Hypertensive Rat Vascular Smooth Muscle Research Articles

Calcilytic NPS2143 promotes proliferation and inhibits apoptosis of spontaneously hypertensive rat vascular smooth muscle cells via activation of the renin-angiotensin system.

Vascular smooth muscle cell (VSMC) proliferation and apoptosis and the renin-angiotensin system (RAS) play critical roles in the development of essential hypertension. The activation of calcium-sensing receptor (CaSR), functionally expressed in VSMCs, inhibits cyclic adenosine monophosphate (cAMP) formation by elevating intracellular calcium ([Ca2+]i) and then suppressing renin release. The present study aimed to investigate the effects of NPS2143-mediated inhibition of CaSR on VSMC proliferation and apoptosis in spontaneously hypertensive rat (SHR) VSMCs and to assess whether these effects were mediated by alterations to RAS signaling. Primary VSMCs were isolated from the aortas of SHRs and Wistar-Kyoto rats. SHR VSMCs were treated with CaSR antagonist NPS2143 and cell proliferation and CaSR and RAS-related protein expression levels were measured to assess the effect. The results indicated that NPS2143 treatment promoted SHR VSMC proliferation, lower CaSR expression levels and higher RAS-related proteins levels when compared with control treatment. Additional measurement of the expression levels of proteins related to proliferation, remodeling, apoptosis and RAS related proteins, as well as cell viability, cell cycle, cell apoptosis ratio, [Ca2+]i, and the concentration of cAMP was performed after treatment with NPS2143, PLC inhibitor U73122, IP3 receptor antagonist 2-aminoethoxydiphenylborane (APB), adenylyl cyclase-V inhibitor MDL12330A, angiotensin converting enzyme inhibitor captopril, angiotensin I receptor (AT1R) inhibitor losartan, NPS2143 + U73122, NPS2143 + 2-APB, NPS2143 + MDL12330A, NPS2143 + captopril and NPS2143 + losartan. The results suggested that NPS2143 promoted cell proliferation, inhibited cell apoptosis, decreased [Ca2+]i and increased the expression of RAS compared with control treatments. NPS2143 + U73122 and NPS2143 + 2-APB enhanced the effects of NPS2143, while NPS2143 + MDL12330A, NPS2143 + captopril, NPS2143 + losartan attenuated the effected of NPS2143 in SHR VSMCs. Furthermore, the knockdown of AT1R by AT1R-short hairpin RNA also attenuated the effects of NPS2143 compared with NPS2143 alone. Collectively, these data indicated that NPS2143 promoted proliferation and inhibited apoptosis of VSMCs in SHRs, the effect of which was achieved by activation of RAS signaling.

Effects of angiotensin III on c-Jun N terminal kinase in Wistar and hypertensive rat vascular smooth muscle cells

Proliferation of vascular smooth muscle cells (VSMCs) and inflammation are well known actions associated with hypertension. Angiotensin (Ang) II mediates these physiological actions through the c-Jun N terminal Kinase (JNK), mitogen-activated proteins kinase (MAPK) pathway. Ang III effects on this pathway in VSMCs are unknown. The aim of this study was to determine whether Ang III activates JNK MAPK in Wistar VSMCs and determined whether the response was different in spontaneously hypertensive rat (SHR) VSMCs. We also ascertained whether this effect leads to VSMC proliferation. Western blots were used to determine the time and concentration effects of Ang II on JNK MAPK phosphorylation in Wistar VSMCs. Similar studies were conducted for Ang III in Wistar and SHR VSMCs. Both peptides induced JNK phosphorylation in a concentration- and time-dependent manner in Wistar VSMCs. Ang III also increased JNK phosphorylation in a concentration- and time-dependent fashion in SHR VSMCs as well. However, the ability of Ang III to induce JNK MAPK was different in SHR VSMCs as the phosphorylation levels of JNK were significantly higher in Wistar VSMCs as compared to SHR VSMCs at several time points and concentrations. Further, Ang III-mediated DNA synthesis, a measure of VSMC proliferation, occurred through activation of JNK MAPK. This study is the first to show Ang III effects on the JNK MAPK pathway in VSMCs and the role of JNK in Ang III-mediated cellular proliferation. These findings impart key information for the understanding of Ang III functions, especially in VSMCs and possible cardiovascular diseases.

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.

Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

Hemin inhibits hypertensive rat vascular smooth muscle cell proliferation through regulation of cyclin D and p21

We tested the hypothesis that HO-1 (heme oxygenase-1) activity varied between vascular smooth muscle cells (VSMC) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. HO-1 levels were measured under baseline and hemin-stimulated conditions and cell proliferation was monitored. Basal HO-1 levels in untreated cells were lower in SHR compared to WKY rats. Treatment with hemin increased HO-1 mRNA and protein levels in the cells obtained from WKY rats compared to that of SHR rats. However, hemin-treatment showed a greater inhibitory effect on VSMC proliferation in SHR rats than in WKY rats. Tin protoporphyrin IX (SnPPIX) showed a greater reversal of the anti-proliferative effect of hemin on cells from SHR rats than WKY. Similarly, VSMC proliferation from SHR was significantly inhibited in VSMC transfected with the HO-1 gene. These inhibitory effects were associated with cell cycle arrest in the G1 phase. The level of cyclin D, and cyclin dependent kinase inhibitor p21 was higher in SHR cells progressing through the G1 phase. Treatment of the cells with hemin down-regulated the expression of cyclin D and up-regulated that of p21. These results indicate that hemin, an HO-1 inducer, may play a more critical role in VSMC proliferation in SHR than WKY.

The effects of antioxidants on insulin signaling in spontaneously hypertensive rat vascular smooth muscle cells

The objective of this study was to investigate the possible role of reactive oxygen species on insulin signaling in a hypertensive rat model. Because Protein kinase B/Akt serves as a link between upstream insulin signals, such as insulin receptor substrate‐1 and their cellular consequences, we sought to determine the phosphorylative characteristics of Akt in the insulin signaling pathway. To elucidate the effects of reactive oxygen species, spontaneously hypertensive vascular smooth muscle cells were treated with 6.25–50 μM hydrogen peroxide (H2O2). Our results indicated that 6.25 μM H2O2 enhanced Akt phosphorylation, while 25 μM decreased insulin‐induced Akt phosphorylation. We also demonstrated that H2O2 decreased insulin receptor substrate‐1 phosphorylation. Further, H2O2‐induced inhibition of insulin signaling was inhibited by the antioxidant, pyrrolidine dithiocarbamate. These studies provide more evidence that reactive oxygen species inhibit insulin signal transduction thereby causing insulin resistance leading to cardiovascular disease.This work was supported by a grant from the Faculty Research Award program of the Division of Research and Sponsored Programs, Tennessee State University.

Prostaglandin D2 Synthase Inhibits the Exaggerated Growth Phenotype of Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells

Lipocalin-type prostaglandin D2 synthase (L-PGDS) has recently been linked to a variety of pathophysiological cardiovascular conditions including hypertension and diabetes. In this study, we report on the 50% increase in L-PGDS protein expression observed in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). L-PGDS expression also increased 50% upon the differentiation of normotensive control cells (WKY, from Wistar-Kyoto rats). In addition, we demonstrate differential effects of L-PGDS treatment on cell proliferation and apoptosis in VSMCs isolated from SHR versus WKY controls. L-PGDS (50 microg/ml) was able to significantly inhibit VSMC proliferation and DNA synthesis and induce the apoptotic genes bax, bcl-x, and ei24 in SHR but had no effect on WKY cells. Hyperglycemic conditions also had opposite effects, in which increased glucose concentrations (20 mm) resulted in decreased L-PGDS expression in control cells but actually stimulated L-PGDS expression in SHR. Furthermore, we examined the effect of L-PGDS incubation on insulin-stimulated Akt, glycogen synthase kinase-3beta (GSK-3beta), and ERK phosphorylation. Unexpectedly, we found that when WKY cells were pretreated with L-PGDS, insulin could actually induce apoptosis and failed to stimulate Akt/GSK-3beta phosphorylation. Insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both cell lines. We propose that L-PGDS is involved in the balance of VSMC proliferation and apoptosis and in the increased expression observed in the hypertensive state is an attempt to maintain a proper equilibrium between the two processes via the induction of apoptosis and inhibition of cell proliferation.

Increased Methylglyoxal and Oxidative Stress in Hypertensive Rat Vascular Smooth Muscle Cells

Methylglyoxal can yield advanced glycation end products via nonenzymatic glycation of proteins. Whether methylglyoxal contributes to the pathogenesis of hypertension has not been clear. The aim of the present study was to investigate whether the levels of methylglyoxal and methylglyoxal-induced advanced glycation end products were enhanced and whether methylglyoxal increased oxidative stress, activated nuclear factor-kappaB (NF-kappaB), and increased intracellular adhesion molecule-1 (ICAM-1) content in vascular smooth muscle cells from spontaneously hypertensive rats. Basal cellular levels of methylglyoxal and advanced glycation end products were more than 2-fold higher (P<0.05) in cells from hypertensive rats than from normotensive Wistar-Kyoto rats. This correlated with levels of oxidative stress and oxidized glutathione that were significantly higher in cells from hypertensive rats, whereas levels of glutathione and activities of glutathione reductase and glutathione peroxidase were significantly lower. Basal levels of nuclearly localized NF-kappaB p65 and ICAM-1 protein expression were higher in cells from hypertensive rats than from normotensive rats. Addition of exogenous methylglyoxal to the cultures induced a greater increase in oxidative stress and advanced glycation end products in cells from hypertensive rats compared with normotensive rats and significantly decreased the activities of glutathione reductase and glutathione peroxidase in cells of both rat strains. Methylglyoxal activated NF-kappaB p65 and increased ICAM-1 expression in hypertensive cells, which was inhibited by N-acetylcysteine. Our study demonstrates an elevated methylglyoxal level and advanced glycation end products in cells from hypertensive rats, and methylglyoxal increases oxidative stress, activates NF-kappaB, and enhances ICAM-1 expression. Our findings suggest that that elevated methylglyoxal and associated oxidative stress possibly contribute to the pathogenesis of hypertension.

Telomerase activation causes vascular smooth muscle cell proliferation in genetic hypertension.

Although abnormal cell growth in arterial vessel walls underpins vascular remodeling in high blood pressure, the molecular basis of the abnormality in hypertension has not been fully defined. Here, we report that in the aorta of spontaneously hypertensive rats, telomerase is selectively activated and telomeres are lengthened, in vivo and in vitro. Down-regulation of telomerase, the ribonucleoprotein complex responsible for the maintenance and elongation of telomeres (the ends of chromosomes) arrests the increased proliferation of spontaneously hypertensive rat vascular smooth muscle cells and induces apoptosis. This apoptosis is reversible by overexpressing telomerase and is prevented by increasing p53 tumor suppressor protein expression and worsened by lowering p53. Telomerase activation, telomere maintenance, and the p53 checkpoint appear to be critical for increased vascular smooth muscle proliferation, thus they represent potential novel therapeutic targets in 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.

A 90-kD Na + -H + Exchanger Kinase Has Increased Activity in Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.

Guanine nucleotide regulatory protein levels and function in spontaneously hypertensive rat vascular smooth-muscle cells

We compared G-protein levels and function in membranes from vascular smooth-muscle cells (VSMC) derived from mesenteric arteries from SHR, WKY and Wistar rats. Basal adenylyl cyclase activity was significantly reduced in SHR membranes compared with Wistar, but was similar to WKY. Isoproterenol stimulation (10 −4 M) was significantly lower in SHR membranes compared to WKY, but was similar to that in Wistar, which was also significantly lower than WKY. Forskolin (10 −4 M) and NaF (10 −2 M), resulted in a higher stimulatory response in SHR membranes. Biphasic effects of GTP on isoproterenol-stimulated membranes demonstrated unaltered G i function in SHR membranes. No significant differences were seen in the levels of G 5α (44- and 42-kDa forms), G i2α and the β-subunit in immunoblotting studies of the membranes. Amounts of G 9α/G 11α and G i3α were also unchanged. In conclusion, there are differences in adenylyl cyclase responses in SHR VSMC membranes which are not a consequence of altered levels of G-proteins, but may reflect genetic differences rather than effects of hypertension.

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.