Estradiol increases apoptosis in human coronary artery endothelial cells by up-regulating Fas and Fas ligand expression

CONTEXT: In animal models, estrogen inhibits atherogenesis by inhibiting many of the early steps of atherosclerotic plaque formation. However, the lack of cardioprotective effect by postmenopausal hormone replacement therapy and possible increase in cardiovascular events observed during the first year after the initiation of hormone replacement therapy may suggest that once the plaque is formed, estrogen may have additional effects that may counteract its beneficial outcomes. Indeed, the effect of estrogen on plaque stability has not been identified. OBJECTIVE: We hypothesized that 17beta-estradiol (E2) may cause increased apoptosis in human coronary artery endothelial cells (HCAECs). This effect would explain an adverse effect on plaque stability in vivo. INTERVENTION(S) AND MAIN OUTCOME MEASURE(S): The effect of E2 on apoptosis, cell proliferation, and expression of proapoptotic molecules Fas and Fas ligand (FasL) in cultured HCAECs was evaluated. RESULTS: HCAECs in culture treated with E2 showed an increase in DNA strand breaks and nuclear fragmentation indicative of apoptosis. E2 treatment also induced a significant concentration-dependent increase in Fas mRNA and protein expressions in HCAECs. Moreover, the expression of FasL mRNA and secretion of FasL protein by HCAECs were enhanced in response to E2 treatments. CONCLUSIONS: E2 increases the apoptosis in cultured HCAECs. Enhanced Fas and FasL expressions in response to E2 suggest that activation of the Fas/FasL pathway may be a mediator of the proapoptotic effects of E2 in these cells.     

Carvedilol prevents epinephrine-induced apoptosis in human coronary artery endothelial cells: modulation of Fas/Fas ligand and caspase-3 pathway

BACKGROUND: Several studies have shown that carvedilol, a multiple action neurohumoral antagonist, reduces mortality in patients with congestive heart failure (CHF). In addition to being a beta-adrenoceptor antagonist, carvedilol is a potent antioxidant. Since there is evidence for elevation of catecholamine levels in plasma and coronary artery endothelial cell injury in CHF, the present study was designed to test the hypothesis that carvedilol inhibits epinephrine-induced apoptosis, and the inhibitory effect is mediated by modulation of Fas, Fas ligand (FasL) and caspase-3 pathway, in cultured human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: HCAECs were exposed to epinephrine alone, carvedilol + epinephrine, or atenolol + epinephrine for 24 h. Epinephrine increased the number of apoptotic cells, measured by in situ nick end-labeling staining (from 4.2 +/- 1.3% to 28.6 +/- 6.0%, P < 0.01, n = 6) and by DNA laddering on agarose gel electrophoresis. Epinephrine also increased Fas and FasL protein expression (P < 0.01 vs. control, n = 6), and activated intracellular protease caspase-3 (P < 0.01 vs. control, n = 6). These effects of epinephrine were completely inhibited by carvedilol. Atenolol in equimolar concentration also attenuated epinephrine-mediated effects, but the effects of atenolol were less marked than those of carvedilol (P < 0.01). To explore the basis of differential effects of carvedilol and atenolol, effects of these agents on epinephrine-induced lipid peroxidation was measured. Lipid peroxidation was completely blocked by carvedilol, whereas equimolar concentration of atenolol had much less (P < 0.05) effect. CONCLUSION: Epinephrine induces apoptosis in HCAECs, and this effect is associated with activation of Fas-FasL and caspase-3 signal transduction pathway. Carvedilol can, more effectively than atenolol, inhibit these effects of epinephrine. The potent antioxidant effect of carvedilol is probably responsible for the superior effect.     

Sensitivity to Fas-mediated apoptosis is determined below receptor level in human vascular smooth muscle cells

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.     

Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions

Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.     

The anti-inflammatory effects of nebivolol in human coronary smooth muscle cells: clinical implications

Given that the majority of hypertensives require more than one drug to control blood pressure, the pleitropic effects (especially anti-inflammatory effects) and favourable metabolic profile of nebivolol could make it an alternative drug regimen for hypertensives requiring beta-blocker treatment. Although standard beta-blockers seem to have a hard time at present, we are convinced that perhaps the new generation beta-blockers such as nebivolol might yet 'rescue' the reputation of beta blockers in the near-future.     

7-Ketocholesterol predisposes human aorta smooth muscle cells to Fas-mediated death

Human vascular smooth muscle cells (HVSMCs) are resistant to Fas-mediated death under normal physiological conditions. However, HVSMC death by activation of the receptor pathway was reported in the atherosclerotic lesions. In this study, we investigated whether 7-ketocholesterol, one of the major cholesterol oxides in the lesions, altered resistance of HVSMC to Fas-mediated death pathway. Cross-linking of Fas receptor with agonistic anti-Fas antibody (CH11) in the presence of 7-ketocholesterol induced death in human aorta smooth muscle cells (HAoSMC) as detected by morphology, viability, and DNA fragmentation. The agonistic anti-Fas antibody, however, did not induce death in the presence of 7alpha-hydroxycholesterol or cholesterol. The HAoSMC death was significantly inhibited by an antagonistic Fas receptor (FasR) antibody and by expression of dominant negative Fas-associated death domain containing protein (DN-FADD) using adenoviruses. Activation of caspase-3 was observed in HAoSMC destined to death. HAoSMC death was significantly inhibited by pharmacological caspase inhibitor, z-VAD and z-DEVD, and baculovirus caspase inhibitor p35. 7-Ketocholesterol impaired mitochondrial transmembrane potential and ATP production. Overexpression of bcl-xL also significantly inhibited HAoSMC death. In dying HAoSMC, bax was translocated from the cytosol to mitochondria and cytochrome c was released from mitochondria into the cytosol. This is the first report demonstrating implication of the oxysterol in Fas-mediated death pathway. The present study proposes that 7-ketocholesterol would contribute to loss of HVSMC in the atherosclerotic lesions by altering resistance to receptor-mediated death pathway.     

Vascular endothelial cells and smooth muscle cells differ in expression of Fas and Fas ligand and in sensitivity to Fas ligand-induced cell death: implications for vascular disease and therapy

Fas ligand (FasL) is a death factor that induces apoptosis in cells bearing its receptor, Fas. Fas and FasL have been detected in the vessel wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the vasculature. Here, we evaluated the expression of Fas in the presence and absence of cytokines on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We also examined the sensitivity of ECs and VSMCs to Fas-mediated apoptosis induced by exposure to multiple Fas agonists: soluble FasL, anti-Fas antibody, and membrane-bound FasL resulting from transduction with a replication-defective adenovirus expressing FasL (Adeno-FasL). Cell-surface FasL expression was detected on human ECs with the use of 4 anti-FasL antibodies, whereas cell-surface FasL expression was not detected on VSMCs. Unstimulated ECs expressed relatively low levels of Fas, but expression was upregulated after treatment with tumor necrosis factor-alpha (TNF-alpha) or interferon gamma (IFN-gamma). In contrast, VSMCs expressed relatively high levels of Fas, and treatment with TNF-alpha or IFN-gamma induced little or no upregulation under the conditions of these assays. ECs were resistant to death after exposure to soluble FasL or agonist anti-Fas antibody and also after infection with Adeno-FasL in the presence or absence of cytokine treatment. In contrast, VSMCs remained viable in the presence of soluble FasL or agonist anti-Fas antibody, but they underwent apoptosis after infection with Adeno-FasL. IFN-gamma enhanced Adeno-FasL-induced death of VSMCs, but TNF-alpha did not. These findings provide insights about the potential role of Fas-mediated apoptosis in the vessel wall and suggest strategies to treat proliferative vascular diseases by exploiting the differential sensitivity of ECs and VSMCs to FasL-induced cell death.     

PGE2 action in human coronary artery smooth muscle: role of potassium channels and signaling cross-talk

Cyclic AMP-stimulating agents are powerful vasodilators, but our knowledge of the signal transduction mechanisms of these agents, particularly in human arteries, is limited. We now report direct molecular effects of prostaglandin E(2) (PGE(2)) on cultured human coronary artery smooth muscle cells (HCASMC). Patch-clamp studies revealed that 10 microM PGE(2) opens a high-conductance (approximately 200 pS), calcium-stimulated potassium (BK(Ca)) channel in intact HCASMC. In contrast, PGE(2) had no direct effect on channels in cell-free patches, indicating involvement of a soluble second messenger. Enzyme immunoassay demonstrated that PGE(2) enhances production of cAMP in HCASMC, but does not increase [cGMP]. Furthermore, forskolin, CPT-cAMP, or CPT-cGMP mimicked the stimulatory effect of PGE(2) on BK(Ca) channel activity. Interestingly, the response to PGE(2) was unaffected by inhibiting the cAMP-dependent protein kinase, but was antagonized by inhibitors of the cGMP-dependent protein kinase (PKG). Furthermore, cAMP-stimulated PKG activity mimicked the effect of PGE(2). These studies suggest a novel PGE(2) action in human arteries: opening of BK(Ca) channels via cAMP cross-activation of PKG in HCASMC. It is proposed that this signaling mechanism may mediate the vasodilatory response to cAMP-dependent agents in the human coronary and other vascular beds.     

Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging

OBJECTIVE: The observation of local 'elementary' Ca2+ release events (Ca2+ sparks) through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) has changed our understanding of excitation-contraction (EC) coupling in cardiac and smooth muscle. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation by activating co-localized Ca2+ activated K+ (K(Ca)) channels (STOCs). However, all prior studies on Ca2+ sparks have been performed in non-human tissues. METHODS: In order to understand the possible significance of Ca2+ sparks to human cardiovascular function, we used high spatial resolution confocal imaging to record Ca2+ sparks in freshly-isolated, individual myocytes of human coronary arteries loaded with the Ca2+ indicator fluo-3. RESULTS: Local SR Ca2+ release events recorded in human myocytes were similar to 'Ca2- sparks' recorded previously from non-human smooth muscle cells. In human myocytes, the peak [Ca2+]i amplitudes of Ca2+ sparks (measured as F/F0) and width at half-maximal amplitude were 2.3 and 2.27 microm, respectively. The duration of Ca2+ sparks was 62 ms. Ca2+ sparks were completely inhibited by ryanodine (10 micromol/l). Ryanodine-sensitive STOCs could be identified with typical properties of K(Ca) channels activated by Ca2+ sparks. CONCLUSION: Our data implies that modern concepts suggesting an essential role of Ca2+ spark generation in EC coupling recently derived from non-human muscle are applicable to human cardiovascular tissue. Although the basic properties of Ca2+ sparks are similar, our results demonstrate that Ca2+ sparks in coronary arteries in humans, have features distinct from non-arterial smooth muscle cells of other species.     

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from monocytes/macrophage

Fas and its ligand (FasL), are a receptor-ligand pair identified as promoting cell death in several tissues. Apoptosis of vascular smooth muscle cells (VSMCs) in human atherosclerotic plaque may contribute to weakening of the fibrous cap, ultimately resulting in plaque rupture. We investigated the ability of monocytes to induce apoptosis of cultured VSMCs through Fas/FasL pathway. In addition, we examined the association of FasL with apoptosis in human coronary plaques. Both activated monocytes and the supernatant obtained from activated monocytes were able to kill cultured VSMCs. The apoptotic response of VSMCs was almost completely blocked by the caspase inhibitor z-VAD-fmk and was partially blocked by incubation with antagonistic anti-Fas IgG1 which suggests that Fas/FasL system was involved in the induction of cell death. An approximate 30 kDa protein, which represents a cleaved, soluble form of FasL, was identified in culture medium from activated monocytes, but not in culture medium from control, unactivated monocytes. Immunohistochemical analysis of human atherosclerotic coronary lesions showed that FasL is expressed by macrophages, and microvessels in the adventitia as well as in the plaque. Finally, double-staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and FasL antibody showed that FasL enriched lesions always included a number of TUNEL-positive cells. These data suggest that Fas/FasL pathway can be employed by monocytes/macrophages to induce VSMC apoptosis in the atherosclerotic lesions.     

Catecholamines block the antimitogenic effect of estradiol on human coronary artery smooth muscle cells

Sequential conversion of estradiol to catecholestradiols and methoxyestradiols by cytochrome-P(450) (CYP450) and catechol-O-methyltransferase (COMT), respectively, contributes to the antimitogenic effects of estradiol on vascular smooth muscle cell (SMC) growth via estrogen receptor-independent mechanisms. Because catecholamines are also substrates for COMT, we hypothesize that catecholamines may abrogate the vasoprotective effects of estradiol by competing for COMT and inhibiting methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic/inhibitory effects of estradiol on human coronary artery SMC growth (cell number, DNA synthesis, collagen synthesis, and SMC migration) and ERK1/2 phosphorylation in the presence and absence of catecholamines. Norepinephrine, epinephrine, isoproterenol, and OR486 (COMT inhibitor) abrogated the inhibitory effects of estradiol on SMC growth and ERK1/2 phosphorylation. The interaction of catecholamines with estradiol was not affected by phentolamine or propanolol, alpha- and beta-adrenoceptor antagonists, respectively. The antimitogenic effects of 2-hydroxy-estradiol, but not 2-methoxyestradiol, were abrogated by epinephrine, isoproterenol, and OR486. Catecholamines inhibited the conversion of both estradiol and 2-hydroxy-estradiol to 2-methoxyestradiol, and SMCs expressed CYP1A1 and CYP1B1. Our findings suggest that catecholamines within the coronary arteries may abrogate the antivasoocclusive effects of estradiol by blocking the conversion of catecholestradiols to methoxyestradiols. The interaction between catecholamines and estradiol metabolism may importantly define the cardiovascular effects of estradiol therapy in postmenopausal women.     

Role of endothelial cell denudation and smooth muscle cell dedifferentiation in neointimal formation of human vein grafts after coronary artery bypass grafting: therapeutic implications

OBJECTIVE—To provide better insights into the genesis of neointimal thickening in human vein grafts early after surgery.
DESIGN—Retrospective study.
SETTING—Tertiary referral centre.
SUBJECTS—18 distal anastomotic sites of patent grafts, obtained at necropsy from eight patients who died over differing periods (ranging from two days to nine months) after the procedure.
MAIN OUTCOME MEASURES—Immunohistochemical evaluation of smooth muscle cell phenotype modulation in relation to proliferative activity.
RESULTS—The earliest changes are characterised by loss of surface lining endothelial cells and insudation of blood corpuscular elements admixed with fibrin-platelet thrombus. At sites of injury vimentin positive and actin negative spindle shaped cells appear in the intima, while the related pre-existent media shows focal absence of actin positive smooth muscle cells. Proliferative activity colocalises at these sites. With time distinct neointimal thickening occurs, associated with disappearance of proliferative activity and a phenotypic shift of the smooth muscle cells.
CONCLUSIONS—The observation that luminal endothelial cell denudation, with insudation of the intima with blood elements, occurs in the very early stages suggests that these phenomena are responsible for the observed dedifferentiation of pre-existent smooth muscle cells, known to be a prerequisite for cell proliferation and the evolution of intimal thickening. It is likely, therefore, that platelet released growth factors play a pivotal role, which thus may provide a target for preventive pharmacological intervention.


Keywords: smooth muscle cell proliferation; vein graft stenosis; platelet derived growth factor; platelet receptor inhibitors     

Thyrotropin regulation of cyclic adenosine monophosphate production in human coronary artery smooth muscle cells.

Thyroid disease has been associated with the occurrence of pathophysiologic changes in the vasculature that may result in part from altered serum thyroid hormone and serum lipid levels. Thyrotropin (TSH) levels are also altered in thyroid disease, but a direct effect of TSH on vascular smooth muscle has not previously been considered. In the present study, human coronary artery smooth muscle cells (CASMC) were induced into two morphologically distinct forms by culturing in either (1) growth factor supplemented, 0.5% serum medium (SmGM-3) or (2) basal medium (SmBM) plus 10% fetal bovine serum (FBS). Intracellular cyclic adenosine monophosphate (cAMP) accumulation was determined by radioimmunoassay after exposure to increasing doses of bovine TSH. Cells grown in SmBM/10% FBS for 3 days exhibited a dose-dependent increase in intracellular cAMP that reached a level 10 times higher than baseline at the highest dose examined (100 mIU/mL). In contrast, cells grown in SmGM-3 medium exhibited no change in intracellular cAMP on exposure to increasing TSII. Low serum (0.5% FBS) reduced the ability of TSH to stimulate cAMP above the control value in CASMC. Pretreatment of CASMC with either transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha) lowered basal levels of cAMP production, but did not inhibit the ability of TSH to stimulate cAMP production. Human, but not rat aortic smooth muscle cells in culture also responded to TSH with a significant increase in cAMP. The results of this study suggest that TSH may exert direct effects on vascular smooth muscle mediated by adenylate cyclase activation that could conceivably affect the progression of vascular disease associated with thyroid dysfunction.     

Effects of insulin-like growth factor-I on cultured human coronary artery smooth muscle cells.

The growth-promoting effects of insulin-like growth factor-I (IGF-I) appear to be different in vascular smooth muscle cells from various segments of the arterial tree. Little information exists on human coronary artery smooth muscle cells (CoSMC), the primary elements of coronary atherosclerosis and post-angioplasty restenosis. In this study we determined the effects of IGF-I on cultured human CoSMC. Type I IGF receptors (IGF-R) were present on CoSMC as assessed by affinity cross-linking of 125I-IGF-I to monolayer cultures. IGF-I was a weak mitogen, 1.5-fold increase in [3H]thymidine incorporation, for CoSMC. However, IGF-I had a potent motility effect on CoSMC with a 314+/-12% increase in cell migration (P<0.001), comparable to that of 5% FBS. IGF-I-stimulated motility was partially inhibited by alphaIR-3, a specific IGF-R inhibitor (P<0.05). Addition of kistrin, a disintegrin, or LM609, a specific alpha(V)beta(3) integrin neutralizing antibody, abolished IGF-I-stimulated migration (P<0.001). This study indicates that IGF-I is a potent motility agent for human CoSMC via the alpha(V)beta(3) integrin receptor, but exerts little mitogenic effect. Because CoSMC migration plays a crucial role in atherosclerosis and restenosis, IGF-I blockade has the potential to limit lumen reduction.