Reduced glutathione prevents nitric oxide-induced apoptosis in vascular smooth muscle cells

The control of medial and neointimal growth, in which vascular smooth muscle (VSM) plays a central role, is most important to the development of hypertension and atherosclerosis, respectively. Growth of vascular smooth muscle cells is regulated by a number of factors, including the vasodilator nitric oxide (NO). In addition, NO modulates intracellular thiol redox states and the thiol redox state of the cell influences NO production. We, therefore, examined the nature of the effect of NO on growth of VSM cells and its modulation by cellular glutathione content. Here, we report that NO, either generated by NO donors or synthesized by iNOS in VSM cells, inhibited DNA synthesis and induced apoptosis in this cell type. NO-induced apoptosis was associated with a significant decrease in the intracellular concentration of reduced glutathione and with an increase in the level of the tumor suppressor gene p53 mRNA. Moreover, addition of glutathione monoethylester to the culture restored the level of reduced glutathione in VSM cells, and prevented the NO-induced increase in p53 expression and programmed cell death. Our findings suggest a role for reduced glutathione in protecting VSM cells exposed to NO from apoptosis.     

Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells

Pulmonary vascular medial hypertrophy in primary pulmonary hypertension (PPH) is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-RII) gene have been implicated in patients with familial and sporadic PPH. The objective of this study was to elucidate the apoptotic effects of BMPs on normal human PASMCs and to examine whether BMP-induced effects are altered in PASMCs from PPH patients. Using RT-PCR, we detected six isoforms of BMPs (BMP-1 through -6) and three subunits of BMP receptors (BMP-RIa, -RIb, and -RII) in PASMCs. Treatment of normal PASMCs with BMP-2 or -7 (100-200 nM, 24-48 h) markedly increased the percentage of cells undergoing apoptosis. The BMP-2-mediated apoptosis in normal PASMCs was associated with a transient activation or phosphorylation of Smad1 and a marked downregulation of the antiapoptotic protein Bcl-2. In PASMCs from PPH patients, the BMP-2- or BMP-7-induced apoptosis was significantly inhibited compared with PASMCs from patients with secondary pulmonary hypertension. These results suggest that the antiproliferative effect of BMPs is partially due to induction of PASMC apoptosis, which serves as a critical mechanism to maintain normal cell number in the pulmonary vasculature. Inhibition of BMP-induced PASMC apoptosis in PPH patients may play an important role in the development of pulmonary vascular medial hypertrophy in these patients.     

Trans fatty acids induce vascular inflammation and reduce vascular nitric oxide production in endothelial cells

Intake of trans fatty acids (TFA), which are consumed by eating foods made from partially hydrogenated vegetable oils, is associated with a higher risk of cardiovascular disease. This relation can be explained by many factors including TFA's negative effect on endothelial function and reduced nitric oxide (NO) bioavailability. In this study we investigated the effects of three different TFA (2 common isomers of C18 found in partially hydrogenated vegetable oil and a C18 isomer found from ruminant-derived-dairy products and meat) on endothelial NF-κB activation and nitric oxide (NO) production. Human endothelial cells were treated with increasing concentrations of Elaidic (trans-C18:1 (9 trans)), Linoelaidic (trans-C18:2 (9 trans, 12 trans)), and Transvaccenic (trans-C18:1 (11 trans)) for 3 h. Both Elaidic and Linoelaidic acids were associated with increasing NF-κB activation as measured by IL-6 levels and phosphorylation of IκBα, and impairment of endothelial insulin signaling and NO production, whereas Transvaccenic acid was not associated with these responses. We also measured superoxide production, which has been hypothesized to be necessary in fatty acid-dependent activation of NF-κB. Both Elaidic acid and Linoelaidic acid are associated with increased superoxide production, whereas Transvaccenic acid (which did not induce inflammatory responses) did not increase superoxide production. We observed differential activation of endothelial superoxide production, NF-κB activation, and reduction in NO production by different C18 isomers suggesting that the location and number of trans double bonds effect endothelial NF-κB activation.     

Sera from patients with sepsis induce nitric oxide production in vascular smooth muscle cells

BACKGROUND: Nitric oxide (NO) is an important physiological mediator of vascular tone and is involved in pathophysiology of septic shock. Although plasma nitrite is a stable end product of NO oxidation derived from endogenous NO, the plasma nitrite level is also easily affected by the intake of various foods, bacterial products and renal functional status. AIMS: We propose an excellent alternative assay technique for measuring endogenous NO production. METHODS: We measured the nitrite level in cultured vascular smooth muscle cells (SMC) treated with serum obtained from patients with sepsis (4 patients), by means of a chemiluminescence detector. RESULTS: The nitrite concentrations in such cells were significantly higher as compared to those in the cells treated with normal serum. Moreover, the increased nitrite levels in the SMC treated with the sera obtained from patients with sepsis were completely inhibited by L-nitroarginine (1 mmol/L), a nitric oxide synthase inhibitor. CONCLUSION: These data suggest that this assay method enable us to know the ability of endogenous NO production in each patient.     

Goniothalamin induces apoptosis in vascular smooth muscle cells

Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.     

Succinobucol induces apoptosis in vascular smooth muscle cells

Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.     

Trans fatty acids induce apoptosis in human endothelial cells.

The present study was designed to investigate the hypothesis that trans fatty acids can induce apoptosis of human umbilical vein endothelial cells (HUVEC). To test this hypothesis apoptosis was measured in HUVEC treated with 0.1, 1.0 or 5.0 mM trans elaidic acid (t-18:1) or linoelaidic acid (t,t-18:2) for 24 hours. For the detection of apoptosis, TdT-mediated dUTP nick end labelling assay (TUNEL), cell binding of annexin V and propidium iodide uptake were measured. Active Caspase-3 and cleaved PARP (poly-ADP-ribose polymerase) were also measured in the cell lysate. Moreover, cellular ability to produce ROS (reactive oxygen species) was measured by DCF fluorescence Both acids studied induce both early (annexin-positive cells) and late stages of apoptosis (cells stained by propidium iodide) in a dose-dependent manner. Also the appearance of TUNEL-positive cells was induced by both trans fatty acids tested, in a dose dependent manner. Both trans acids induce apoptosis through their effect on Caspase-3 activity and on intracellular ROS production. It is worth emphasising that linoelaidic acid proved to be a more potent inducer of apoptosis and ROS production in endothelial cells than elaidic acid. The present studies suggest that trans fatty acids may play a role in damaging and death of vascular endothelial cells in atherosclerosis.     

Potentiation of nitric oxide-induced apoptosis in p53-/- vascular smooth muscle cells

The functionalrole of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell(VSMC) apoptosis remains unknown. In this study, VSMC fromp53^/ and p53^+/+ murine aortas were exposedto exogenous or endogenous sources of NO. Unexpectedly,p53^/ VSMC were much more sensitive to theproapoptotic effects of NO than were p53^+/+ VSMC.Furthermore, this paradox appeared to be specific to NO, because otherproapoptotic agents did not demonstrate this differential effect onp53^/ cells. NO-induced apoptosis inp53^/ VSMC occurred independently of cGMP generation.However, mitogen-activated protein kinase (MAPK) pathways appeared toplay a significant role. Treatment of the p53^/ VSMCwith S-nitroso-N-acetylpenicillamine resulted ina marked activation of p38 MAPK and, to a lesser extent, of c-JunNH₂-terminal kinase, mitogen-activated protein kinasekinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase,ERK). Furthermore, basal activity of the MEK-p42/44 (ERK)pathway was increased in the p53^+/+ VSMC. Inhibition of p38MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-inducedapoptosis. Therefore, p53 may protect VSMC against NO-mediatedapoptosis, in part, through differential regulation of MAPK pathways.

     

The oxidized phospholipids POVPC and PGPC inhibit growth and induce apoptosis in vascular smooth muscle cells

Oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are typically present in oxidatively modified low density lipoprotein (oxLDL) and have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses like inflammation, proliferation, or cell death. The aim of this study was to elicit the type and outcome of the cellular response of vascular smooth muscle cells (VSMC) upon treatment with POVPC and PGPC. Both oxidized phospholipids led to inhibition of cell proliferation and showed cytotoxic effects in VSMC. Several morphological criteria, the presence of typical DNA fragments, and a phosphatidylserine shift towards the outer leaflet of the cell membrane revealed that apoptosis was the predominant mode of cell death. In all experiments, POVPC was found to be a more potent inducer of apoptosis than PGPC. Interestingly, in the presence of high levels of serum in the growth media the proapoptotic but not the antiproliferative effects of both oxidized phospholipids were abolished. Thus, we conclude that under low serum conditions both intact POVPC and PGPC are proapoptotic mediators. Under high serum conditions, these lipids are hydrolyzed and the resultant lipid mixture containing the degradation products is no longer apoptotic but antiproliferative. Thus, the direct and indirect effects of POVPC and PGPC on cell viability may account for the detrimental actions of oxLDL on VSMC.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF- mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-, when added to these cells in the presence of serum-containing media, increases p73 expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH₂-terminally deleted p73 plasmid results in markedly decreased TNF--induced apoptosis. Thus p73 is likely a mediator of the apoptotic effect of TNF- in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies. atherosclerosis; inflammation; plaque     

Release of nitric oxide from human vascular smooth muscle cells

It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NO-release from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10(-9) moles nitric oxide per 10(8) cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10(-5) M) to 3% of the control levels (p less than 0.02, n = 4), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine.     

Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells

Methylglyoxal (MG) is a metabolite of glucose. Our previous study demonstrated an elevated MG level with an increased oxidative stress in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. Whether MG causes the generation of nitric oxide (NO) and superoxide anion (O2*-), leading to peroxynitrite (ONOO-) formation in VSMCs, was investigated in the present study. Cultured rat thoracic aortic SMCs (A-10) were treated with MG or other different agents. Oxidized DCF, reflecting H2O2 and ONOO- production, was significantly increased in a concentration- and time-dependent manner after the treatment of SMCs with MG (3-300 microM) for 45 min-18 h (n = 12). MG-increased oxidized DCF was effectively blocked by reduced glutathione or N-acetyl-l-cysteine, as well as L-NAME (p < 0.05, n = 12). Both O2*- scavenger SOD and NAD(P)H oxidase inhibitor DPI significantly decreased MG-induced oxidized DCF formation. MG significantly and concentration-dependently increased NO and O2*- generation in A-10 cells, which was significantly inhibited by L-NAME and SOD or DPI, respectively. In conclusion, MG induces significant generation of NO and O2*- in rat VSMCs, which in turn causes ONOO- formation. An elevated MG level and the consequential ROS/RNS generation would alter cellular signaling pathways, contributing to the development of different insulin resistance states such as diabetes or hypertension.     

Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 μM of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-α-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 μM), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.     

Detection of conjugated dienoic fatty acids in human vascular smooth muscle cells treated with conjugated linoleic acid

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recent studies demonstrated that conjugated metabolites of CLA are found in tissues of CLA-fed animals and cultured cells treated with CLA. This observation has gained in importance since it has recently been shown that these metabolites of CLA exert specific biological activities. Therefore, the present study aimed to explore the potential formation of metabolites of cis-9, trans-11 CLA, trans-10, cis-12 CLA and trans-9, trans-11 CLA in cells of the vascular wall, which has not yet been shown. Examination of fatty acid composition of total cell lipids using Ag+-HPLC, GC-FID and GC-MS analysis revealed a significant isomer-specific formation of conjugated metabolites of CLA such as CD16:2, CD20:2 and CD22:2 in human coronary artery smooth muscle cells treated with various CLA isomers. Different CD16:2/CLA ratios between various CLA isomers as observed in the present study indicate that fatty acid metabolism is differently affected by the configuration of the double bonds. In conclusion, the observation from the present study suggests that the effects of CLA in vascular cells might not only be mediated by CLA itself but also by its conjugated metabolites. Future studies using highly purified conjugated metabolites of CLA are necessary to study their role in mediating biological effects of CLA in cell culture systems.     

Arachidonic acid-derived oxidation products initiate apoptosis in vascular smooth muscle cells

The mechanism of arachidonic acid (AA)-induced apoptosis in vascular smooth muscle cells (VSMCs) was studied in the A-10 rat aortic smooth muscle cell line. Treatment of serum-deprived VSMCs with 50 microM AA for 24 h resulted in a loss of cell viability. The apoptotic effect of AA was characterized by annexin V binding, sub-G1 population of cells, cell shrinkage and chromatin condensation. AA-induced VSMC death was attenuated by antioxidants alpha-tocopherol and glutathione, the hydrogen peroxide (H2O2) scavenger catalase and by serum proteins, albumin and gamma globulins. Moreover, the AA peroxidation products, 12(S)-hydroperoxyeicosatetraenoic acid (HPETE), 15(S)-HPETE, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) caused VSMC apoptosis. These data suggest an oxidative mechanism of AA-induced VSMC death. The apoptotic effect of AA was pH-dependent, being inhibited by extracellular alkalinization to pH 8.0. AA inhibited serum-stimulated cell cycle progression in quiescent cells, but not in proliferating cells. In conclusion, AA, through its oxidation products causes VSMC apoptosis. Antioxidants, by inhibiting VSMC apoptosis, may prevent consequent pathological events such as atherosclerotic plaque rupture.     

Induction of apoptosis in vascular smooth muscle cells by mechanical stretch

We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun NH(2)-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-alpha (TNF-alpha) receptor-1 and its association with TNF-alpha receptor-associated factor-2 (TRAF-2). Overexpressing the wild-type TRAF-2 in PVSMCs caused an increase in apoptosis. In contrast, the expression of a dominant-negative mutant of TRAF-2 attenuated stretch-induced apoptois. These results support the hypothesis that circumferential overload under hypertensive conditions induces a clustering of death receptors that cause vascular smooth muscle cell apoptosis.     

Overexpression of p73 causes apoptosis in vascular smooth muscle cells

Abnormal vascular smooth muscle (VSM) cell proliferation contributes to the development of atherosclerosis and its associated disorders, including angioplasty restenosis. The tumor-suppressor protein p53 has been linked to the development of atherosclerotic lesions, and its homolog, p73, is proving to have contrasting functions in a variety of tissues. As an outgrowth of our previous finding that p73 is increased in serum-stimulated VSM cells and human atherosclerotic tissue, we examined p73 overexpression in VSM cells to elucidate causality of p73 expression with growth response. Overexpression of p73 results in decreased cell cycle transit and is accompanied by apoptosis. The apoptotic changes in p73 overexpressing VSM cells are independent of p53 and are associated with a decrease in levels of p21(waf1/cip1). In conjunction with our previous data finding that p73 is increased in serum-stimulated VSM cells, this work suggests a role for p73 in vascular proliferative diseases.     

Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells.

Growing evidence suggests that a pressure-induced increase in the synthesis of endothelin (ET-1) is involved in arterial remodeling and, as a consequence, in the manifestation of chronic hypertension. To study potential stretch-induced changes in gene expression and their functional consequences, we have cultured rat aortic smooth muscle cells (raSMC) and porcine aortic endothelial cells (PAEC) on flexible elastomer membranes. The cells were periodically stretched (up to 20% elongation, 0.5 Hz, 6 h) and the expression of prepro-ET-1 and that of the endothelin A and B receptors (ET(A)-R and ET(B)-R) were analyzed by semi-quantitative RT-PCR analysis and ELISA (ET-1). In contrast to PAEC where ET-1 synthesis was up-regulated up to eightfold on exposure to cyclic stretch, ET-1 synthesis in raSMC was decreased by more than 80% under these conditions. ET(A) R -mRNA expression in stretched raSMC declined to 50% whereas ET(B) R -mRNA levels were increased up to 10-fold. One functional consequence of this apparent shift in receptor abundance was an apoptosis-promoting action of exogenous ET-1 (10 nM), as judged by the appearance of subdiploid peaks during FACS analysis, caspase-3 activation and chromatin condensation. This ET-1-induced apoptosis appeared to be ET(B)-R mediated, as it was completely suppressed by the ET(B)-R antagonist BQ 788 but not by the ET(A)-R antagonist BQ 123. Moreover, raSMC derived from homozygous spotting lethal rats, which lack a functional ET(B)-R, showed no signs of apoptosis after exposure to cyclic strain and exogenous ET-1. These findings suggest a central role for the endothelin system in the onset of hypertension-induced remodeling in conduit arteries, which may proceed via an initial stretch-induced apoptosis of the smooth muscle cells.