an inhibitor of complex I in the mitochondrial respiratory chain, and it was also found to almost completely suppress the induction of NOX1 mRNA and the phosphorylation of activating transcription factor (ATF-1) by PGF2α or PDGF in a rat VSMC cell line. In this study, we found that the Ald-induced phosphorylation of ATF-1 and NOX1 expression was significantly suppressed by DPI. Silencing of ATF-1 gene expression attenuated the induction of NOX1 mRNA expression, and over-expression of ATF-1 restored Ald-induced NOX1 expression. On the basis of this data, we show that the mitochondria mediate aldosterone-induced NOX1 gene expression in an ATF-1-dependent manner.

Pharmacology in Health Food: Metabolism of Quercetin In Vivo and Its Protective Effect Against Arteriosclerosis

Quercetin, a member of the bioflavonoids family, has been proposed to have antiatherogenic, anti-inflammatory, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. It was recently demonstrated that quercetin 3-O-β-D-glucuronide (Q3GA) is one of the major quercetin conjugates in human plasma, in which the aglycone could not be detected. Although most of the in vitro pharmacological studies have been carried out using only the quercetin aglycone form, experiments using Q3GA would be important to discover the preventive mechanisms of cardiovascular diseases by quercetin in vivo. Therefore we examined the effects of the chemically synthesized Q3GA, as an in vivo form, on vascular smooth muscle cell (VSMC) disorders related to the progression of arteriosclerosis. Platelet-derived growth factor–induced cell migration and proliferation were inhibited by Q3GA in VSMCs. Q3GA attenuated angiotensin II–induced VSMC hypertrophy via its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by the electron paramagnetic resonance method. In addition, immunohistochemical studies with monoclonal antibody 14A2 targeting the Q3GA demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may have preventative effects on arteriosclerosis relevant to VSMC disorders.

Vascular effects of cardiotrophin-1: a role in hypertension?

To investigate cardiotrophin-1 (CT-1) effects and regulation in vascular smooth muscle cells (VSMCs) in vitro and in aortic tunica media ex vivo in normotensive Wistar rats and spontaneously hypertensive rats (SHRs). CT-1 expression was quantified by real-time reverse-transcription PCR and western blotting. CT-1-activated intracellular pathways were assessed by western bloting analysis. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ki67 immunodetection, and cell hypertrophy by planimetry. Extracellular matrix components were quantified by real-time reverse-transcription PCR and western blot, and metalloproteinases activities by zymography. VSMCs from Wistar rats and SHRs expressed spontaneously CT-1 at the mRNA and the protein level, with a two-fold more increase in SHRs. CT-1 phosphorylated p42/44 mitogen-activated protein kinase, p38 mitogen-activated protein kinase, Akt and Stat-3 in both strains. CT-1 stimulated VSMCs proliferation and hypertrophy in both strains, with an enhanced stimulation in SHRs. CT-1 increased the secretion of collagen type I and fibronectin in VSMCs and aortic tunica media of Wistar rats and SHRs, with greater magnitude in SHRs. In SHRs VSMCs in vitro and ex vivo, CT-1 increased the secretion of collagen type III and elastin and the expression of tissue inhibitors of metalloproteinases, without altering metalloproteinase activity. These effects were blocked by CT-1 receptor antibodies. Aldosterone treatment increased CT-1 expression in VSMCs and aortic tunica media from both strains, with a greater magnitude in SHRs. CT-1 induces VSMCs proliferation, hypertrophy and extracellular matrix production, and is upregulated in hypertension and by aldosterone. CT-1 may represent a new target of vascular wall remodeling in hypertension.

Involvement of ERK1/2 Kinase in Insulin-and Thrombin-Stimulated Vascular Smooth Muscle Cell Proliferation

It is well recognized that the proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42- and 44-kDa isoforms (ERK1/2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS) and thrombin (Thr). However, understanding of the intracellular signal transduction pathways involved is incomplete. This review considers the recent findings in INS and Thr signaling mechanisms that modulate the proliferation of VSMCs with particular emphasis on the ERK1/2 signaling pathway, an important mediator of VSMCs hypertrophy and vascular disease. Moreover, because the ERK1/2 pathway have been acknowledged as an important mediator of VSMCs hypertrophy, ERK1/2 is identified as a key target for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis.

P21-Activated Kinase 1 Participates in Vascular Remodeling In Vitro and In Vivo

Vascular smooth muscle cell hypertrophy, proliferation, or migration occurs in hypertension, atherosclerosis, and restenosis after angioplasty, leading to pathophysiological vascular remodeling. Angiotensin II and platelet-derived growth factor are well-known participants of vascular remodeling and activate a myriad of downstream protein kinases, including p21-activated protein kinase (PAK1). PAK1, an effector kinase of small GTPases, phosphorylates several substrates to regulate cytoskeletal reorganization. However, the exact role of PAK1 activation in vascular remodeling remains to be elucidated. Here, we have hypothesized that PAK1 is a critical target of intervention for the prevention of vascular remodeling. Adenoviral expression of dominant-negative PAK1 inhibited angiotensin II–stimulated vascular smooth muscle cell migration. It also inhibited vascular smooth muscle cell proliferation induced by platelet-derived growth factor. PAK1 was activated in neointima of the carotid artery after balloon injury in the rat. Moreover, marked inhibition of the neointima hyperplasia was observed in a dominant-negative PAK1 adenovirus-treated carotid artery after the balloon injury. Taken together, these results suggest that PAK1 is involved in both angiotensin II and platelet-derived growth factor–mediated vascular smooth muscle cell remodeling, and inactivation of PAK1 in vivo could be effective in preventing pathophysiological vascular remodeling.

PGC-1α Serine 570 Phosphorylation and GCN5-mediated Acetylation by Angiotensin II Drive Catalase Down-regulation and Vascular Hypertrophy

Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including aging, cardiovascular and renal diseases, and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H(2)O(2). The molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1 alpha serine 570 phosphorylation, which is required for the binding of the histone acetyltransferase GCN5 (general control nonderepressible 5) to PGC-1 alpha and for its lysine acetylation. These sequential post-translational modifications suppress PGC-1 alpha activity and prevent its binding to the catalase promoter through the forkhead box O1 transcription factor, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation-defective mutant PGC-1 alpha (S570A) prevents Ang II-induced increases in H(2)O(2) levels and hypertrophy ([(3)H]leucine incorporation). Knockdown of PGC-1 alpha by small interfering RNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by polyethylene glycol-conjugated catalase. Thus, endogenous PGC-1 alpha is a negative regulator of vascular hypertrophy by up-regulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases.

Aldosterone up‐regulates 12‐ and 15‐lipoxygenase expression and LDL oxidation in human vascular smooth muscle cells

Several lines of evidence suggest that aldosterone excess may have detrimental effects in the cardiovascular system, independent of its interaction with the renal epithelial cells. Here we examined the possibility that aldosterone modulates 12- and/or 15-lipoxygenase (LO) expression/activity in human vascular smooth muscle cells (VSMC), in vitro, thereby potentially contributing to both vascular reactivity and atherogenesis. Following 24 h treatment of VSMC with aldosterone (1 nmol/L), there was a approximately 2-fold increase in the generation rate of 12 hydroxyeicosatetraenoic acid (12-HETE), 70% increase in platelet type 12-LO mRNA expression (P < 0.001) along with a approximately 3-fold increase in 12-LO protein expression, which were blocked by the mineralocorticoid receptor (MR) antagonists spironolactone (100 nmol/L) and eplerelone (100 nmol/ml). Additionally, aldosterone (1 nmol/L; 24 h) increased the production of 15-HETE (50%; P < 0.001) and the expression of 15-LO type 2 mRNA (50%; P < 0.05) (in VSMC). Aldosterone also increased the 12- and 15-LO type 2 mRNA expression in a line of human aortic smooth muscle cells (T/G HA-VSMC) (60% and 50%, respectively). Aldosterone-induced 12- and 15-LO type 2 mRNA expressions were blocked by the EGF-receptor antagonist AG 1478 and by the MAPK-kinase inhibitor UO126. Aldosterone-treated VSMC also showed increased LDL oxidation, (approximately 2-fold; P < 0.001), which was blocked by spironolactone. In conclusion, aldosterone increased 12- and 15-LO expression in human VSMC, in association with increased 12- and 15-HETE generation and enhanced LDL oxidation and may directly augment VSMC contractility, hypertrophy, and migration through 12-HETE and promote LDL oxidation via the pro-oxidative properties of these enzymes.

Role of PI3K/AKT, cPLA2 and ERK1/2 Signaling Pathways in Insulin Regulation of Vascular Smooth Muscle Cells Proliferation

Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.

Spleen tyrosine kinase mediates angiotensin II‐ and vascular endothelial growth factor‐induced angiogenesis

Angiotensin II (AngII) and vascular endothelial growth factor (VEGF) stimulate angiogenesis. A 72 kDa non‐receptor tyrosine kinase, spleen tyrosine kinase (Syk) expressed in endothelial cells has been implicated in their migration and proliferation and in vasculogenesis. Syk also mediates AngII‐induced vascular smooth muscle cell hypertrophy. This study was conducted to determine the contribution of Syk to the angiogenic effect of AngII and VEGF. Angiogenesis was determined in Matrigel by tube formation from endothelial cells EA.hy926, and sprouting of rat aortic rings into microvessels. EA.hy926 cells were allowed to form tubular structures for 16 h and aortic rings to sprout into microvessel for 5 days and then phase contrast images were recorded. Both tube length and numbers were calculated. AngII (10 nM) and VEGF (50 ng/ml) stimulated EA.hy926 cells to organize into tubular network and increased aortic sprouting. The effect of VEGF but not AngII to stimulate tube formation and aortic sprouting was inhibited by VEGFR2 blocker SU1498 (10 µM). Both AngII‐ and VEGF‐induced angiogenesis and Syk phosphorylation, as determined by Western blot analysis, was inhibited by piceatannol (10 µM), a Syk kinase inhibitor. These data suggest that AngII‐induced angiogenesis is independent of VEGFR2 phosphorylation and that both AngII‐ and VEGF‐induced angiogenesis is mediated by Syk.

CaMKII mediates Ang‐II induced vascular smooth muscle cell hypertrophy by a pathway involving HDAC4/MEF2

IntroductionThe calcium/calmodulin‐dependent kinase II has been implicated in VSM growth and is activated by Ang‐II. Ang‐II activates the HDAC4/MEF2 dependent gene transcription which is important in muscle growth and hypertrophy. CaMKII specifically phosphorylates HDAC4 S467,632. We assessed the hypothesis that CaMKII mediates Ang‐II induced smooth muscle hypertrophy by a pathway involving HDAC4/MEF2.Methods and ResultsThe medial hypertrophy induced by Ang‐II over 10 days in C57Bl/6 mice was significantly reduced when the CaMKII inhibitor KN93 was given daily i.p.Infection of rat aortic smooth muscle cells with adenovirus expressing CaMKIIδ2 resulted in a 108% increase in 3H‐Leucine uptake with Ang‐II compared to 50% (p&lt;0.05) increase in controls, whereas overexpression of the CaMKII peptide inhibitor CaMKIIN resulted in a 14 % increase.The phosphorylation of HDAC4 in response to Ang‐II was increased with the overexpression of CaMKIIδ2. CaMKII overexpression resulted in increased nuclear export of HDAC4, MEF2‐promoter activity and nucleoprotein complex formation in EMSA.HDAC4 overexpression led to decreased MEF2‐promoter activity and hypertrophy in response to Ang‐II. The dominant negative mutant HDAC4 S467,632A abrogated the activation.ConclusionCaMKII mediates Ang‐II induced VSM hypertrophy by derepressing HDAC4 and activating MEF2.Supported by: AHA, Carver Trust.

Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

Abstract 1357: PGC-1 α as a Novel Regulator for Angiotensin II-induced Reactive Oxygen Species Production and Hypertrophy in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) increases H 2 O 2 production and vascular smooth muscle cell (VSMCs) hypertrophy, in part through redox-sensitive PI3K/Akt, which is inhibited by catalase overexpression. The relevant molecular mechanism remains unclear. Peroxisome proliferator-activated receptor gamma coactivator-1 α (PGC-1 α ) is reported to protect from oxidative stress by regulating expression of antioxidant enzymes such as catalase. We thus hypothesized that PGC-1 α may be important mediator for Ang II-induced H2O2 production and vascular hypertrophy. Here we show that Ang II stimulation increases serine phosphorylation of PGC-1 α (2.2 folds) with a peak at 15 min, which is inhibited by LY294002, a specific PI3 kinase inhibitor (98% decrease), and by Akt inhibitor-2/Triciribine (95% decrease). Ang II promotes PGC-1 α phosphorylation mainly at Ser 570 in an Akt-dependent manner. Ang II significantly suppresses Gal4-fused PGC-1 α transcriptional activity in a dose dependent manner, which is partially reversed by PI3K/Akt inhibition. Chromatin immunoprecipitation (ChIP) assay shows that PGC-1 α associates with the catalase promoter and this association is blocked by Ang II in a PI3K/Akt-dependent manner. Consistent with these results, Ang II stimulation time-dependently decreases endogenous catalase expression at both messenger RNA and protein levels. Ang II-induced downregulation of catalase at protein level at 24 hrs is prevented by Akt inhibitor (86%) and by overexpression of PGC-1 α S570A, an Akt phosphorylation site mutant, (75%). Moreover, overexpression of PGC-1 α S570A significantly inhibits Ang II-induced increase in H2O2 production (&gt;80%) and leucine incorporation (&gt;90%) as measured at 12 and 24 hrs, respectively. In summary, Akt-dependent serine phosphorylation of PGC-1 α by Ang II plays an important role for Ang II-induced downregulation of catalase, thereby increasing H2O2 production, which may contribute to ROS-dependent VSMC hypertrophy. These findings provide insight into a novel mechanisms by which Ang II promotes long-term H2O2 production to increase oxidative stress via targeting PGC-1alpha, and mediates metabolic abnormalities.

Abstract 1363: Novel Role of Early Endosome Antigen 1 in Reactive Oxygen Species-dependent AT1 Receptor-mediated Signaling Linked to Hypertrophy in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) stimulates hypertrophy in vascular smooth muscle cells (VSMCs) primarily through the G protein-coupled receptor (GPCR) Ang II type 1 receptor (AT1R) at the cell membrane and after its internalization. Major outputs of AT1R leading to vascular hypertrophy are triggered by reactive oxygen species (ROS)-dependent transactivation of the EGF receptor (EGFR), followed by activation of p38MAPK, Akt, p70S6K, and ERK1/2. However, underlying regulatory mechanisms are largely unknown. The small GTPase Rab5 and its downstream effector early endosome antigen 1 (EEA1) regulate endosomal internalization of GPCR. We hypothesized that EEA1 could be a scaffold facilitating the multiple outputs of AT1R signaling including those involved in hypertrophy. We found that Ang II (0.1 μ M, 5 min) and H2O2 (100 μ M, 5 min) induce formation of a complex composed of EEA1, Rab5, AT1R and EGFR that is inhibited by PEG-catalase (100 U/ml) and PEG-superoxide dismutase (SOD, 200 U/ml), inferring the redox sensitivity of the process. To explore this possibility further, we showed that Ang II and H2O2 stimulate EEA1 threonine phosphorylation (3.0- and 2.0-fold, respectively) co-temporaneously with Ang II- and H2O2-induced complex formation. Inhibition of p38MAPK by inhibitors (SB202190, PD169316; 1 μ M) or treatment with PEG-catalase and PEG-SOD prevents EEA1 phosphorylation and its recruitment to the complex. To gain further insight, we used EEA1 siRNA to knock down EEA1 (80%). EEA1 siRNA significantly inhibits (79% control) Ang II-stimulated [3H]leucine incorporation in VSMC and is associated with decrease in Akt phosphorylation and its downstream p70S6K (80% and 50%, respectively) without affecting ROS-independent Erk1/2 phosphorylation. EEA1 siRNA does not affect Ang II-induced EGFR phosphorylation. Conclusion: ROS- and p38MAPK-dependent Ang II-induced EEA1 phosphorylation is involved in formation of a signaling complex (AT1R, EGFR, EEA1, Rab5) that is related to Ang II stimulated, AT1R- and EGFR-mediated vascular hypertrophy. Akt-dependent phosphorylation of the p70S6K kinase is also EEA1-dependent. These findings provide insight into a novel role for EEA1 in Ang II signaling in VSMC hypertrophy.

Role of ERK1/2 Activation In Thrombin-Induced Vascular Smooth Muscle Cell Hypertrophy

It is well recognized that the proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. It is generally considered that the phosphorylation/dephosphorylation reactions of a variety of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including thrombin. ERK1/2 activation by G-protein-coupled receptors (GPCRs) has been shown to be Ca2+-dependent and to require the transactivation of epidermal growth factor receptor (EGFR). In addition, it is generally admitted that variations of the intracellular Ca2+ concentration ([Ca2+] i) play an important role in the transduction of mitogenic signal. Recently, we have shown that in thrombin-stimulated VSMCs, EGFR-independent activation of ERK1/2 activation could occur when agonist-induced ([Ca2+] i) elevation was reduced. This review examines recent findings in ERK1/2 signaling pathway that have been identified as critically important mediator of VSMCs hypertrophy and vascular diseases. Future investigations should now focus on the mechanisms of MAPK activation which might therefore represent a new mechanism involved in the antiproliferative effect revealed in this review.

Triple Twist Theory of Rho Inhibition by the Angiotensin II Type 2 Receptor

Although there are some exceptions,1,2 numerous publications support the counterregulatory roles of the angiotensin II type 2 receptor (AT2) against the AT1 receptor functions, such as inhibition of vascular contraction and hypertrophy.3–5 However, it is still very uncertain as to how the AT2 receptor signals interfere with those of the AT1 receptor in the cardiovascular system.5,6 Past findings suggest that the signal transduction of AT1 inhibition by the AT2 receptor may involve multiple distinct mechanisms. Some of these mechanisms appear to be indirect, such as production of nitric oxide through bradykinin opposing the vasoconstrictor actions of the AT1 receptor.3 The direct inhibitory crosstalk of the 2 receptors occurs proximal to the receptor heterodimerization, as well as downstream from the receptors between AT1-activated protein kinases epidermal growth factor receptor kinase and extracellular signal-regulated kinase (ERK)1/2/p42/44 mitogen-activated protein kinase (MAPK), etc and AT2-activated protein phosphatases protein phosphatase 2A, SHP-1, and MAPK phosphatase-1.7,8 The activation of the protein phosphatases by the AT2 receptor may or may not require heterotrimeric G proteins (Gi or Gs) and/or the recently identified AT2 receptor C-terminal tail–interacting proteins.4–6 Given that induction …

Central Role of Gqin the Hypertrophic Signal Transduction of Angiotensin II in Vascular Smooth Muscle Cells

The angiotensin II (AngII) type 1 receptor (AT(1)) plays a critical role in hypertrophy of vascular smooth muscle cells (VSMCs). Although it is well known that G(q) is the major G protein activated by the AT(1) receptor, the requirement of G(q) for AngII-induced VSMC hypertrophy remains unclear. By using cultured VSMCs, this study examined the requirement of G(q) for the epidermal growth factor receptor (EGFR) pathway, the Rho-kinase (ROCK) pathway, and subsequent hypertrophy. AngII-induced intracellular Ca(2+) elevation was completely inhibited by a pharmacological G(q) inhibitor as well as by adenovirus encoding a G(q) inhibitory minigene. AngII (100nm)-induced EGFR transactivation was almost completely inhibited by these inhibitors, whereas these inhibitors only partially inhibited AngII (100nm)-induced phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1. Stimulation of VSMCs with AngII resulted in an increase of cellular protein and cell volume but not in cell number. The G(q) inhibitors completely blocked these hypertrophic responses, whereas a G protein-independent AT(1) agonist did not stimulate these hypertrophic responses. In conclusion, G(q) appears to play a major role in the EGFR pathway, leading to vascular hypertrophy induced by AngII. Vascular G(q) seems to be a critical target of intervention against cardiovascular diseases associated with the enhanced renin-angiotensin system.

Angiotensin II‐induced migration of vascular smooth muscle cells (VSMCs) is mediated by both 72‐KDa spleen tyrosine kinase (Syk) via p38‐MAPK activated c‐Src and by ERK1/2 via c‐Src‐induced EGFR transactivation

We have previously shown that angiotensin II (Ang II)‐induced VSMC hypertrophy is mediated by activation of Syk via p38‐MAPK stimulated phosphorylation of c‐Src. However, Ang II‐induced proliferation of VSMCs is mediated through transactivation of EGFR. The purpose of this study was to examine the contribution of Syk and EGFR in Ang II‐induced migration of aortic VSMCs using the wound healing technique. Ang II (200nM) treatment for 24 hours increased VSMC migration by 100% (p &lt; 0.05). Ang II‐inducedmigration of VSMC and Syk phosphorylation, as determined by WB analysis, was inhibited by piceatannol (10μM), a tyrosine kinase inhibitor, and by transfection of VSMCs with dominantnegative (DN) but not wild type (WT) Syk plasmids. Ang II‐induced VSMC migration and Syk phosphorylation was attenuated by inhibitors of p38‐MAPK (SB202190) (20μM) and c‐Src (PP2) (10μM) and by DN c‐Src but not partially active c‐Src. Ang II‐induced VSMC migration and EGFR phosphorylation was also inhibited by EGFR blocker AG1478 (2μM). ERK1/2 inhibitor U0126 (10μM) attenuated Ang II‐induced cell migration, and ERK1/2 but not EGFR phosphorylation. c‐Src inhibitor PP2, reduced EGFR and ERK1/2 phosphorylation. These data suggest that Ang II stimulates VSMC migration via Syk through p38‐MAPK activated c‐Src, and via ERK1/2 through c‐Src induced EGFR transactivation (Supported by NIH Grant 079109 from HLBI).

Does Increased Oxidative Stress Cause Hypertension?

Hypertension is associated with increased vascular oxidative stress; however, there is still a debate whether oxidative stress is a cause or a result of hypertension. Animal studies have generally supported the hypothesis that increased blood pressure is associated with increased oxidative stress; however, human studies have been inconsistent. Oxidative stress promotes vascular smooth muscle cell proliferation and hypertrophy and collagen deposition, leading to thickening of the vascular media and narrowing of the vascular lumen. In addition, increased oxidative stress may damage the endothelium and impair endothelium-dependent vascular relaxation and increases vascular contractile activity. All these effects on the vasculature may explain how increased oxidative stress can cause hypertension. Treatment with antioxidants has been suggested to lower oxidative stress and therefore blood pressure. However, to date, clinical studies investigating antioxidant supplements have failed to show any consistent benefit. It is noteworthy that lowering blood pressure with antihypertensive medications is associated with reduced oxidative stress. Therefore, it seems that oxygen stress is not the cause, but rather a consequence, of hypertension.

Novel Role of Protein Kinase C-δ Tyr 311 Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by Angiotensin II

We have shown previously that activation of protein kinase C-delta (PKC delta) is required for angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC delta phosphorylation at Tyr(311) plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC delta phosphorylation at Tyr(311), and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC delta was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr(311) by Ang II. This phosphorylation was markedly blocked by an Src family kinase inhibitor and dominant-negative Src but not by an epidermal growth factor receptor kinase inhibitor. Ang II-induced Akt phosphorylation and hypertrophic responses were significantly enhanced in VSMCs expressing PKC delta wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC delta Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC delta K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC delta kinase activation but also the Src-dependent Tyr(311) phosphorylation contributes to Akt activation and subsequent VSMC hypertrophy induced by Ang II, thus signifying a novel molecular mechanism for enhancement of cardiovascular diseases induced by Ang II.

Thromboxane Receptor Activates the AMP-Activated Protein Kinase in Vascular Smooth Muscle Cells via Hydrogen Peroxide

Thromboxane A2 receptor (TPr) stimulation induces cellular hypertrophy in vascular smooth muscle cells (VSMCs); however, regulation of VSMC hypertrophy remains poorly understood. Here we show that TPr stimulation activates AMP-activated kinase (AMPK), which in turn limits TPr-induced protein synthesis in VSMCs. Exposure of cultured VSMCs to either TPr agonists, IBOP and U46619, or exogenous hydrogen peroxide (H2O2) caused time- and dose-dependent AMPK activation, as evidenced by increased phosphorylation of both AMPK-Thr172 and acetyl-coenzyme A carboxylase-Ser79, a downstream enzyme of AMPK, whereas SQ29548, a selective TPr antagonist, significantly attenuated TPr-enhanced AMPK activation. In parallel, both IBOP and U46619 significantly increased the production of reactive oxygen species such as H2O2. Furthermore, adenoviral overexpression of catalase (an H2O2 scavenger) abolished, whereas superoxide dismutase (which catalyzes H2O2 formation) enhanced, IBOP-induced AMPK activation, suggesting that TPr-activated AMPK was mediated by H2O2. Consistently, exposure of VSMCs to either TPr agonists or exogenous H2O2 dose-dependently increased the phosphorylation of LKB1 (at serines 428 and 307), an AMPK kinase, as well as coimmunoprecipitation of AMPK with LKB1. In addition, direct mutagenesis of either Ser428 or Ser307 of LKB1 into alanine, like the kinase-dead LKB1 mutant, abolished both TPr-stimulated AMPK activation and coimmunoprecipitation. Finally, genetic inhibition of AMPK significantly accentuated IBOP-enhanced protein synthesis, whereas adenoviral overexpression of constitutively active AMPK abolished IBOP-enhance protein synthesis in VSMCs. We conclude that TPr stimulation triggers reactive oxygen species-mediated LKB1-dependent AMPK activation, which in return inhibits cellular protein synthesis in VSMCs.