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.

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.

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.

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.

Acceleration of arteriosclerosis of the rat aorta allograft by insulin growth factor-I.

We demonstrate here, for the first time, the mitogenic effect of insulin-like growth factor-I (IGF-I) on the development of transplant arteriosclerosis in a rat orthotopic aorta allotransplantation model (Brown Norway to Lewis). 125I-IGF-I uptake by the abdominal aorta of male Brown Norway rats occurred within 30 min. Consequently, we exposed the donor abdominal aorta to 0, 200, or 500 ng/ml IGF-I at 37 degrees C for 30 min ex vivo (n=7 per group), before transplantation. Fourteen days after transplantation, intimal thickening of the allografts in each of the three groups was 0.18+/-0.02 (IGF-I at 0 ng/ml), 0.23+/-0.03 (IGF-I at 200 ng/ml), and 0.30+/-0.03 (IGF-I at 500 ng/ml), respectively (mean+/-SEM, P<0.005 for 500 ng/ml vs. 0 ng/ml). [3H]thymidine incorporation (cpm/microg protein) in the transplanted grafts at 7 days after transplantation (n=4 per group) was 40.6+/-7.6, 78.5+/-12.3, and 66.9+/-10.1, respectively (P<0.01 for 200 ng/ml vs. 0 ng/ml). [3H]thymidine incorporation in the native thoracic aorta of the recipient was 23.4+/-4.4. We conclude that acceleration of allograft myointimal proliferation and intimal thickening was induced directly by IGF-I.

HMG-CoA reductase inhibitors in organ transplantation.

Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors reduces serum cholesterol in patients with high cholesterol blood levels including organ transplant recipients. HMG-CoA reductase inhibitor also inhibits a series of immune responses and thus have the potential of exerting immunosuppressive effect in patients with organ allografts. Experimentally, HMG-CoA reductase inhibitors reduces transplant arteriosclerosis. Whether this is linked to an immunosuppressive effect or not is unknown. There is little evidence that post-transplant hyperlipidemia directly increases the risk of cardiovascular diseases. Lipid lowering with HMG-CoA reductase inhibitor is not indicated for all allograft recipients but should be used if other cardiovascular risk factors are present.

Angiopeptin, a somatostatin analogue, inhibits rat coronary artery and aorta smooth muscle cell proliferation induced by the thromboxane A2 mimetic U46619.

Thromboxane A2 (TXA2) is a potent mitogenic agent. Its synthesis is increased in transplant patients during rejection episodes, which is the suspected etiology of accelerated transplant arteriosclerosis. Angiopeptin, a stable analogue of somatostatin, inhibits arterial myointimal thickening in a number of vascular balloon injury models of angioplasty and in vivo models of transplant arteriosclerosis. In this study, we investigated whether TXA2-induced smooth muscle cell proliferation is inhibited by Angiopeptin in vitro. Primary rat coronary and aorta smooth muscle cells were cultured in the presence of U46619, a TXA2 mimetic. Proliferation induced by U46619, as determined by 3H-thymidine incorporation, was abrogated by two specific thromboxane receptor antagonists, SQ 30741 and SQ 29548, indicating that the effect of U46619 on smooth muscle cells is a specific receptor-mediated response. We found Angiopeptin to inhibit proliferation following exposure of both coronary and aorta smooth muscle cells to varying concentrations of U46619 for 3 and 6 days. This study demonstrates that U46619 exerts a specific receptor-mediated response stimulating the rat coronary and aorta smooth muscle cell proliferation. This mitogenic effect is obtained by increasing the G1 to S transition rate. Angiopeptin inhibits thromboxane-induced cell proliferation to the same extent as a thromboxane antagonist. This inhibition is obtained by maintaining the noncycling fraction in that Angiopeptin prevents a progression from G0-G1 to S phase.

Urinary thromboxane B2 in cardiac transplant patients as a screening method of rejection.

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A2 (TXA2) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA2 metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B2 (i-TXB2) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB2 was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 +/- 372 vs. 526 +/- 57 pg/mL). The urine i-TXB2 (704 +/- 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB2 were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 +/- 11 vs. 39 +/- 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB2 is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB2 may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.