Cellular adhesion to elements of the extracellular matrix in the hypertensive rat modulates the effect of angiotensin II]

This study was to assess the role of different components of the extracellular matrix (ECM) on the mobilization of Cai++ induced by angiotensin II in vascular smooth muscle cells (VSMC) from hypertensive (SHR) and normotensive (WKY) rats. The effect of AII (10-6 M) on Cai++ release was studied in VSMC isolated from the aorta of 5-week-old WKY and SHR using fluorescent imaging microscopy (fura-2). Cai++ mobilization was characterized by amplitude, slope of Cai++ increase and total amount of Cai++. Cells were cultured on glass coverslips (control) or coated with either collagen I, collagen IV, vitronectin, fibronectin and extracellular matrix (ECM) and studied at confluence between passage 3 and 9. A significant increase of Cai++ released by AII has been observed with cells from WKY cultured on collagen I (meam +/- SEM, amplitude: 192 +/- 12% of control values, slope: 194 +/- 13%, total amount Cai++: 173 +/- 12%, n = 270, p < or = 0.0001 for each, unpaired t-test). Conversely, response with SHR was not significatively modified. Cai++ mobilization was not significatively modified after culture of VSMC from SHR and WKY on collagen IV. A significative decrease of the slope (WKY: 66 +/- 6%, p < or = 0.0001; SHR: 83 +/- 5%, p < or = 0.03) and of the amount of Cai++ (WKY: 74 +/- 7%, p < or = 0.01; SHR: 74 +/- 5%, p < or = 0.01) has been observed after culture of VSMC from the 2 strains on vitronectin. A decrease in amplitude (53 +/- 3%, p < or = 0.0001), slope (38 +/- 4%, p < or = 0.0001) and Cai++ release (69 +/- 5%, p < or = 0.004, n = 106) has been observed in VSMC from SHR seeded on fibronectin. Conversely, in VSMC from WKY, Cai++ mobilisation has not been modified compared with control cells. Culture of VSMC from SHR on ECM induced a significative decrease of amplitude (49 +/- 2%), slope (54 +/- 4%) and Cai++ release (53 +/- 3%, p < or = 0.0001 for each, n = 122), while in WKY, ECM induced a significative stimulation of these parameters (amplitude: 157 +/- 11%, slope: 149 +/- 13% and Cai++ release: 130 +/- 9%, p < or = 0.0001 for each, n = 247). These results show that the Cai++ mobilization induced by AII is modified by the adhesion of cells to different ECM components. This suggests a modulation of the A II-associated signalling events via the focal adhesion points. Furthermore, a difference in this modulation is observed between SHR and WKY when cells are seeded on collagen I, fibronectin or ECM. These modulations of Cai++ mobilization could play a role in the regulation of growth and differentiation of cells during the development of hypertension.     

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.     

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.     

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.     

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.     

Alterations of calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats.

We examined the possible alterations in calcium handling through the calcium channels of spontaneously hypertensive rats (SHR) using 45Ca2+ uptake measurements in cultured aortic cells. Primary cultures of vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the thoracic aortas from 8-week-old SHR and age-matched Wistar-Kyoto rats (WKY). The functions of voltage sensitive calcium channels (VSCC) and receptor operated calcium channels (ROCC) were estimated from the activated 45Ca2+ uptake in VSMC with high K+ depolarization and arginine vasopressin (AVP), respectively. Compared to basal conditions, depolarization with 55 mM KCl increased 45Ca2+ uptake at 20 min by 94 +/- 17 (SE) % in SHR and 38 +/- 6% in WKY. The activated 45Ca2+ uptake was significantly greater in SHR than in WKY (p < 0.01). There was no significant difference in 45Ca2+ uptake at 20 min in the presence of 5 x 10(-8)M AVP between SHR and WKY. These results suggest that calcium uptake, at least through VSCC, is increased in VSMC of SHR. This enhanced activity may be implicated in the hypertensive mechanisms in this model of hypertension.     

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.     

Gender-dependent difference in cell calcium handling in VSMC isolated from SHR: the effect of angiotensin II

OBJECTIVE: To investigate gender-dependent difference in the free cytosolic calcium concentration ([Ca2+ ]i ) response to angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). To further evaluate this gender-dependent difference by studying the role of thapsigargin-sensitive intracellular calcium stores and calcium influx in VSMC isolated from male and female SHR. DESIGN AND METHODS: Confluent primary cultures of VSMC isolated from male (n = 14) and female (n = 14) SHR aged 10 weeks were used in this study. [Ca2+ ]i was measured by image analysis of single myocytes loaded with Fura-2. [Ca2+ ]i response of VSMC to Ang II was measured in the presence and absence of extracellular Ca2+, to evaluate the influence of Ca2+ influx. To characterize inositol triphosphate (IP3 )-sensitive sarcoplasmic reticulum calcium stores, thapsigargin-sensitive calcium stores were measured in VSMC isolated from SHR of both genders. RESULTS: VSMC isolated from male SHR were characterized by an augmented [Ca2+ ]i response to angiotensin II in comparison with VSMC isolated from female SHR. Surprisingly, the thapsigargin-stimulated [Ca2+ ]i rise was found to be significantly greater in VSMC isolated from female SHR compared with VSMC isolated from male SHR. On the other hand, the gender-dependent difference in [Ca2+ ]i response to angiotensin II was abolished in the absence of extracellular calcium. CONCLUSIONS: We demonstrated in VSMC isolated from SHR of both genders that a greater [Ca2+ ]i response to angiotensin II in male than female VSMC is dependent on Ca2+ influx.     

Activation of the Na(+)-H+ exchanger modulates angiotensin II-stimulated Na(+)-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension.

This study investigated the role of the Na(+)-H+ exchanger (NHE) on angiotensin II (Ang II)-induced activation of Na(+)-dependent Mg2+ transport in vascular smooth muscle cells (VSMCs) from Wistar-Kyoto rats (WKY; n=20) and spontaneously hypertensive rats (SHR; n=20). Intracellular free concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) and intracellular pH (pHi) were measured with the specific fluorescent probes mag-fura 2-AM, SBFI-AM, and BCECF-AM, respectively. Na+ dependency of Mg2+ transport was assessed in Na(+)-free buffer, and the role of the NHE was determined with the highly selective NHE blocker 5-(N-methyl-N-isobutyl) amiloride (MIA). Basal [Mg2+]i was lower in SHR than WKY (0.59+/-0.01 versus 0.71+/-0.01 mmol/L, P<0.05). Basal pHi and [Na+]i were not different between the 2 groups. Ang II dose dependently increased [Na+]i and pHi and decreased [Mg2+]i. Responses were significantly greater (P<0.05) in SHR versus WKY ([Na+]i E(max)=37.5+/-1.1 versus 33.7+/-1.9 mmol/L; pHi E(max)=7.35+/-0.04 versus 7.20+/-0.01; [Mg2+]i E(min)=0. 28+/-0.09 versus 0.53+/-0.02 mmol/L, SHR versus WKY). In Na(+)-free buffer, Ang II-elicited [Mg2+]i responses were inhibited. MIA (1 micromol/L) inhibited Ang II-stimulated responses in WKY and normalized responses in SHR ([Mg2+]i E(min)=0.49+/-0.02). Ang II-stimulated activation of NHE was significantly increased (P<0.05) in SHR (0.07+/-0.002 DeltapH(i)/s) compared with WKY (0.05+/-0.004 DeltapH(i)/s). These data demonstrate that in VSMCs [Mg2+]i regulation is Na+ dependent, that activation of NHE modulates Na(+)-Mg2+ transport, and that increased activity of NHE may play a role in altered Na(+)-dependent regulation of [Mg2+]i in SHR.     

Thapsigargin-insensitive calcium pools in vascular smooth muscle cells.

Since sarcoplasmic Ca2+-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca2+]i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats of the Münster strain (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated. VSMC were cultured on glass cover slips and [Ca2+]i was measured using the fluorescent dye fura2. To exclude transplasmamembrane calcium influx all experiments were performed in a calcium free medium. Thapsigargin, a selective inhibitor of the sarcoplasmic Ca2+-ATPase, and bradykinin, that is known to induce inositol trisphosphate release, dose dependently caused an increase of [Ca2+]i by emptying intracellular Ca2+ stores. The peak increase of [Ca2+]i after addition of saturation doses of thapsigargin (1 micromol/L) was not significantly different in the two strains (SHR: 69 +/- 11 nmol/L, n=24; WKY: 58 +/- 12 nmol/L, n=20; mean +/- SEM). When 10 micromol/L bradykinin was added after depletion of the thapsigargin-sensitive pools, still a release of [Ca2+]i could be observed. The bradykinin-induced [Ca2+]i increase was similar in the absence and presence of thapsigargin in VSMC from SHR (62 +/- 12 nmol/L, n=20; vs 52 +/- 18 nmol/L, n=22). In contrast, in the VSMC from WKY a significant reduction of the bradykinin induced [Ca2+]i-increase could be observed after the depletion of the thapsigargin sensitive calcium pools (70 +/- 8 nmol/L, n=21, vs. 33 +/- 7, n=20; p<0.002). It is concluded that bradykinin releases calcium from a pool that is not refilled by the common, thapsigargin-sensitive Ca2+-ATPase. In contrast to VSMC from normotensive WKY, in VSMC from spontaneously hypertensive rats thapsigargin and bradykinin sensitive pools may be regulated separately.     

Role of c-Src in the regulation of vascular contraction and Ca2+ signaling by angiotensin II in human vascular smooth muscle cells

OBJECTIVE: Tyrosine kinases, typically associated with growth-signaling pathways, also play a role in Ang II-stimulated vascular contraction. However the specific kinases involved are unclear. We hypothesize here that c-Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascular smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contraction induced by Ang II. METHODS: Cultured VSMCs from resistance arteries of healthy subjects were studied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-deficient VSMCs from small arteries of c-Src knockout mice (Src-/-mVSMCs) were also investigated. Intracellular free Ca2+ concentration ([Ca2+]i), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioimmunoassay respectively. Contraction was examined in intact rat small arteries. RESULTS: Ang II rapidly increased VSMC c-Src activity, with peak responses obtained at 1 min. Ang II induced a biphasic [Ca2+]i response (Emax = 636 +/- 123 nmol/l). The initial [Ca2+]i transient, mediated primarily by Ca2+mobilization, was dose-dependently attenuated by the selective Src inhibitor, PP2, but not by PP3 (inactive analogue). Ang II-elicited [Ca2+]i responses were blunted in cells electroporated with anti-c-Src antibodies and in c-Src-/-mVSMCs. Src inhibition decreased Ang II-induced generation of IP3 in human VSMCs. Ang II dose-dependently increased vascular contraction (Emax = 40 +/- 6.5%). These responses were attenuated by PP2 (Emax = 7.8 +/- 0.08%) but not by PP3 (Emax = 35 +/- 4.5%). CONCLUSIONS: Our findings identify c-Src as an important regulator of VSMC [Ca2+]i signaling and implicate a novel contractile role for this non-receptor tyrosine kinase in Ang II-stimulated vascular smooth muscle.     

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.     

Silencing regulator of G protein signaling-2 (RGS-2) increases angiotensin II signaling: insights into hypertension from findings in Bartter's/Gitelman's syndromes

OBJECTIVE: Regulator of G-protein signaling (RGS)-2 is a regulator of angiotensin II (Ang II) signaling. In Bartter's syndrome/Gitelman's syndrome (BS/GS), we have demonstrated increased RGS-2 levels and blunted Ang II signaling which contribute to their reduced vasomotor tone and remodeling.The present study investigates the effect of silencing RGS-2 in fibroblasts from six BS/GS patients on intracellular Ca2+ (CaI2+) mobilization and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, established Ang II-mediated responses. METHODS: Fibroblasts were RGS-2 silenced by transfecting chemically synthesized small interfering RNA. Silencing efficiency and Ang II-induced ERK 1/2 phosphorylation were evaluated by western blot and Ang II-induced Cai2+ using Fura-2 AM. RESULTS: RGS-2 expression in not silenced BS/GS fibroblasts from patients is increased compared with healthy controls [0.34 +/- 0.02 vs. 0.19 +/- 0.01 densitometric units (d.u.), P = 0.0005]. Silencing RGS-2 in BS/GS patients was achieved to the level of controls. Ang II-induced Cai2+ release and ERK 1/2 phosphorylation were reduced in not silenced cells from BG/GS patients compared with controls (112.16 +/- 13.2 vs. 130.33 +/- 13.64 mmol/l, P = 0.011 and 0.64 +/- 0.08 vs. 0.91 +/- 0.03 mmol/l, P < 0.006, respectively). Silencing RGS-2 in BS/GS patients increased Ang II-induced Cai2+ release and ERK 1/2 phosphorylation in silenced cells compared with not silenced cells [59.3 +/- 10.8 (peak-basal) vs. 40.5 +/- 14.1 nmol/l, P = 0.017 and 0.84 +/- 0.06 vs. 0.64 +/- 0.08 nmol/l, P < 0.03, respectively], whereas they were not different compared with controls (60.1 +/- 4.3 and 0.91 +/- 0.03 nmol/l). Integrating the Cai2+ response over time showed increased Cai2+ area under the curve (AUC) of BS/GS silenced cells compared with that of not silenced cells (P = 0.013). CONCLUSION: This is the first report of silencing RGS-2 effect on Ang II signaling in a human clinical condition of altered vascular tone regulation and remodeling and establishes RGS-2 as a key regulatory element of Ang II signaling in humans.     

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.     

Inhibition of mitogen-activated protein/extracellular signal-regulated kinase improves endothelial function and attenuates Ang II-induced contractility of mesenteric resistance arteries from spontaneously hypertensive rats

OBJECTIVES: Extracellular signal-regulated kinases (ERK1/2) modulate vascular smooth muscle cell (VSMC) growth and contractility, important factors in blood pressure regulation. In the present in vivo study, we investigated whether short-term inhibition of ERK1/2-dependent signaling pathways influences vascular function and blood pressure (BP) in spontaneously hypertensive rats (SHR). METHODS: SHR and Wistar-Kyoto (WKY) rats were injected subcutaneously with either PD98059, selective MEK1/2 inhibitor (20 mg/kg), or vehicle. BP was measured by telemetry. Rats were killed 24 h after injection and small mesenteric arteries mounted as pressurized systems for morphometric analysis and assessment of endothelial function and angiotensin II (Ang II)-induced contractility. ERK1/2 phosphorylation was measured by Western blots, using protein extracts from mesenteric arteries, aorta, heart and kidneys. RESULTS: BP was higher (P < 0.01) in SHR than in WKY rats. PD98059 did not influence BP in either group. Endothelial-dependent relaxation (acetylcholine-induced), which was impaired in SHR, was improved by PD98059 (P < 0.05). Ang II increased contraction, with greater responses in SHR (Emax = 25 +/- 4%) than WKY (Emax = 9 +/- 3%) (P < 0.01). PD98059 reduced Ang II-induced contraction in SHR (Emax = 5.8 +/- 0.4%) and WKY (Emax = 4 +/- 0.4%). Vascular structure was unaltered by PD98059. Vascular and renal ERK1/2 phosphorylation, which was higher in SHR than WKY, was decreased by PD98059 in SHR. CONCLUSION: Short-term treatment with PD98059 improves endothelial function and vascular contractility without influencing BP in SHR. These findings provide evidence that vascular ERK1/2 activity is upregulated and that MEK1/2-sensitive signaling pathways play an important role in the regulation of vascular function in SHR. Acute inhibition of MEK1/2 does not alter blood pressure despite improved endothelial function and reduced arterial reactivity to Ang II.     

Differential effects of antihypertensive agents on proliferation of vascular smooth muscle cells from spontaneously hypertensive rats.

We have previously shown that Ca-antagonists and alpha-blockers substantially inhibit the cellular proliferation of cultured rat vascular smooth muscle cells (VSMC). This study explored whether these inhibitory effects on cellular proliferation differ between cultured VSMC from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). SHR VSMC proliferated much faster than WKY VSMC in 10% FCS. Cellular proliferation, determined by both cell number count and 3H-thymidine incorporation, was significantly blunted in the presence of either nifedipine (Nif) or bunazosin (Bun). The magnitude of these inhibitory effects was more pronounced for SHR cells than WKY cells (% reduction of 3H-thymidine uptake with Nif: 62.1 +/- 7.8% for SHR vs 75.3 +/- 10.2% for WKY, n = 6, p less than 0.05, and with Bun: 70.2 +/- 7.8% for SHR vs 82.1 +/- 9.9% for WKY, n = 6, p less than 0.05). In contrast, the intracellular water volume was unaffected by these antihypertensive agents based on equilibrium distribution of 3-O-methyl-D-glucose14C. It is concluded that SHR VSMC grow much faster than WKY VSMC and that this abnormality is innate to the SHR cells. It is also concluded that both Ca-antagonists and alpha-blockers exerted a substantial inhibitory effect on cellular proliferation of the cultured VSMC of either SHR or WKY. Furthermore, the greater inhibition of proliferation in the SHR VSMC suggests that Ca mediated- and/or alpha-receptor mediated processes of cellular proliferation of SHR could differ from that of WKY and that these abnormalities may contribute to the hyperproliferative changes of VSMC in this model.     

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.)     

Activation of vascular p38MAPK by mechanical stretch is independent of c-Src and NADPH oxidase: influence of hypertension and angiotensin II

Little is known about vascular MAPK regulation in response to mechanical strain. Whether mechanically-sensitive pathways are altered in hypertension is unclear. We examined effects of stretch and Ang II on activation of p38MAPK in vascular smooth muscle cells (VSMC) from WKY and SHR. The role of c-Src and redox-sensitive pathways in stretch-induced effects were examined. VSMC from mesenteric arteries were plated onto flexible silastic plates and exposed to acute or chronic cyclic stretch (10%, 1 Hz) with or without Ang II (0.1 uM). Acute stretch stimulated p38MAPK activation in WKY and SHR, independently of c-Src and reactive oxygen species (ROS), since PP2 (c-Src inhibitor) and apocynin (NADPH oxidase inhibitor), failed to alter stretch-mediated p38MAPK. Chronic stretch blunted p38MAPK phosphorylation in WKY and increased phosphorylation in SHR. Stretch, in the presence of Ang II, induced an increase in procollagen-1 expression. This was blocked by SB203580 (p38MAPK inhibitor). Accordingly, vascular p38MAPK is a mechano-sensitive MAPK, differentially regulated by acute and chronic stretch in WKY and SHR. Functionally, stretch and Ang II, amplify profibrotic responses in a p38MAPK-dependent manner, responses that are perturbed in SHR. Such molecular process may influence vascular fibrosis in hypertension and appear to be independent of c-Src and ROS.