Coculture of Vascular Endothelial Cells and Smooth Muscle Cells from Spontaneously Hypertensive Rats
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Abstract:
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.Keywords:
Vasoactive
Spontaneously hypertensive rat
The aim of the present study was to investigate the role of caveolin-1 in the inhibition of endothelin-1 induced proliferation of vascular smooth muscle cells (VSMCs) by 17beta-estradiol. In the cultured rat thoracic aortic VSMCs, proliferation of VSMCs was determined by using [(3)H]-thymidine incorporation and the expression of caveolin-1 protein was measured by immunofluorescence assays and Western blotting. The measurement demonstate VSMCs exposed to various concentrations of endothelin-1 (1-100 nmol/L) for 24 h induced an increase in [(3)H]-thymidine incorporation. Pretreament with various concentrations of 17beta-estradiol (0.1-10 nmol/L) for 24 h inhibited the proliferation effect of endothelin-1. Immunofluorescence assays showed that after 24 h treatment of VSMCs with endothelin-1 (100 nmol/L), the expression of caveolin-1 in VSMCs was significantly increased, whereas pretreament with 17beta-estradiol (10 nmol/L) for 24 h inhibited the effect. Western blotting results further proved that endothelin-1 inhibited and 17beta-estradiol increased the expression of caveolin-1 in VSMCs. These results demonstrate that 17beta-estradiol inhibits the VSMCs proliferation induced by endothelin-1, and that the effect of estradiol is probably mediated by caveolin-1.
Immunofluorescence
Caveolin
Caveolin 1
Thymidine
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According to previous studies, endothelin-1 (ET-1) is the most potent growth factor in the regulation of vascular smooth muscle cell (VSMC) proliferation in spontaneously hypertensive rats (SHR). To evaluate if the dominant effect of ET-1-induced VSMC proliferation is achieved by autocrine regulation, aortic smooth muscle cells from four-week-old SHR and WKY (Wistar-Kyoto) rats were cultured in 24-well dishes, and the effects of ET-1 on VSMC proliferation were determined by (a) 3H-thymidine incorporation assays with different ET-1 blocking treatments, including a specific anti-ET-1 antibody; BQ-123, an ETA receptor blocker; and BQ-788, an ETB receptor blocker; and (b) examining the ET-1 blockade on the effects of treatment with other growth factors, including thrombin and angiotension II (AT-II). These results demonstrated that the anti-ET-1 antibody, BQ-123, BQ-788, and BQ-123 plus BQ-788 all caused dose-dependent inhibition of proliferation. A 90% inhibitory effect was observed at the maximum doses used except for BQ-123. The ET-1 receptor blockers inhibited thrombin-induced VSMC growth; however, they did not efficiently inhibit AT-II-induced VSMC growth. These results indicate that the autocrine effects of ET-1 play a predominant role in the proliferation of VSMCs from SHR and WKY rats. They also suggest that thrombin-induced VSMC growth is mediated by the autocrine effects of ET-1, and angiotensin II-induced VSMC growth is mediated by other signal pathways.
Endothelin 3
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Aim:to explore the proliferative effect of angiotensin Ⅱ(AngⅡ), endothelin 1 (ET 1) on the aortic smooth muscle cells (VSMC) from SHR and WKY. Methods:VSMC were cultured by explant method. Proliferation of VSMC was assessed by 3H TdR incorporation and cell number. Results: 3H TdR incorporation of VSMC from SHR and WKY were increased , dependent of the concentration of AngⅡ and ET 1. 3H TdR incorporation of VSMC from SHR were increased 0.9,1.7 times by 10 7 mol/L AngⅡ, ET 1 respectively. 3H TdR incorporation of VSMC from WKY were only increased 0.3, 0.6 times. AngⅡ did not induced hyperplasia of VSMC. The cell number of VSMC from SHR and WKY were increased 209±27%, 97±13% by 10 7 mol/L ET 1, respectively. 3H TdR incorporation of VSMC from SHR and WKY were increased 6.0, 3.2 times, The cell number of VSMC from SHR and WKY were increased 3.0, 1.3 times by 10 -7 mol/L AngⅡ and 10 -7 mol/L ET 1. Conclusions:AngⅡ and ET 1 had synergistic effect on the proliferation of VSMC. VSMC from SHR were sensitive to growth factors and inclined to hypertrophy and hyperplasia.
Spontaneously hypertensive rat
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Objective To investigate the effect of S-nitrosoglutahione(GSNO) on SD rat vascular smooth muscle cells(VSMCs) proliferation induced by endothelin(ET). Methods In vitro, the rat thoracic aortic VSMCs were cultured and induced by ET, and then treated with different dose of GSNO. The change of VSMCs proliferation was determined by MTT and BrdU-ELISA incorporation methods. The proliferation cycle of VSMCs was observed by flow cytometric analysis. Results ET significantly accelerated DNA synthesis and the VSMCs proliferation. Compared with ET group,the A value and BrdU-ELISA incorporation were significantly reduced in the GSNO groups with different dose(P0.05). The percentage of cells in DNA synthetic( S ) and mitotic phase ( G2/M ) groups was lower than that of ET group(P005). With the increasing dose of GSNO, the inhibition became more significant.Conclusion To certain extent,GSNO could inhibit the proliferation of VSMCs induced by ET in dose-dependence manner.
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AIM: To explore the effects of hydrogen sulfide (H_2S) on proliferation of vascular smooth muscle cells (VSMC) stimulated by endothelin (ET-1, 10 -7 mol/L ) and mitrogen-activated protein kinase (MAPK) activity in VSMCs. METHODS: Cultured VSMCs were divided into six groups: (1) control group, (2) serum group, (3) endothelin group, (4) NaHS groups, (5) serum+NaHS group, and (6) endothelin+NaHS group. VSMC proliferation was measured by [ 3H]-TdR incorporation and MAPK activity in VSMC was determined by radioactivity assay. RESULTS: ET-1 increased VSMC [ 3H]-TdR incorporation by 2.39 times ( P 0.01) and MAPK activity by 1.62 times( P 0.01), as compared with control. H_2S (5×10 -5 -5×10 -4 mol/L) decreased VSMC [ 3H]-TdR incorporation and MAPK activity by 16.8%-37.4% and 7.4%-33.6%, respectively ( P 0.05 or P 0.01). CONCLUSION: This study demonstrates that H_2S inhibits ET-1-induced proliferation of VSMC,which might be mediated by the inhibition of MAPK.
Sodium hydrosulfide
Urotensin II
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Hypertension can lead to mood disorders that may worsen or ameliorate depending on the type of antihypertensive prescribed. Depression is associated with modifications in basal brain asymmetry particularly that of the frontal cortex, which is involved in blood pressure control. Furthermore, different vasoactive drugs may change the brain's asymmetry in a manner that contributes to cognition status. We studied the bilateral activity of several neuropeptidases in frontal cortex as a reflect of the functional status of certain neuropeptides involved in mood.Using arylamide derivatives as substrates, we fluorometrically analysed the activity of these enzymes in the left and right frontal cortex of control untreated Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs) and compared their activities with WKY or SHR treated with the antihypertensive drugs captopril (CAP) and propranolol (PRO) or with the hypertensive N (G)-nitro-L-arginine methyl ester. SBP was also measured in all WKY and SHR groups.Untreated WKY, WKY treated with CAP or PRO and SHR treated with CAP exhibited normotensive values of SBP. However, WKY treated with N (G)-nitro-L-arginine methyl ester as well as untreated SHR and SHR treated with PRO and N(G)-nitro-L-arginine methyl ester demonstrated hypertensive values of SBP. Changes in the bilateral distribution of neuropeptidases were depending on the strain, the enzyme analysed and the drug used. Normotensive WKY groups (WKY, CAP, PRO) revealed intrahemispheric correlations mainly in the left hemisphere. In contrast, WKY treated with N(G)-nitro-L-arginine methyl ester and SHR groups demonstrated intrahemispheric correlations mainly in the right hemisphere. Interhemispheric correlations were mostly observed in WKY as well as in SHR groups with antihypertensive treatments (CAP, PRO).Our results suggest specific brain bilateral patterns of neuropeptidase activities in WKY that change in SHR. This observation may be related to the cognitive disorders that have been described in these animals and that change under antihypertensive or hypertensive drug's treatments.
Vasoactive
Spontaneously hypertensive rat
Frontal cortex
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Aim To investigate the effect of endothelin-1(ET-1) on osteopontin expression of rat aortic vascular smooth muscle cell(VSMC). Methods Rat aortic vascular smooth muscle cell were cultured in vitro with DMEM and 10 percent calf plasma.The cell were incubated with different concentration of ET-1 for different hours,then RT-PCR and Western bloting analysis were used to observe the effect of H_2O_2 on osteopontin expression of rat VSMC. Results Different concentration of ET-1 could obviously induce gene expression and protein synthesis of osteopontin,without exhibiting dose-dependent promotion effect.Cell incubated with ET-1 for different hours also obviously induce gene expression and protein synthesis of osteopontin except 6 h,and exhibited time-dependence. Conclusions These results demonstrate that ET-1 could stimulate osteopontin expression of rat VSMC.
Osteopontin
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This study was to investigate the effect of sodium ferulate (SF), one of the principal components of chinese rhizoma ligustici wallichi, on the migration induced by endothelin and platelet derived growth factor (PDGF) in vascular smooth muscle cells. Cultured vascular smooth muscle cells derived from spontaneously hypertensive rats(SHR) were used. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium([Ca2+]i) was measured with fluorescent [Ca2+]i indicator Fura-2/AM. It was showed that endothelin and PDGF significantly induced a migration of vascular smooth muscle cells in a dose-dependent manner, which was dose-dependently inhibited by pre-treatment with 10-7-10-3mol/L sodium ferulate . The peak inhibition rate of migration induced by endothelin and PDGF were 85.04% and 81.92% (P<0.01) respectively. endothelin and PDGF provoked the rise of [Ca2+]i in vascular smooth muscle cells, which was significantly suppressed by 10-3mol/L sodium ferulate (P<0.01). It is concluded that migration and rise of [Ca2+]i induced by endothelin and PDGF in vascular smooth muscle cells from SHR may be suppressed by ferulate. Am J Hypertens (2004) 17, 173A–174A; doi: 10.1016/j.amjhyper.2004.03.457
Fura-2
Platelet-derived growth factor
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Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-β1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-β1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-β1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-β1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-β1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-β1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-β1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy.
Gq alpha subunit
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Summary: Endothelin-1 (ET-1) produced by vascular endothelial cells has been proposed to act in a paracrine manner on adjacent smooth muscle cells (SMCs) in vivo, exerting a variety of short- and long-term effects. Although some of the in vitro ET-1-mediated effects are related to growth-promoting events, the physiological significance of these observations remains to be clarified. Reported discrepancies of the mitogenic potential of ET-1 may relate to differences in culturing conditions (submitogenic levels of serum in combination with ET-1). Because ET-1 has been implicated in proliferation of vascular SMCs (VSMCs) at sites of vascular injury, as well as pathological events during atherogenesis, a clarification of the mitogenic effects of ET-1 is important. This study demonstrates the possible autocrine role for ET-1 in the regulation of the vasculature, its influence on VSMC cell cycle, and autocrine and phenotypic regulation of VSMCs. Stimulation of quiescent VSMCs with a variety of peptides resulted in the secretion of biologically active ET-1 by VSMCs. In contrast to previous reports, long-term exposure (12-15 days) of VSMCs to ET-1 in nonmitogenic medium did not promote cycling of cells. On the contrary, ET-1 attenuated the cycling of VSMCs in the S and G2/M phases and interrupted progression through the cell cycle at late G1/early S phase. Subsequent to ET-1 exposure, VSMCs expressed increased levels of smooth muscle-specific α-actin. Therefore, autocrineproduced ET-1 may contribute to phenotypic differentiation of VSMCs.
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