Wistar-Kyoto Vascular Smooth Muscle Cells Research Articles

Age and Blood Pressure Contribute to Aortic Cell and Tissue Stiffness Through Distinct Mechanisms.

Aortic stiffening is strongly associated with both aging and hypertension, but the underlying mechanisms remain unclear. We hypothesized that aging-induced aortic stiffness is mediated by a mechanism differing from hypertension. We conducted comprehensive in vivo and in vitro experiments using multiple rat models to dissect the different mechanisms of aortic stiffening mediated by aging and hypertension. A time-course study in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive rats showed more pronounced aging-associated aortic stiffening in SHR versus WKY. Angiotensin II-induced hypertension was associated with more significant aortic stiffening in older versus young WKY rats. Hypertension aggravated aging effects on aortic wall thickness and extracellular matrix content, indicating combinational effects of aging and hypertension on aortic stiffening. Intrinsic stiffness of isolated aortic vascular smooth muscle cells (VSMCs) increased with age in WKY rats, although no significant difference between older SHR and older WKY VSMCs was observed in 2-dimensional culture, reconstituted 3-dimensional tissues were stiffer for older SHR versus older WKY. A selective inhibitor that reduced hypertension-mediated aortic stiffening did not decrease age-related stiffening in aortic VSMCs and aortic wall. Integrin β1 and SM22 (smooth muscle-specific SM22 protein) expression were negligibly changed in WKY VSMCs during aging but were markedly increased by hypertension in older versus young WKY VSMCs. A notable shift of filamin isoforms from B to A was detected in older WKY VSMCs. Our results indicate distinct mechanisms mediating aging-associated aortic VSMC and vessel stiffness, providing new insights into aortic stiffening and the pathogenesis of hypertension in the elderly.

Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats.

SummaryAortic stiffening is an independent risk factor that underlies cardiovascular morbidity in the elderly. We have previously shown that intrinsic mechanical properties of vascular smooth muscle cells (VSMCs) play a key role in aortic stiffening in both aging and hypertension. Here, we test the hypothesis that VSMCs also contribute to aortic stiffening through their extracellular effects. Aortic stiffening was confirmed in spontaneously hypertensive rats (SHRs) vs. Wistar‐Kyoto (WKY) rats in vivo by echocardiography and ex vivo by isometric force measurements in isolated de‐endothelized aortic vessel segments. Vascular smooth muscle cells were isolated from thoracic aorta and embedded in a collagen I matrix in an in vitro 3D model to form reconstituted vessels. Reconstituted vessel segments made with SHR VSMCs were significantly stiffer than vessels made with WKY VSMCs. SHR VSMCs in the reconstituted vessels exhibited different morphologies and diminished adaptability to stretch compared to WKY VSMCs, implying dual effects on both static and dynamic stiffness. SHR VSMCs increased the synthesis of collagen and induced collagen fibril disorganization in reconstituted vessels. Mechanistically, compared to WKY VSMCs, SHR VSMCs exhibited an increase in the levels of active integrin β1‐ and bone morphogenetic protein 1 (BMP1)‐mediated proteolytic cleavage of lysyl oxidase (LOX). These VSMC‐induced alterations in the SHR were attenuated by an inhibitor of serum response factor (SRF)/myocardin. Therefore, SHR VSMCs exhibit extracellular dysregulation through modulating integrin β1 and BMP1/LOX via SRF/myocardin signaling in aortic stiffening.

Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive rats.

Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium-dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar-Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration-dependent manner. The Ang II-induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium-dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired endothelium in SHRs.

Role of Complement 3a in the Synthetic Phenotype and Angiotensin II-Production in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

BackgroundSpontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMCs) show exaggerated growth with a synthetic phenotype and angiotensin II (Ang II)-production. To evaluate the contribution of complement 3 (C3) or C3a toward these abnormalities in SHR, we examined effects of a C3a receptor inhibitor on proliferation, phenotype, and Ang II-production in VSMCs from SHR and Wistar–Kyoto (WKY) rats.MethodsExpression of pre-pro-C3 messenger RNA (mRNA) and C3 protein was evaluated by reverse transcription-PCR and western blot analyses, and C3a receptor mRNA was evaluated by reverse transcription-PCR analysis in quiescent VSMCs from SHR and WKY rats. We examined the effects of the C3a inhibitor, SB290157, on proliferation and the expression of phenotype-marker and Krueppel-like factor 5 (KLF-5) mRNAs in VSMCs from SHR and WKY rats. We examined effects of C3a receptor inhibitor, SB290157, on Ang II-production in conditioned medium of VSMCs from SHR and WKY rats by a radioimmunoassay.ResultsExpression of pre-pro-C3 mRNA and C3 protein was significantly higher in SHR VSMCs than WKY VSMCs. SB290157 significantly inhibited proliferation of VSMCs from SHR, but not in cells from WKY rats. Relative to WKY VSMCs, SB290157 significantly increased the low expression of SM22α mRNA and decreased the high expression of osteopontin mRNA in SHR VSMCs. SB290157 significantly decreased the high expression of KLF-5 and Ang II-production in VSMCs from SHR, but not in cells from WKY rats.ConclusionsC3a induces exaggerated growth, a synthetic phenotype and Ang II-production in SHR-derived VSMCs. C3a may be primarily involved in cardiovascular remodeling in hypertension.

Mechanical stretch potentiates angiotensin II-induced proliferation in spontaneously hypertensive rat vascular smooth muscle cells

Angiotensin II (AngII) stimulates vascular smooth muscle cell (VSMC) proliferation; however, the effect of AngII on cell proliferation in the presence of mechanical force is not clear. We investigated the mechanism of AngII-induced cell proliferation mediated by mechanical stretch in VSMCs of both normotensive and hypertensive rats. VSMCs obtained from the thoracic aortas of 8-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were stretched by a Flex culture system. Mechanical stretch significantly upregulated protein expression of AngII type 1 (AT₁) receptor, epidermal growth factor (EGF) receptor and mitogen-activated protein kinase phosphatase-1 in both SHR and WKY VSMCs; however, there was no significant difference in these changes between the cells from SHR and WKY. Mechanical stretch attenuated AngII-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, ERK kinase (MEK) and EGF receptor; it also attenuated [³H] thymidine incorporation and cell proliferation in VSMC of WKY. In contrast, the effects of AngII were augmented by mechanical stretch in VSMC of SHR. AngII-induced ERK 1/2 phosphorylation and cell proliferation in SHR were inhibited by pretreatment with an AT₁ receptor blocker, candesartan and an inhibitor of MEK, PD98059. Moreover, pretreatment with an EGF receptor tyrosine kinase inhibitor, AG1478, also blocked upregulation of AngII-induced ERK 1/2 phosphorylation induced by stretch in SHR VSMCs. This study demonstrates that mechanical stretch augments SHR VSMC proliferation through an AT₁/EGF receptor/ERK-dependent pathway. These findings may provide new insights into the signaling mechanisms whereby AngII exerts its growth-promoting effects on vasculature in a hypertensive state.

ANGIOTENSIN II-INDUCED TRANSGLUTAMINASE 2 REGULATES P-38 MAP-KINASE LONG TERM EXPRESSION IN SHR VASCULAR SMOOTH MUSCLE CELLS: 2C.02

Objective: Transglutaminase 2 (TG2) is ubiquitously expressed and induces transamidation and cross-linking of proteins. This process is important for cell-matrix interactions and vascular remodeling, and is modulated by angiotensin II (Ang II). We hypothesized that Ang II through the angiotensin type 1 receptor (AT1R) modulates TG2 expression, which differentially regulates MAP kinase expression in rat vascular smooth muscle cells (VSMCs). Methods: Mesenteric artery-derived VSMCs from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were studied. Cells were stimulated with Ang II (100 nM) ± the AT1R blocker valsartan (Val 10 μM) or ± the TG2 inhibitor cystamine (CYS 10 μM). Expression of TG2, p38 MAPK, AT1R and AT2R was evaluated by immunoblotting. Results: Basal TG2 expression in SHR VSMCs was significantly higher compared to basal TG2 expression in WKY VSMCs (+28 ± 12%, p < 0.05). Ang II significantly increased TG2 expression only in SHR VSMCs compared to vehicle (+57 ± 19%, p < 0.05). Val significantly reduced Ang II-induced TG2 expression in SHR VSMCs. Basal AT1R expression in cells from WKY was lower compared to SHR (1.15 ± 0.2 AU vs 1.8 ± 0.1 AU, p < 0.05). Basal AT2R expression was 2-fold higher in SHR cells than in WKY (p < 0.05). Ang II had no effect on AT1R expression in either WKY or SHR. In presence of Ang II, AT2R expression was significantly increased in WKY and reduced in SHR (224.6 ± 65.13% vs 47.87 ± 11.95%, p < 0.05). After one-hour stimulation, Ang II increased p38 MAPK expression only in SHR (3-fold increase vs vehicle, p < 0.05). Ang II-induced p38 MAPK expression was significantly reduced by CYS. Conclusions: Ang II positively regulates TG2 expression in rat VSMCs via AT1R. TG2 plays a role in Ang II-induced p38 MAPK long-term production. These effects are particularly evident in SHR VSMCs which presented high basal levels of Ang II receptors and TG2.

Cadmium Toxicity on Arterioles Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats

Cadmium (Cd) is frequently used in various industrial applications and is a ubiquitous environmental toxicant, also present in tobacco smoke. An important route of exposure is the circulatory system whereas blood vessels are considered to be main stream organs of Cd toxicity. Our previous results indicate that cadmium chloride (CdCl2) affects mean arterial blood pressure in hypertensive rats. We hypothesized that Cd alters the intracellular calcium transient mechanism, by cadmium-induced stimulation of MAPKs (ERK 1 & 2) which is mediated partially through calcium-dependent PKC mechanism. To investigate this hypothesis, we exposed primary cultures of vascular smooth muscle cells (VSMCs) from wistar kyoto (WKY) and spontaneously hypertensive rats (SHR) to increased concentrations of CdCl2 on cell viability, expression of mitogen-activated protein kinases (MAPKs/ERK 1 & 2), and protein kinase C (PKC) which are activated by Cd in several cell types. The results from these studies indicate that CdCl2 decreased cell viability of both SHR and WKY VSMCs in a concentration dependent-manner. Viability of both cell types decreased 33+/-5.3 (SHR) and 39+/-2.3% (WKY) when exposed to 1 microM CdCl2, whereas, 8 and 16 microM reduced viability by 66+/-3.1 and 62+/-4.5% in SHR cells. CdCl2 increased ERK 1 & 2 in a biphasic manner with maximum increase occurring when cells are exposed to 1 and 4 muM in SHR VSMCs, whereas, a reduction in ERK 1 and 2 is observed when WKY cells are treated with 2 microM. The results also indicate that CdCl2 increased PKC a/Beta in both SHR and WKY VSMCs with a greater increase in expression in SHR VSMCs. In addition, the [Ca2+]i chelator, BAPTA, suppressed the CdCl2 effect, whereas, the PKC inhibitor, GF109203X, reduced the CdCl2 induced-effect on PKC expression. The present studies support the hypothesis that Cd can be a risk factor of hypertension through dysfunction of vascular smooth muscle cells under certain conditions.

Transforming Growth Factor β Suppresses Human Telomerase Reverse Transcriptase (hTERT) by Smad3 Interactions with c-Myc and the hTERT Gene

Telomerase underpins stem cell renewal and proliferation and is required for most neoplasia. Recent studies suggest that hormones and growth factors play physiological roles in regulating telomerase activity. In this report we show a rapid repression of the telomerase reverse transcriptase (TERT) gene by transforming growth factor beta (TGF-beta) in normal and neoplastic cells by a mechanism depending on the intracellular signaling protein Smad3. In human breast cancer cells TGF-beta induces rapid entry of Smad3 into the nucleus where it binds to the TERT gene promoter and represses TERT gene transcription. Silencing Smad3 gene expression or genetically deleting the Smad3 gene disrupts TGF-beta repression of TERT gene expression. Expression of the Smad3 antagonist, Smad7, also interrupts TGF-beta-mediated Smad3-induced repression of the TERT gene. Mutational analysis identified the Smad3 site on the TERT gene promoter, mediating TERT repression. In response to TGF-beta, Smad3 binds to c-Myc; knocking down c-Myc, Smad3 does not bind to the TERT gene, suggesting that c-Myc recruits Smad3 to the TERT promoter. Thus, TGF-beta negatively regulates telomerase activity via Smad3 interactions with c-Myc and the TERT gene promoter. Modifying the interaction between Smad3 and TERT gene may, thus, lead to novel strategies to regulate telomerase.

MKP-1 expression and stabilization and cGK Ialpha prevent diabetes- associated abnormalities in VSMC migration.

Diabetes mellitus is a major risk factor in the development of atherosclerosis and cardiovascular disease conditions, involving intimal injury and enhanced vascular smooth muscle cell (VSMC) migration. We report a mechanistic basis for divergences between insulin's inhibitory effects on migration of aortic VSMC from control Wistar Kyoto (WKY) rats versus Goto-Kakizaki (GK) diabetic rats. In normal WKY VSMC, insulin increased MAPK phosphatase-1 (MKP-1) expression as well as MKP-1 phosphorylation, which stabilizes it, and inhibited PDGF-mediated MAPK phosphorylation and cell migration. In contrast, basal migration was elevated in GK diabetic VSMCs, and all of insulin's effects on MKP-1 expression and phosphorylation, MAPK phosphorylation, and PDGF-stimulated migration were markedly inhibited. The critical importance of MKP-1 in insulin inhibition of VSMC migration was evident from several observations. MKP-1 small interfering RNA inhibited MKP-1 expression and abolished insulin inhibition of PDGF-induced VSMC migration. Conversely, adenoviral expression of MKP-1 decreased MAPK phosphorylation and basal migration rate and restored insulin's ability to inhibit PDGF-directed migration in GK diabetic VSMCs. Also, the proteasomal inhibitors lactacystin and MG132 partially restored MKP-1 protein levels in GK diabetic VSMCs and inhibited their migration. Furthermore, GK diabetic aortic VSMCs had reduced cGMP-dependent protein kinase Ialpha (cGK Ialpha) levels as well as insulin-dependent, but not sodium nitroprusside-dependent, stimulation of cGMP. Adenoviral expression of cGK Ialpha enhanced MKP-1 inhibition of MAPK phosphorylation and VSMC migration. We conclude that enhanced VSMC migration in GK diabetic rats is due at least in part to a failure of insulin-stimulated cGMP/cGK Ialpha signaling, MKP-1 expression, and stabilization and thus MAPK inactivation.

Coculture of Vascular Endothelial Cells and Smooth Muscle Cells from Spontaneously Hypertensive Rats

The effect of endothelium‐released vasoactive factors on vascular smooth muscle cell (VSMC) proliferation was studied in a coculture system. Isolated aortic endothelial cells and smooth muscle cells from 4‐week‐old spontaneously hypertensive rats (SHR) and age‐matched Wistar–Kyoto (WKY) rats were cocultured. After coculture, the VSMC proliferation rate was examined by 3H‐thymidine incorporation assay and the levels of the vasoactive factors in medium were determined by enzyme immunoassay (EIA). The results indicate that the proliferation rate of VSMCs in SHR was significantly higher than in WKY rats when VSMCs were cultured alone. When SHR vascular endothelial cells (VECs) were cocultured with VSMCs, the proliferation rate of SHR VSMCs was enhanced; however, there was no growth promoting effect in WKY VSMCs. When WKY VECs were cocultured with VSMCs, no VSMC proliferation effect was observed. When VSMCs were cultured alone, the endothelin‐1 (ET‐1) secretion in SHR was significantly higher than in WKY rats. When VECs and VSMCs were cocultured, the ET‐1 concentration increased in both SHR VEC and WKY VEC coculture groups in a similar manner; but the SHR VECs tended to release more thromboxaneA2 (TXA2) and less PGI2 than WKY VECs. These results suggest that some kind of interaction between SHR VSMCs and SHR VECs is responsible for the high proliferation of SHR VSMCs but not the effects of SHR VECs per se.

DNA synthesis and apoptosis in smooth muscle cells from a model of genetic hypertension.

The present study was designed to assess vascular smooth muscle cell (VSMC) proliferation and apoptosis in primary cultured VSMCs prepared from the aortic tunica media of adult (4 to 5 months old) age- and gender-matched groups of stroke-prone spontaneously hypertensive rats (SHRSP) and the normotensive reference strain, Wistar-Kyoto (WKY) rats. In the present study, VSMC proliferation was assessed with measurement of DNA synthesis in response to stimulation of G(0)/G(1) arrested VSMCs with 10% serum, whereas apoptosis was measured in response to serum deprivation. Apoptosis in aortic VSMCs was assessed in vitro with the technique of Annexin V binding in combination with propidium iodide exclusion with bivariate flow cytometric analysis. The percentage of necrotic VSMCs in the cell populations was assessed simultaneously. The light-scattering properties of the cells were assessed to provide further information on cell shrinkage and chromatin condensation. Results of the present study have shown enhanced DNA synthesis in VSMCs from SHRSP (n=10; 5.2+/-0.9 cpmx10(3)/mg protein) compared with WKY (n=12; 2.4+/-0.7 cpmx10(3) /mg protein; P<0.05, 95% CI, -5271 to -296). In addition, the results of the present study have demonstrated the role of serum in the survival of VSMCs in vitro, because SHRSP VSMCs underwent significantly more apoptosis in response to insult by serum deprivation (n=13; 10.21+/-1.8%) than WKY VSMCs (n=7; 3.44+/-1.4%; P<0.01, 95% CI, -11.5 to -2.0). Thus, it appears that both proliferation and apoptosis are enhanced in synthetic phenotype aortic medial VSMCs from the SHRSP in vitro.

GENDER DIFFERENCES IN GROWTH OF VASCULAR SMOOTH MUSCLE CELLS ISOLATED FROM HYPERTENSIVE AND NORMOTENSIVE RATS

Higher male sensitivity to atherosclerotic and hypertensive events was a reason to study sex differences in migration and proliferation of vascular smooth muscle cells (VSMC) isolated from male and female spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls. Outgrowth of cells from explants, doubling time, curves of cumulative labeling and the length of cell cycle were measured in aortic VSMC. Systolic and mean arterial pressures were higher in males than in females of the two strains. The migration of cells from male explants was significantly faster than those from female aortas in both strains. The doubling time was always shorter in male VSMC than in those from females and this was more apparent in the late exponential phase of growth. The thymidine incorporation into newly synthesized DNA, which was enhanced in SHR compared to WKY cells, was also higher in male cells compared to female ones. Cell cycle was always shorter in male than in female VSMC due to the shorter G1 phase. In contrast, shorter S phase caused shorter cell cycle in SHR compared to WKY VSMC. Consequently, the shortest cell cycle was found in VSMC from SHR males with the highest blood pressure. It can be concluded that gender and genotype are two independent factors participating in the control of migration and proliferation of VSMC.

Mechanisms of Increased NGF Production in Vascular Smooth Muscle of the Spontaneously Hypertensive Rat

The spontaneously hypertensive rat (SHR) was developed as a genetic model of essential hypertension.In vivoandin vitroevidence demonstrates that vascular smooth muscle cells (VSMCs) from the SHR produce more nerve growth factor (NGF) than the normotensive Wistar-Kyoto (WKY) control strain. This increased NGF production is accompanied by excessive innervation of target tissues in the SHR. In the present study, a sensitive, competitive, quantitative, reverse-transcriptase polymerase chain reaction (C Q RT-PCR) assay is characterized and used to analyze levels of NGF mRNA in cultured VSMCs derived from the SHR and WKY strains as well as bladder tissue. Differences in NGF secretion rates between SHR and WKY VSMCs were partially due to an increased stability of NGF mRNA in SHR VSMCs. Following treatment with platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β1) to elevate NGF production, the half-life of the NGF mRNA was 104.5 ± 18.0 min in SHR VSMCs, compared to only 36.5 ± 11.6 min in WKY VSMCs. Sequence analysis of the 3′ untranslated region (UTR) revealed no strain differences incis-acting sequences potentially involved in determining mRNA stability. Thus, it seems unlikely to be a 3′UTR mutation that prolongs mRNA lifetime. Rather, differential regulation of an RNA-binding protein may play a role in the abnormal NGF mRNA stability in SHR VSMCs. SHR VSMCs also demonstrate an increased translational efficiency of NGF protein; more NGF protein is synthesized per unit of NGF mRNA. The use of a C Q RT-PCR assay has allowed the determination that abnormal NGF mRNA stabilization as well as altered translational efficiency may contribute to excess NGF synthesis and progressive hypertension in the SHR.

Transforming Growth Factor-β and Receptor Tyrosine Kinase–Activating Growth Factors Negatively Regulate Collagen Genes in Smooth Muscle of Hypertensive Rats

Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-beta (TGF-beta) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-beta on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-beta isoforms -beta1, -beta2, or -beta3 induced rapid, transient elevations in mRNAs encoding collagens alpha1(I), alpha2(I), and alpha1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen alpha1(III)] to ninefold [collagen alpha1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-beta isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-beta1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-beta isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-beta did not affect the time course or magnitude of the changes in collagens alpha1(I), alpha2(I), or alpha1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-beta1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-beta, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-beta responsiveness.

Endothelium stimulates the vascular smooth muscle cell Na/K pump of normotensive and hypertensive rats by a protein kinase C pathway.

To investigate the mechanisms involved in the endothelial stimulation of the vascular smooth muscle cell Na/K pump and their possible alteration by hypertension. The Na/K pump activity of vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) was studied using the radioactive analogue of K+ 86Rb+. Conditioned medium of bovine endothelial aortic cells was used to investigate the endothelial modulation of VSMC Na/K pump activity. Conditioned medium enhanced VSMC Na/K pump activity (ouabain-sensitive 86Rb+ uptake), this effect being higher in SHR cells. This stimulatory effect was neither modified in Na(+)-loaded cells from both rat strains nor inhibited by the Na/H exchange blocker amiloride. Permeable analogues of cyclic adenosine and guanosine monophosphates did not modify the baseline VSMC Na/K pump activity of WKY rats and SHR, and subsequently the guanylate cyclase inhibitor methylene blue did not alter the conditioned medium-induced stimulation of the pump. However, the Ca(2+)-channel inhibitor nifedipine reduced the Na/K pump stimulation by conditioned medium, this decrease being higher in WKY rat than in SHR VSMC. Moreover, treatment with phorbol 12,13-dibutyrate for 24 h or with the protein kinase C inhibitor staurosporine for 15 min reduced the conditioned medium-induced Na/K pump activation in both VSMC cultures. Na/K pump stimulation by conditioned medium of endothelial cells is mediated mainly via activation of protein kinase C in VSMC from either WKY or SHR VSMC. However, SHR VSMC show some alterations in their intracellular signalling pathways.

Inducible nitric oxide synthase in vascular smooth muscle cells from prehypertensive spontaneously hypertensive rats

The inducible nitric oxide synthase (iNOS) present in vascular smooth muscle cells (VSMC) may play a role in the generation of nitric oxide (NO) in the vascular wall, regulating blood vessel tone in normotension and hypertension. In this study the effect of interleukin (IL)-1β, a cytokine that induces iNOS, on NO generation (measured as nitrite), cyclic guanosine monophosphate (cGMP) generation, and steady-state abundance of iNOS mRNA were examined in VSMC from 3 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, during the period preceding the elevation of blood pressure. With cell density dependent variations in nitrite production eliminated, VSMC from SHR and WKY did not differ in NO generation except after prolonged incubation (30 h), when SHR cells produced less NO. However, cGMP concentrations associated with IL-1β stimulation were significantly smaller in SHR VSMC than in cells from WKY. IL-1β stimulation resulted in increased abundance of iNOS mRNA to the same extent in both WKY and SHR VSMC. Inhibitors of NOS, N G-monomethyl- l-arginine (L-NMMA) and N ω-nitro- l-arginine methyl ester (L-NAME), did not block the induction of iNOS mRNA, although nitrite production and cGMP generation were inhibited. The protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin-D almost completely blocked the production of nitrite in cells from both strains of rats. Actinomycin-D completely blocked the induction of iNOS mRNA by IL-1β in cells from both strains of rats, whereas cycloheximide partially blocked its synthesis in WKY, but had no significant effect on IL-1β induced expression of iNOS mRNA in SHR VSMC. Thus, IL-1β controls iNOS gene expression at the transcriptional level, and an intermediate labile protein, whose synthesis is inhibited by cycloheximide, is required for IL-1β stimulated induction of iNOS mRNA transcription in WKY cells but not in SHR. We conclude that although iNOS is expressed to similar extent in VSMC of prehypertensive SHR and WKY and similar amounts of NO are initially generated, there are differences between the VSMC of SHR and WKY in the regulation of the transcription of iNOS mRNA, there is a lower sustained production of NO, and there is a reduced generation of cGMP in response to IL-1β stimulated NO production. These differences between VSMC from prehypertensive SHR and WKY may indicate a pathophysiological role of iNOS in early blood pressure elevation in SHR.

Ca(2+)-dependent mitogen-activated protein kinase activation in spontaneously hypertensive rat vascular smooth muscle defines a hypertensive signal transduction phenotype.

The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities in VSMC function, including increased growth, Na(+)-H+ exchange, and increased signal transduction by protein kinase C. The family of kinases termed mitogen-activated protein (MAP) kinases has recently been shown to be essential mediators of growth factor signal transduction. In the present study, alterations in MAP kinase function in the hypertensive phenotype were investigated using early-passage SHR and Wistar-Kyoto (WKY) VSMCs stimulated with angiotensin II (Ang II, 100 nmol/L) or platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Two differences between SHR and WKY rats were observed for Ang II-mediated MAP kinase activation: (1) Inactivation after Ang II stimulation was more rapid in SHR than WKY VSMCs. (2) Activity in SHR VSMCs showed a greater dependence on Ca2+ mobilization, since chelation of intracellular Ca2+ with BAPTA inhibited maximal activity by 95% in SHR VSMCs but by only 50% in WKY VSMCs. In contrast to the results with Ang II, no differences in PDGF-stimulated MAP kinase activity were observed. These findings establish activation of MAP kinase by Ang II as a feature that distinguishes SHR VSMCs from WKY VSMCs and suggest that differences in regulation of MAP kinase signaling may alter cellular events that are increased in the SHR genetic model of hypertension.

Altered signalling in vascular smooth muscle from spontaneously hypertensive rats may link medial hypertrophy, vessel hyperinnervation and elevated nerve growth factor.

1. Secretion of nerve growth factor (NGF) by cultured vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto (WKY) strain was measured via two site immunoassay (ELISA). 2. Basal NGF secretion rates of quiescent SHR VSMC in serum-free culture medium were elevated compared to similar WKY VSMC. 3. SHR VSMC displayed increased NGF secretion in response to activation of sympathetic neurotransmitter receptors while VSMC of WKY were largely unresponsive to the agents (phenylephrine, isoproterenol, alpha-beta-methyl ATP, neuropeptide Y). 4. Mitogenic stimulation with platelet-derived growth factor (PDGF) raised SHR NGF secretion rates almost three times more than PDGF increased WKY secretion. 5. SHR VSMC also failed to demonstrate normal inhibitory control over NGF secretion seen in WKY and previously in Sprague-Dawley and Wistar strain VSMC with adenylate cyclase activation and down-regulation of protein kinase C. High concentrations of forskolin stimulated, instead of inhibiting, secretion in SHR. Stimulation was also seen after pretreatment with phorbol ester for 24 h while this inhibited secretion in the WKY. 6. These results confirm that the SHR VSMC are hyperresponsive to growth stimuli such as contractile agonists and mitogens. This hyperresponsiveness includes an abnormal control over NGF secretion such that normally inhibitory treatments stimulate NGF output in the SHR. 7. Because the SHR demonstrates important defects in the major intracellular growth-signalling systems that also regulate NGF output and vessel innervation, the predicted result of the defects is a destructive feed-forward cycle of growth and innervation. This is the SHR phenotype in vivo.

Altered regulation of nerve growth factor secretion by cultured VSMCs from hypertensive rats.

Vascular tissues from spontaneously hypertensive rats (SHR) exhibit increased nerve growth factor (NGF) levels and increased density of sympathetic innervation compared with those from normotensive Wistar-Kyoto (WKY) rats. The present study asked whether basal NGF secretion or secretion elicited by agents analogous to sympathetic neurotransmitters differ in cultured vascular smooth muscle cells (VSMCs) from SHR and WKY rats. VSMCs were maintained in serum-free medium (SFM) for 72 h and then treated and sampled at 4, 6, 8, and 24 h. Conditioned medium was assayed for NGF using a two-site enzyme-linked immunoassay. NGF secretion by SHR (19.2 +/- 4.6 pg.well-1.48 h-1) and WKY VSMCs (16.7 +/- 5.4 pg.we..-1.48 h-1) was similar in cultures grown in serum-containing medium, whereas SHR VSMCs maintained in SFM secrete more NGF than WKY VSMCs (9.1 +/- 1.9 vs. 2.9 +/- 0.4 pg.well-1.24 h-1, respectively). Treatment of cultures with phenylephrine (0.1-10 microM), neuropeptide Y (1-1,000 nM), or alpha beta-methyleneadenosine 5'-triphosphate (10 and 100 microM) had no effect on NGF secretion by WKY VSMCs, while increasing NGF secretion by SHR VSMCs. Treatment with isoproterenol (0.1-10 microM) decreased NGF secretion by WKY VSMCs but not SHR VSMCs. These data indicate that the regulation of NGF secretion by sympathetic neurotransmitter receptors is different for cultured VSMCs from SHR and WKY rats. If similar differences exist in vivo, they could account for the alterations in NGF levels and sympathetic innervation that are observed.

Kinetics of Na+/H+ exchange in vascular smooth muscle cells from WKY and SHR: effects of phorbol ester.

The kinetic properties of Na+/H+ exchange were investigated in vascular smooth muscle cells (VSMC) in culture from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Antiport activity was measured in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells after nigericin-induced cytosolic acidification. Studies were performed without (control) and with pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA; 200 nM). Na+/H+ exchange markedly differed between the two strains with lower Hill coefficients [1.56 +/- 0.17 (SE) vs. 2.62 +/- 0.36] and higher maximal activity (Vmax) values (55.85 +/- 5.24 vs. 31.11 +/- 2.38 mmol H+.l-1.min-1) in SHR compared with WKY cell lines. PMA markedly altered the antiport kinetics in WKY VSMC with a decrease in the Hill coefficient (1.75 +/- 0.14) without affecting Vmax (31.88 +/- 1.55 mmol H+.l-1.min-1). In VSMC from SHR, PMA had no effect on the kinetic variables investigated. Thus two kinetic abnormalities are present with respect to Na+/H+ antiport activity in VSMC from SHR compared with WKY, i.e., increased Vmax and decreased Hill coefficient. The observation that PMA does not affect the kinetics of the Na+/H+ antiport in VSMC from SHR suggests a marked degree of antiporter prestimulation in this animal model of genetic hypertension.