Phenotypic modulation by fibronectin enhances the angiotensin II-generating system in cultured vascular smooth muscle cells

We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II-generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0. 2% calf serum for 48 hours. Immunofluorescence and protein levels of alpha-smooth muscle (SM) actin and expression of SM22alpha mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 microg/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II-like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II-generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-beta1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.     

Angiotensin II regulates the cell cycle of vascular smooth muscle cells from SHR

We have demonstrated that spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show the exaggerated growth and produce angiotensin II (Ang II). In the current study, we investigated the role of endogenous Ang II in the regulation of the cell cycle in VSMC from SHR. Levels of Ang II in conditioned medium from SHR-derived VSMC cultured without serum were significantly higher than levels in conditioned medium from Wistar-Kyoto (WKY) rat-derived VSMC. Basal DNA synthesis was higher in quiescent VSMC from SHR than that in cells from WKY rats. An Ang II type 1 receptor antagonist, CV11974, significantly inhibited the elevation in DNA synthesis in quiescent VSMC from SHR but did not affect it in cells from WKY rats. Cellular DNA content analysis by flow cytometry revealed that the proportion of cells in S phase was higher, whereas the proportion of cells in G1+G0 phase was lower in VSMC from SHR than those in cells from WKY rats. CV11974 significantly decreased the proportion of cells in S phase and correspondingly increased the proportion of cells in G1+G0 phase in VSMC from SHR, but it did not affect the proportion in cells from WKY rats. Cyclin-dependent kinase 2 (CDK2) activity, which is known to induce the progression from G1 to S phase, was higher in VSMC from SHR than in cells from WKY rats. Expression of CDK2 inhibitor p27(kip1) mRNA was markedly higher in VSMC from SHR than in cells from WKY rats. CV11974 decreased expression of p27(kip1) mRNA in VSMC from SHR, whereas CV11974 increased it in cells from WKY rats. These findings indicate that enhanced production of endogenous Ang II regulates the cell cycle especially in the progression from G1 to S phase, and increases CDK2 activity, which is independent of p27(kip1) in VSMC from SHR.     

Troglitazone inhibits growth and improves insulin signaling by suppression of angiotensin II action in vascular smooth muscle cells from spontaneously hypertensive rats

Troglitazone, a thiazolizidinedione, has recently been reported to possess anti-arteriosclerotic properties. To evaluate mechanisms underlying the anti-arteriosclerotic effects of troglitazone, we examined the effect of troglitazone on growth, expression of growth factors, and insulin signaling in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) which produce angiotensin II (Ang II) in a homogeneous culture. Troglitazone inhibited basal and serum-stimulated DNA synthesis and inhibited increases in the number of VSMC from SHR and normotensive Wistar-Kyoto (WKY) rats. Its inhibition was greater in VSMC from SHR. Troglitazone abolished DNA synthesis in response to Ang II in VSMC from both rat strains and markedly inhibited DNA synthesis in response to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AA in VSMC from SHR. Troglitazone did not alter the expression of transforming growth factor (TGF)-beta1, PDGF A-chain, or basic fibroblast growth factor (bFGF) mRNAs in VSMC from WKY rats, but it markedly decreased expression of these growth factor mRNAs in VSMC from SHR. Troglitazone markedly decreased basal and Ang II-stimulated expression of extracellular signal-regulated kinase proteins in VSMC from both rat strains. Troglitazone abolished Ang II-induced suppression of phosphatidilinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and IRS-1 associated p85 levels in VSMC from WKY rats. Basal PI3-kinase activity, tyrosine phosphorylation of IRS-1, and IRS-1 associated p85 levels were lower in VSMC from SHR than in cells from WKY rats. Troglitazone significantly increased PI3-kinase activity, IRS-1 associated tyrosine phosphorylation, and IRS-1 associated p85 levels in VSMC from SHR. These results indicate that troglitazone produce its anti-arteriosclerotic effects through suppression of the action of growth-promoting factors including Ang II, and that troglitazone inhibits Ang II-induced suppression of insulin signaling in VSMC from SHR, suggesting that tissue Ang II may lead to insulin resistance and to arteriosclerosis in hypertension. Troglitazone may be useful in the treatment of insulin resistance as well as of hypertensive vascular diseases.     

Endogenous angiotensin II suppresses insulin signaling in vascular smooth muscle cells from spontaneously hypertensive rats

BACKGROUND : Angiotensin II (Ang II) has been reported to inhibit insulin signaling at multiple levels in vascular smooth muscle cells (VSMC) in vitro. We have demonstrated that VSMC from spontaneously hypertensive rats (SHR) produce Ang II in a homogeneous culture. OBJECTIVE : In the current study, we investigated influences of endogenous Ang II on insulin signaling in VSMC from SHR. DESIGN AND METHODS : Phosphatidylinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were measured in VSMC from SHR and normotensive Wistar-Kyoto (WKY) rats in the absence and presence of Ang II type 1 receptor antagonist RNH6270 and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor U0126. RESULTS : Insulin treatment increased PI3-kinase activity in VSMC from WKY rats in a dose-dependent manner. In contrast, insulin treatment of VSMC from SHR did not affect PI3-kinase activity. However, co-treatment of VSMC from SHR with RNH6270 and insulin, increased PI3-kinase activity. PI3-kinase activity, IRS-1-associated tyrosine phosphorylation and p85 subunit of PI3-kinase in VSMC from WKY rats decreased in response to treatment with Ang II and returned to control levels upon co-treatment with U0126. Basal levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were significantly lower in VSMC from SHR than in cells from WKY rats. U0126 treatment of VSMC from SHR significantly increased levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase. CONCLUSION : These results indicate that endogenous Ang II suppresses insulin signaling in VSMC from SHR by activating extracellular signal-regulated kinase. These findings suggest that tissue Ang II may play a role in insulin resistance in hypertension.     

Upregulation of interleukin-8/CXCL8 in vascular smooth muscle cells from spontaneously hypertensive rats.

Chemokines promote vascular inflammation and play a pathogenic role in the development and maintenance of hypertension. In the present study, the expression of the chemokine interleukin-8/CXCL8 (IL-8/CXCL8) was investigated in cultured vascular smooth muscle cells (VSMC) obtained from the thoracic aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). IL-8/CXCL8 expression in thoracic aorta tissue and VSMC in SHR were significantly higher than in WKY. However, the expression of CXCR1 mRNA in VSMC from WKY was higher than that in VSMC from SHR. Angiotensin II (Ang II) induced a higher level of IL-8/CXCL8 mRNA expression in VSMC from SHR than in VSMC from WKY. The time course of Ang II-induced IL-8/CXCL8 expression in VSMC from SHR correlated with those of Ang II-induced CXCL1 and Ang II type 1 (AT1) receptor expression, and the expression of IL-8/CXCL8 by Ang II was inhibited by the AT1 receptor antagonist losartan. The effect of Ang II on IL-8/CXCL8 expression was not dependent on nuclear factor-kappaB (NF-kappaB) activation, but was mediated by an extracellular signal-regulated kinase (ERK) signaling pathway. Although Ang II directly induced IL-8/CXCL8 expression, expression of Ang II-induced IL-8/CXCL8 decreased in VSMC transfected with heme oxygenase-1. These results suggest that IL-8/CXCL8 plays an important role in the pathogenesis of Ang II-induced hypertension and vascular lesions in SHR.     

Role of complement 3a in the growth of mesangial cells from stroke-prone spontaneously hypertensive rats

Vascular smooth muscle cells (VSMCs) derived from spontaneously hypertensive rats (SHR) show exaggerated growth with a synthetic phenotype and angiotensin II (Ang II) production associated with increased production of complement (C3). We hypothesized that C3 is involved in the growth of mesangial cells (MCs) from hypertensive rats. We examined the effects of a C3a receptor inhibitor on proliferation, phenotype and Ang II generation in MCs from stroke prone-spontaneously hypertensive rats (SHR)-SP, SHR and Wistar-Kyoto (WKY) rats. Expression of C3 and C3a receptor were evaluated by immunohistochemical staining of the renal cortex. We examined the effects of the C3a inhibitor, SB290157, on proliferation, the expression of phenotype-marker mRNAs and Ang II production in cells from SHR-SP, SHR and WKY rats. Immunostaining of C3 was stronger in SHR and SHRSP glomeruli. MCs from SHR-SP and SHR abundantly express pre-pro C3 mRNA. SB290157 significantly inhibited basal DNA synthesis and proliferation of MCs from SHR-SP and SHR. Expression of osteopontin mRNA in MCs from SHR-SP and SHR was decreased with SB290157 treatment, whereas MC basal expression of α-SMA mRNA was decreased. SB290157 significantly decreased the production of Ang II in MCs from SHR-SP and SHR. Endogenous C3a promotes exaggerated growth with a synthetic phenotype and the production of Ang II in MCs from SHR-SP and SHR. The C3 and C3a receptor system may primarily be involved in the pathogenesis of renal remodeling in hypertensive rats.     

The Mechanism of Distinct Diurnal Variations of Renin-Angiotensin System in Aorta and Heart of Spontaneously Hypertensive Rats

Diurnal variations in plasminogen activator inhibitor-1 mRNA expression are different between the spontaneously hypertensive rats (SHRs) and the Wistar-Kyoto (WKY) rats, and between the aorta and the heart. To elucidate the mechanisms, we examined diurnal changes in the circulating renin-angiotensin system in the SHR and WKY rats. Diurnal variations in plasma renin activity (PRA), plasma angiotensin I, and aldosterone concentrations were similar between the SHR and WKY rats. On the other hand, plasma angiotensin II (Ang II) concentration in the SHR was lower than that in the WKY rats at most time points, but increased to the level of the WKY rats in the late light phase. Treatment with AT1 receptor antagonist candesartan increased plasma Ang II concentration except at ZT 8 and lessened its diurnal variation in the SHR. At the peak in plasma Ang II in the SHR, Ang II regulated genes such as transforming growth factor-β1 and p22phox were upregulated in the aorta. On the other hand, these genes were upregulated throughout the day in the heart of SHR. Candesartan treatment increased AT1a receptor mRNA expression in the heart but not in the aorta of SHR. These findings suggest that an AT1 receptor-mediated mechanism might cause a surge in plasma Ang II concentration at the late light phase in the SHR. Homologous down-regulation of AT1a receptor by Ang II may dampen the effect of a surge in plasma Ang II concentration in the heart of SHR.     

Increased expression of osteoprotegerin in vascular smooth muscle cells from spontaneously hypertensive rats

Osteoprotegerin (OPG) is a secreted protein of the tumor necrosis factor receptor family,whichregulates bone mass by inhibiting osteoclast differentiation and activation.Although OPG is expressed ubiquitouslyand abundantly in many tissues and cell types including vascular cells,the role of OPG in other tissues is unknown.Our previous studies demonstrated that OPG was highly expressed in vascular smooth muscle cells (VSMC) andupregulated during vascular lesion formation.Methods and Results We documented,by Northern blot analysis,that the expression of OPG was more prevalent in the aorta and cultured VSMC from spontaneously hypertensive rats(SHR) compared to Wistar-Kyoto rats (WKY).In addition,we found that the expression of Angiotensin Ⅱ (Ang Ⅱ)type Ⅰ receptor (AT1R) in SHR VSMC was at significantly increased levels than in WKY VSMC.Furthermore,AngⅡ potently induced the expression of OPG in VSMC in a time- and dose-dependent manner through the AT1Rsignaling pathway.Conclusions OPG expression was substantially greater in SHR VSMC,suggesting that OPGmay be an important determinant of vascular remodeling in SHR.(J Ceriatr Cardiol 2004;1:49-54.)     

Transforming growth factor-beta1 modulates angiotensin II-induced calcium release in vascular smooth muscle cells from spontaneously hypertensive rats

OBJECTIVES: To investigate the role of transforming growth factor-beta1 (TGF-beta1) on Ca2+-dependent mechanisms elicited by angiotensin II in aortic vascular smooth muscle cells (VSMC) of Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Cai2+ release induced by angiotensin II (1 micromol/ l) was studied in cultured VSMC isolated from the aortas of 6-week-old WKY rats and SHR. Intracellular Ca2+ (Cai2+) was assessed in Fura-2 loaded cells using fluorescent imaging microscopy. Angiotensin II receptors were analysed by binding studies. RESULTS: Pretreatment of VSMC for 24 h with TGF-beta1 significantly increased angiotensin II-induced Cai2+ mobilization from internal stores in SHR, while Ca2+ influx was not altered. This effect involves tyrosine kinase and is not due to an increase in angiotensin II binding sites, or a change in the affinity of the receptors. By contrast, TGF-beta1 did not modify the response of VSMC from WKY rats to angiotensin II. CONCLUSIONS: These results help our understanding of the interactions between the pathways activated by TGF-beta1 and the G protein-coupled receptor signalling pathway, and their role in genetic hypertension.     

Differential effect of low-dose thiazides on the renin angiotensin system in genetically hypertensive and normotensive rats

Fifty years since thiazide diuretics were introduced, they are established as first-line antihypertensive therapy. Because the thiazide dosing profile lessened, the blood pressure lowering mechanism may lie outside their diuretic properties. We evaluated this mechanism in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) by examining the effects of low-dose hydrochlorothiazide (HCTZ) administration on renin-angiotensin system components. The 7-day, 1.5 mg/kg per day HCTZ did not change systolic pressure (SBP) in WKY, but decreased SBP by 41 ± 2 mm Hg (P < .0001) in SHR, independent of increased water intake, urine output, or alterations in electrolyte excretion. HCTZ significantly increased the plasma concentrations of angiotensin I (Ang I) and angiotensin II (Ang II) in both WKY and SHR while reducing angiotensin-converting enzyme (ACE) activity and the Ang II/Ang I ratio (17.1 ± 2.9 before vs. 10.3 ± 2.9 after, P < .05) only in SHR. HCTZ increased cardiac ACE2 mRNA and activity, and neprilysin mRNA in WKY. Conversely in SHR, ACE2 activity was decreased and aside from a 75% increase in AT₁ mRNA in the HCTZ-treated SHR, the other variables remained unaltered. Measures of cardiac mas receptor mRNA showed no changes in response to treatment in both strains, although it was significantly lower in untreated SHR. These data, which document for the first time the effect of low-dose thiazide on the activity of the ACE2/Ang-(1-7)/mas receptor axis, suggest that the opposing arm of the system does not substantially contribute to the antihypertensive effect of thiazides.     

Impaired ceramide signalling in spontaneously hypertensive rat vascular smooth muscle: a possible mechanism for augmented cell proliferation

OBJECTIVES: In hypertension, the vascular wall undergoes morphological changes that alter mechanical responses to vasoactive substances. Ceramide is a recently identified second messenger synthesized in response to cytokines such as tumour necrosis factor alpha (TNF-alpha). It has been previously demonstrated that vascular smooth muscle cells (VSMC) from genetically hypertensive rats proliferate at a higher rate than those of normotensive origin. We tested the hypothesis that the ceramide pathway is impaired in VSMC from spontaneously hypertensive rats (SHR). DESIGN: VSMC were isolated from aortae of SHR and from Wistar-Kyoto (WKY) rats. Ceramide levels were measured under baseline and agonist-stimulated conditions and cell proliferation was monitored. METHODS: Cell proliferation was determined by cell counting. Ceramide levels were determined via radioactive labelling, high-performance thin-layer chromatography and phosphorimaging. Relative mRNA levels of neutral sphingomyelinase were determined using semi-quantitative polymerase chain reaction (PCR). RESULTS: Basal ceramide levels in untreated cells were lower in cells from SHR compared to WKY rats. During chronic treatment with TNF-alpha, ceramide levels increased in WKY rat cells but remained unchanged in cells from SHR. TNF-alpha treatment had an inhibitory effect on WKY rat VSMC proliferation, but stimulated proliferation in cells from SHR. Short-term incubation with TNF-alpha resulted in a greater increase in ceramide in cells from WKY rats than those from SHR. Semiquantitative PCR analysis indicated that neutral sphingomyelinase mRNA may be reduced in SHR VSMC. CONCLUSIONS: We conclude that ceramide synthesis is impaired in vascular smooth muscle from SHR and may contribute to increased VSMC proliferation in hypertension.     

Alterations in sarcoplasmic reticulum and angiotensin II receptor type 1 gene expression in spontaneously hypertensive rat hearts

Left ventricular hypertrophy (LVH) is an adaptive change in response to hypertensive pressure overload. Some evidence indicates that the decrease in sarcoplasmic reticulum (SR) Ca2+-ATPase mRNA expression, which may contribute to a diastolic dysfunction of the heart, occurs in the experimental pressure overload model. Also, recent studies have demonstrated that angiotensin II (Ang II) and angiotensin II receptor type 1 (AT1) play important roles in LVH. The purpose of this study was to investigate the function of the SR and the role of AT1 in genetic hypertension in spontaneously hypertensive rats (SHR) at ages 10 and 18 weeks. In SHR, cardiac hypertrophy has already developed at 10 weeks of age. SR Ca2+-ATPase activity and mRNA expression were significantly lower in SHR than in Wistar-Kyoto rats (WKY). Plasma renin activity in SHR was unchanged compared with WKY, whereas the Ang II concentration in SHR was significantly higher than that in WKY. AT1 mRNA expression in SHR was similar to that in WKY. These results suggest that in the early stage of hypertension in SHR Ang II may stimulate hypertrophy in the cardiomyocytes through the AT1, which is not downregulated by a high concentration of Ang II.     

Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells

This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (10(-12) to 10(-6) mol/L) in the absence and presence of PD98059 (10(-5) mol/L). [Ca2+]i and pHi were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]i and pHi and contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9+/-5.1% reduction in cell length relative to basal length) than in WKY (28.1+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]i and pHi effects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 576+/-24 versus 413+/-43 nmol/L; Emax of pHi, 7.33+/-0.01 versus 7.27+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax,19. 3+/-3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i (Emax, 294+/-55 nmol/L) and pHi (Emax, 7. 27+/-0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.     

Inhibition of vascular smooth muscle cell proliferation by DNA-RNA chimeric hammerhead ribozyme targeting to rat platelet-derived growth factor A-chain mRNA

BACKGROUND: Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show exaggerated growth and increasingly express platelet-derived growth factor (PDGF) A-chain mRNA compared to VSMC from normotensive Wistar-Kyoto (WKY) rats. OBJECTIVE: To investigate the effects of designed DNA-RNA chimeric hammerhead ribozyme to rat PDGF A-chain on exaggerated growth of VSMC from SHR. DESIGN AND METHODS: We designed and synthesized a 38-base DNA-RNA chimeric hammerhead ribozyme with two phosphorothioate linkages at the 3' terminal to cleave rat PDGF A-chain mRNA at the GUC sequence at nucleotide 921. We confirmed the cleavage activity of designed ribozyme by in vitro cleavage reaction and by lipofectin-mediated transfection of ribozyme into VSMC. RESULTS: Doses of 0.1 and 1 micromol/l DNA-RNA chimeric ribozyme dose-dependently inhibited basal DNA synthesis in VSMC from SHR. A dose of 1 micromol/l DNA-RNA chimeric ribozyme time-dependently inhibited basal DNA synthesis in VSMC from SHR. However, the same doses of all-RNA ribozyme had no effects on DNA synthesis in VSMC from SHR. Fluorescein isothiocyanate-labeled DNA-RNA chimeric ribozyme was recognized in cytosol at 30 min, and in nucleus at 60 min after lipofectin transfection. A dose of 1 micromol/l DNA-RNA chimeric ribozyme significantly inhibited expressions of both PDGF A-chain mRNA and PDGF-AA protein in VSMC from SHR, but not from WKY rats. CONCLUSION: These results indicated that the designed DNA-RNA chimeric ribozyme to PDGF A-chain mRNA effectively and specifically inhibited the exaggerated growth of VSMC from SHR at low concentrations, which were mediated by the reduction of PDGF A-chain mRNA and PDGF-AA protein expressions.