Effects of estrogen on nitric oxide synthase expression in rat aorta allograft and smooth muscle cells.

BACKGROUND: We find that chronic estradiol treatment inhibits the development of transplant arteriosclerosis (TA). The mechanism of this inhibition remains unclear. The objective of this study is to investigate in a non-cyclosporin-requiring TA model whether estradiol-17beta treatment modulates the expression of both endothelial nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) in the early phase following transplantation. METHODS: Orthotopic abdominal aorta allograft transplantation was performed in male rats using Brown-Norway rats as donors and Lewis rats as recipients. The recipients (n = 50) were treated with estradiol 20 microg/kg/day or placebo by osmotic minipump from 2 days prior to surgery until sacrifice on post-operative days 1, 3, 7, 14, and 21. The allografts were harvested and cross-sections of the vascular tissues were used for immunohistochemical staining of ecNOS and iNOS. The effects of estradiol on cytokine-induced (tumor necrosis factor-alpha and interleukin-1 beta iNOS protein and messenger RNA (mRNA) expression were also evaluated on rat aorta smooth muscle cells by Western blotting and RT-PCR in vitro, respectively. RESULTS: The expression of ecNOS and iNOS was graded semiquantitatively from 0 to +3. Estrogen elevates ecNOS expression in the intima in the early phase following transplantation, 0.85 +/- 0.14 (day 7) and 1.08 +/- 0.11 (day 14) vs 1.53 +/- 0.25 (day 7) and 1.60 +/- 0.17 (day 14) for placebo and estradiol treated groups respectively, p < 0.01. Estrogen suppresses iNOS expression in neointima (0.67 +/- 0.17 vs 0.24 +/- 0.04, p < 0.01, day 14), media (1.03 +/- 0.15 vs 0.4 +/- 0.09, p < 0.01, day 7), and adventitia (1.55 +/- 0.12 vs 1.02 +/- 0.10, p < 0.05, day 14) in the same phase. Estradiol treatment inhibits cytokine-induced iNOS mRNA expression in cultured smooth muscle cells. CONCLUSIONS: Chronic estrogen treatment modulates both ecNOS and iNOS expression in the early phase following transplantation. This is associated with the estrogen-protective effects on TA.

Growth and phenotypic characterization of porcine coronary artery smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation significantly contributes to atherosclerotic plaque formation and limits the success rate of percutaneous transluminal coronary angioplasty. We derived a population of porcine coronary artery SMCs to characterize VSMC proliferation and phenotype in preparation to study the molecular actions of VSMC mitogens and antiproliferative agents. Growth assays were designed to minimize the estrogen content in the culture medium, since this steroid hormone significantly influences VSMC growth and the expression of VSMC mitogens and their receptors. Culture conditions were identified such that this criterion was achieved while maintaining a significant VSMC growth rate. Cells cultured in serum-free medium, regardless of growth factor supplements, did not remain adherent to a plastic culture substrate, nor did they proliferate. Dextran-coated charcoal (DCC)-treated sera, including fetal bovine, calf, and porcine, supported VSMC adhesion, but not growth. Whole fetal bovine serum (FBS) produced the best proliferative response. A type-I collagen-coated culture surface significantly enhanced VSMC growth, but only in culture medium containing non-DCC-treated FBS. Flow cytometry analyses confirmed the mitogenic effects of this substrate. The VSMCs exhibited a morphological change on type-I collagen, but this was not accompanied by a change in VSMC phenotype. Our data indicate that culture of these porcine coronary artery SMCs in 2.5% FBS plus 10 ng platelet-derived growth factor-BB per ml in phenol red-free medium on type-I collagen may be the optimal conditions for studying the molecular aspects of VSMC mitogens and antiproliferative agents.

Inhibition of estrogen receptor function promotes porcine coronary artery smooth muscle cell proliferation.

Estradiol-17beta (E2) can inhibit vascular smooth muscle cell (VSMC) proliferation probably through its ability to activate its nuclear estrogen receptors (ER). Activation or inhibition of the ER by cognate permissive or non-permissive ligands, respectively, would indicate whether ER action is critical for this vascular protective effect. We investigated a previously characterized population of cultured porcine coronary artery SMCs for ER expression and for the response of these cells to estrogens and antiestrogens. Reverse transcription-polymerase chain reaction and Western blot analyses demonstrated ER mRNA and protein, respectively, in these cells. While the culture conditions required may have prevented the demonstration of physiological effects of E2, the antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, stimulated VSMC proliferation. The data suggest that, by interrupting ER function, antiestrogens significantly increased the VSMC mitotic rate. This model may be used to identify ER-regulated genes that function to control the growth of these coronary artery SMCs.

Oestrogen and transplant vascular disease.

1. The aetiology of chronic rejection is clearly multifactorial and relates to both immunological and non-immunological factors. 2. Our studies suggest that the insulin-like growth factor (IGF)-I ligand and receptor genes are rate limiting in smooth muscle proliferation in the development of transplant arteriosclerosis. 3. Suppressing growth factor ligand or receptor expression could be effective strategies for the prevention or treatment of transplant arteriosclerosis. 4. We consistently find chronic oestradiol treatment of transplant recipients inhibits arteriosclerosis by attenuating both IGF-I expression and the immune response, particularly major histocompatibility complex class II expression. 5. Thus, a cell- or tissue-specific oestrogen with minimal feminizing properties may be an ideal drug for prevention of one of the major causes of loss of transplant function.

Estradiol inhibits allograft-inducible major histocompatibility complex class II antigen expression and transplant arteriosclerosis in the absence of immunosuppression.

BACKGROUND: The etiology of transplant arteriosclerosis is unknown, but current data point to the alloimmune response. Previously, we found that estradiol-17beta (E2) with immunosuppressant cyclosporine abolishes major histocompatibility complex (MHC) class II expression in the allograft. This study determines the effect of E2 on MHC class II antigen expression in the allograft, in the absence of immunosuppression. METHODS: Lewis male rats received orthotopic abdominal aorta allografts from male Brown-Norway rats. The recipients were treated continuously subcutaneously with either 20 microg x kg(-1) x day1 of E2 (n=20) or placebo (n=20), from 2 days before transplantation until death on posttransplant days 1, 3, 7, and 14. The allografts were harvested and processed for morphometry and for immunohistochemical staining of MHC class II antigens, macrophages, CD4 and CD8 T lymphocytes, interferon-gamma (IFN-gamma), and IFN-gamma receptor. RESULTS: With E2 treatment, we observed that inducible MHC class II antigen expression is abolished in the media of the vascular allograft; the expression of IFN-gamma and IFN-gamma receptor is unaffected; and macrophage infiltration of the vascular allograft is inhibited significantly (P<0.01), whereas the CD4 and CD8 T lymphocytes are not significantly (P=0.07) suppressed. The myointimal hyperplasia in the allografts from E2-treated-recipients was 3-4-fold less than that from the placebo-treated recipients. CONCLUSIONS: Without immunosuppression, E2 inhibition of transplant arteriosclerosis is still associated with inhibition of inducible MHC class II antigen expression in the allografts. The estradiol-17beta abolition of inducible MHC class II antigen expression in the aorta allograft occurs in spite of up-regulation of IFN-gamma ligand and receptor protein.

Upregulation of estrogen receptor-alpha expression in rabbit cardiac allograft.

Estrogen receptor (ER) expression has been detected in different tissues, and estradiol-17beta treatment protects against experimental transplant arteriosclerosis. In this study, ER-alpha expression in the rabbit hearts and attached aortas before and after cardiac-aorta allograft transplantation was examined. Ten male New Zealand White rabbits were transplanted with cardiac-aorta allografts from male Dutch Belted rabbits. This transplant arteriosclerosis model uses a 0.5% cholesterol diet and immunosuppression with cyclosporin A (10 mg . kg-1 . d-1) until euthanatization 42 days later. The cardiac grafts with the attached aorta were harvested. Strong staining of ER-alpha protein was shown in the coronary arteries of the cardiac allografts by immunohistochemistry with the use of a mouse anti-human ER-alpha monoclonal antibody (ID5). In contrast, both the nongrafted hearts of the recipients and donor hearts expressed only weak staining. RNase protection assay with the use of a 32P-labeled ER-alpha antisense riboprobe (pOR 300) proved that the basal expression of ER-alpha mRNA is similar in the nongrafted aorta of both recipients and donors. A marked increase of ER-alpha mRNA was observed in the allograft aorta compared with the nongrafted aorta (289+/-69%, P<0. 02) by reverse transcription and polymerase chain reaction. The DNA sequence analysis confirmed that the polymerase chain reaction-amplified fragment corresponded to ER-alpha. This is the first observation of ER-alpha upregulation in the allograft vasculature and may relate to the allograft cardiovascular protective effects of estrogen.

Estradiol 17-beta represses insulin-like growth factor I receptor expression in smooth muscle cells from rabbit cardiac recipients.

BACKGROUND: A crucial step in cell cycle progression is the activation of the insulin-like growth factor I (IGF-I) receptor (IGF-IR) by its ligand. Earlier, we found estradiol 17-beta treatment of cardiac allograft recipients attenuates transplant arteriosclerosis; this was associated with inhibition of vascular cell proliferation induced by IGF-I. The current study demonstrates regulation of IGF-IR by estradiol 17-beta in vivo and in vitro in recipient native and allograft aorta and in aorta smooth muscle cells (SMCs). METHODS: Twenty cardiac transplant recipient rabbits were treated with estradiol 17-beta (100 microg/kg/day) or placebo for 6 weeks. IGF-IR expression in the coronary arteries of rabbits was demonstrated by immunohistochemistry. Reverse transcription-polymerase chain reaction and RNase protection assay were used to detect IGF-IR mRNA in rabbit aortas and cultured aortic SMCs in the presence or absence of estradiol 17-beta in vitro. IGF-I-induced cell proliferation was performed with the aorta explants and aorta SMCs from estradiol- or placebo-treated rabbits. RESULTS: Estradiol 17-beta treatment of rabbits significantly inhibited IGF-IR expression in the allograft coronary arteries and abrogated cell proliferation induced by IGF-I in the allograft aorta compared with placebo-treated recipients (65.4+/-5% vs. 500+/-139%, P<0.002). Expression of IGF-IR mRNA in the allograft aorta of placebo-treated recipients was significant higher than that of the native aorta (286+/-56%, P<0.02). Estradiol treatment significantly inhibited IGF-IR mRNA expression in the aorta versus that of the placebo-treated recipients (65+/-8.5% vs. 140+/-23%, P<0.02). Repression of IGF-IR mRNA expression in aortic SMCs by estradiol in vitro was in a concentration-dependent manner (P<0.02). CONCLUSION: Repression of IGF-IR protein and mRNA by estradiol 17-beta in vivo and in vitro suggest that one of the mechanisms of estradiol inhibition of SMC proliferation and transplant arteriosclerosis is down-regulation of IGF-IR.

17-Beta estradiol regulation of myocardial glutathione and its role in protection against myocardial stunning in dogs.

We studied the effect of 2-week treatment with estradiol 17beta on myocardial glutathione concentration in dogs and isolated perfused rat heart subjected to brief coronary ischemia and reperfusion. Estradiol protected against ischemia/reperfusion-induced myocardial systolic shortening and malonylaldehyde production and increased myocardial glutathione concentration and glucose-6-phosphate dehydrogenase enzyme activity. Reduction of myocardial glutathione with buthionine sulfoximine to levels seen in the absence of estrogen reversed the protective effect of estradiol against myocardial dysfunction and lipid peroxidation associated with ischemia/reperfusion. These results suggest that the antioxidant effect of estradiol in ischemia/reperfusion may be mediated by regulation of myocardial glutathione metabolism.

Exposure of vascular allografts to insulin-like growth factor-I (IGF-I) increases vascular expression of IGF-I ligand and receptor protein and accelerates arteriosclerosis in rats.

BACKGROUND: Accelerated arteriosclerosis limits the survival of transplanted hearts. We hypothesized that insulin-like growth factor-I (IGF-I) is crucial in accelerating transplant arteriosclerosis. Recently, we reported that exposure to IGF-I prior to transplantation accelerates transplant arteriosclerosis in the rat aorta allograft model. Here, we studied the mechanism whereby IGF-I exposure accelerates transplant arteriosclerosis. METHODS: The abdominal aorta was harvested from male Brown Norway rats and exposed to 0, 200, or 500 ng/ml of IGF-I at 37 degrees C for 30 min prior to transplantation to the abdominal position of male Lewis rats. The allografts were harvested 14 days later and processed for immunohistochemical staining for alpha-actin, growth factors (IGF-I, IGF-I receptor, platelet-derived growth factor-BB, and basic fibroblast growth factor), and immunological markers (major histocompatibility complex class II antigen, macrophage, and CD4- and CD8-positive T cells). RESULTS: By 14 days, the ex vivo IGF-I donor aorta treatment with IGF-I increased in a concentration-dependent manner the expression of IGF-I and IGF-I receptor in both the intima and the adventitia. In contrast, the expression of platelet-derived growth factor-BB was decreased in a concentration-dependent manner in the intima while basic fibroblast growth factor remained unchanged. The cell-mediated immune response was not affected by IGF-I at 14 days after transplantation, which suggests that the immune events associated with acceleration of transplant arteriosclerosis may occur at an earlier time. CONCLUSION: Acceleration of transplant arteriosclerosis by exposure to IGF-I is associated with increased IGF-I ligand and receptor expression in the allograft vascular wall. These data further suggest that IGF-I may be a major factor in mediating graft arteriosclerosis.

Stereoisomer-specific inhibition of superoxide anion-induced rat aortic smooth-muscle cell proliferation by 17beta-estradiol is estrogen receptor dependent.

An in vitro xanthine/xanthine oxidase reaction system was used to generate superoxide anions that significantly stimulated tritiated [3H]thymidine incorporation into endothelium-removed (denuded) male rat aortic explants. Tritiated thymidine uptake was used as an index of vascular smooth-muscle cell (VSMC) proliferation. Superoxide dismutase (SOD) significantly attenuated the oxygen free radical-induced proliferative response of these cells. 17Beta-estradiol (17beta-E) significantly inhibited superoxide anion-induced VSMC proliferation. In contrast, the growth-modifying effects of 17beta-E were not mimicked by 17alpha-estradiol (17alpha-E), progesterone, or testosterone. The pure estrogen receptor (ER) antagonist, ICI 164,384, reversed the growth-inhibitory effect of 17beta-E. 17Beta-estradiol failed directly to reduce in vitro superoxide anion production or to modify xanthine oxidase activity. Therefore, these data indicate that 17beta-E, through an ER-dependent mechanism, specifically and significantly inhibited superoxide anion-mediated SMC proliferation in denuded rat aortic explants.

Cardiovascular effects of estrogen: implications of the discovery of the estrogen receptor subtype beta.

In addition to the traditional estrogen receptor-alpha, a second estrogen receptor was recently discovered, namely estrogen receptor-beta. This review focuses on the cardiovascular significance of the differential ligand activation by these nuclear transcription factors and emphasizes the need for re-evaluation of all previously described estrogen receptor mediated estrogen and antiestrogen effects.

Specific effects of estrogen on growth factor and major histocompatibility complex class II antigen expression in rat aortic allograft.

OBJECTIVE: Transplant arteriosclerosis is the major determinant for long-term survival of cardiac transplants. Estradiol treatment inhibits transplant arteriosclerosis. The objective of this study is to determine, in the absence of immunosuppression, the temporal effect of estradiol treatment on the expression of insulin-like growth factor, platelet-derived growth factor, basic fibroblast growth factor, and major histocompatibility complex class II antigen in rat aortic allografts. METHODS: Orthotopic abdominal aortic allograft transplantation was performed in male rats with Brown-Norway rats used as donors and Lewis rats as recipients. The recipients (n = 50) were treated with estradiol 20 micrograms/kg per day or placebo by osmotic minipump for 2 days before the operation and until they were put to death on postoperative days 1, 3, 7, 14, or 21. The allografts were harvested and insulin-like growth factor, platelet-derived growth factor, basic fibroblast growth factor, and major histocompatibility complex class II antigen expression were determined by immunohistochemical staining. Myointimal thickening was measured by morphometric analysis. RESULTS: In the placebo-treated group, insulin-like growth factor protein progressively increased in all three layers of the allograft, whereas platelet-derived growth factor protein peaked at day 3 and basic fibroblast growth factor protein increased only moderately. Estradiol treatment inhibited the continuous increase in insulin-like growth factor expression, the peak in platelet-derived growth factor expression at day 3, the moderate-basic fibroblast growth factor increase at day 21, and major histocompatibility complex class II antigen expression in all three layers of the allograft at day 21. Intimal thickening of allografts from estradiol-treated recipients was twofold to threefold less than that of the placebo-treated recipients at day 21. CONCLUSION: The development of transplant arteriosclerosis is associated with an early alloimmune response involving sustained increase in insulin-like growth factor expression. Estradiol treatment of the recipient inhibits transplant arteriosclerosis and suppresses insulin-like growth factor and major histocompatibility complex class II antigen expression but not platelet-derived growth factor or basic fibroblast growth factor in all three layers of the allograft during the early posttransplantation alloimmune rejection phase.

Estrogen effects on insulin-like growth factor-I (IGF-I)-induced cell proliferation and IGF-I expression in native and allograft vessels.

BACKGROUND: Estrogen protects against cardiovascular disease in both patients and animal models and regulates insulin-like growth factor-I (IGF-I), an important cell-cycle progression factor. METHODS AND RESULTS: Smooth muscle cells and tissues were harvested from male recipient rabbits that 6 weeks earlier had received a cardiac allograft transplant consisting of a donor heart and ascending aorta. Segments of the ascending aorta from the native and allograft hearts from 9 placebo-treated and 8 estradiol-treated recipients were compared by using IGF-I-stimulated [3H]thymidine incorporation. The responses of the native vessel segments were similar (175.3+/-32% and 166.9+/-41%, respectively; P>.05) whether or not the recipients had been treated for 6 weeks with estradiol. In the grafts, however, estradiol markedly inhibited vascular cell thymidine incorporation (328.04+/-56% compared with 67.3+/-11%; P<.02). Smooth muscle cells were derived from the native aorta of the placebo-treated rabbits to study the effect of estradiol in vitro. IGF-I increased cell counts in a concentration-dependent manner. In serum-starved cells estradiol further decreased cell proliferation; this effect was blocked by the specific estrogen receptor antagonist ZK-119.010. Immunohistochemistry staining for IGF-I protein in the coronary arteries and ascending aorta of the cardiac allograft from the placebo-treated recipients revealed extensive IGF-I expression in the myointima. In contrast, IGF-I protein was not expressed in the coronary arteries and ascending aorta of the cardiac allograft from the estradiol-treated recipients. The IGF-I protein was extensively expressed only in the placebo-treated graft vessels. Myointimal thickening of the coronary arteries was significantly reduced by estradiol treatment (17.9+/-1.5% versus 44.3+/-3.7%; P<.02). CONCLUSIONS: In vivo estradiol treatment abolishes both IGF-I mitogenic effects and IGF-I protein expression in the vascular wall, which may be causally related to the inhibitory effect of estradiol on transplant arteriosclerosis.

Angiopeptin inhibits thymidine incorporation by explants of porcine coronary arteries.

Angiopeptin, a stable octapeptide analog of somatostatin, inhibits proliferation in a variety of cancer cell lines. We studied the effect of angiopeptin on 3H-thymidine uptake into ring segments from the porcine coronary tree. The incorporation of 3H-thymidine into segments of porcine left anterior descending (LAD) coronary artery was time dependent and reached a plateau after 48 h. The addition of angiopeptin (48.1 and 96.2 nM) to the culture medium significantly inhibited 3H-thymidine incorporation into the segments by 36.7 +/- 10.1% and 48.3 +/- 2.3% of the control, respectively. Forskolin (100 microM), inhibited 3H-thymidine incorporation (52.7 +/- 10.1%) to the same degree as did angiopeptin (96.2 nM). Incubation of the segments with 125I-labeled angiopeptin, for 2 h at 37 degrees C, showed angiopeptin uptake to be time dependent and exhibited a first-order kinetics, reaching equilibrium after 30 min. Autoradiographic studies showed a uniform distribution of angiopeptin within the endothelium, media, and adventitia. Most of the labeling was associated with the nuclei of the cells. Angiopeptin, after 30-min incubation, did not significantly modify the basal levels of cyclic adenosine monophosphate (cAMP). In contrast, forskolin (100 microM) elicited a 50-fold increase of the basal levels of cAMP. These results indicate that in addition to its endocrine effects, angiopeptin reduces the rate of proliferation by acting directly on the vessel wall.

L-arginine reverses the adverse pregnancy changes induced by nitric oxide synthase inhibition in the rat.

OBJECTIVE: Inhibition of nitric oxide synthase with N omega-nitro-L-arginine methyl ester (L-NAME) induces a preeclampsia-like syndrome of hypertension, proteinuria, intrauterine growth restriction, and renal glomerular capillary endothelial lesions in pregnant rats. We attempted to reverse these changes with late-pregnancy administration of L-arginine. STUDY DESIGN: Sprague Dawley rats with timed pregnancies received infusions of either saline solution (n = 12) (group SC) or L-NAME (n = 12) (group LC) (160 mg/kg per day) on gestational day 10 through term. On gestational day 16 half of the saline solution group (group SA) and half of the L-NAME group (group LA) received L-arginine (21 mg/kg per day) through delivery. Systolic blood pressures were determined via tail cuff on days 10, 16, and 21. Pup weights were assessed at delivery, serum and urine were collected and analyzed for nitrites and nitrates, and renal tissue was processed for histologic examination. Data were analyzed with the one-way analysis of variance and the Newman-Keuls test for multiple comparisons. RESULTS: In the L-NAME-treated animals L-arginine significantly lowered systolic blood pressure at late pregnancy (125 +/- 2.42 vs 153 +/- 3.0 mm Hg) (p < 0.01), increased mean pup weight (5.6 +/- 0.11 gm in group LA vs. 5.0 +/- 0.02 gm in group LC) (p < 0.001), decreased the degree of proteinuria (2+ vs trace), and decreased the proportion of injured glomeruli (7% vs 64%) (p < 0.001). CONCLUSIONS: Lesions induced by chronic inhibition of endothelium-derived nitric oxide synthesis (hypertension, intrauterine growth restriction, proteinuria, renal glomerulus injury) are reversed by treatment with L-arginine. These findings lend support to the potential for use of nitric oxide donors in the treatment and prevention of preeclampsia.

The vascular protective effects of estrogen.

There is now strong epidemiological evidence that estrogen replacement therapy has a protective effect in postmenopausal women. The cardiovascular protective action of estrogen is reported to be mediated indirectly by an effect on lipoprotein metabolism and by a direct effect on the vessel wall itself. Estrogen is active both in vascular smooth muscle and endothelium. Functionally competent estrogen receptors have been identified in vascular smooth muscle cells, and specific binding sites have been demonstrated in endothelium. Estrogen administration promotes vasodilation both in human and experimental animals, in part by stimulating] prostacyclin and nitric oxide synthesis. Both the prostaglandin synthase and the constitutive nitric oxide synthase were recently reported to be induced by estrogen treatment. In vitro, estrogen exerts a direct inhibitory effect on the smooth muscle by inhibiting calcium influx. In addition, estrogen inhibits vascular smooth muscle cell proliferation. In vivo, estradiol-17 beta prevents neointimal thickening after balloon injury and in rabbit cardiac transplant allografts. These data are consistent with in vitro studies wherein estrogen inhibits [3H]thymidine uptake by arterial segments from porcine coronary artery as well as proliferation of rabbit aortic vascular smooth muscle cells induced by hyperlipedemic serum. Recent studies have also reported an effect of estrogen on directed vascular smooth muscle cell migration. Furthermore, like other steroids, the effect of estrogen on the vessel wall has a rapid nongenomic component involving membrane phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, as well as the classical genomic effect involving estrogen receptor activation and gene expression. The nature of these estrogen response genes in the vessel wall and their relation to vasodilation and antiproliferation remain to be determined.