Modulation Of Long-term Estrogen Replacement Treatment On Circulating And Cardiac Renin-Angiotensin System And Effect On Structure Of Aorta And Myocardium

Cardiovascular diseases are the leading cause of death in postmenopausal women. Heart disease develops in women on average 10 years later in life compared with men. The incidence of cardiovascular diseases is 4-fold higher in postmenopausal women than in women of the same age who are premenopausal. The decrease of the estrogen level is the main physiological change of the postmenopausal women. Much research indicates that estrogen plays an important role in preventing the cardiovascular diseases. Unfortunately, the WHI studies showed that HRT caused a higher incidence of myocardial infarction, ischemic stroke and pulmonary emboli than placebo in 2002. The "estrogen only" arm of WHI was also recently stopped because of the slightly high storke risk and the lack of any beneficial CVD effects. The study report of WHI causes a great panic of the upholders and receivers of hormone replacement therapy. Many researchers believe that although the test of WHI is scientific and rigorous, there is some defect in it. For example, random and 'double blind' are not guaranteed in this design of the experiment, and the patients are prone to be older. In 2005, Mendelsohn et al suggested that resolving this controversy would require a more complete understanding of the cardiovascular biololgy that exist between perimenopausal and older women. It is necessary to explore the interactions between estrogen and cardiovascular disease by continuous basic study and new clinical experiment.The renin-angiotensin system (RAS) is a critical regulator of blood pressure and fluid homeostasis. Studies suggest that: (1) The gene promoter of angiotensinogen contains an estrogen response element; (2) Plasma renin activity is higher in men than in women; (3) The majority of human studies document a mild to moderate suppression of angiotensin-converting enzyme (ACE) activity with estrogen replacement therapy. These studies suggest that estrogen maybe influence blood pressure and cardiovascular systems through affecting renin-angiotensin system. It has been described that all of the components of the renin-angiotensin system are present in cardiac tissue and that angiotensinⅡ(AngⅡ) is produced locally which regulated independent of the circulating RAS. Activation of cardiac RAS is related to the development of heart diseases such as hypertensively myocardial hypertrophy, myocardial infarction, arrhythmia, ischemia-reperfusion injury. But there are few studies on the effect of estrogen on cardiac renin-angiotensin system.There is a close relationship between the function and structure of blood vessel and heart. Estrogen may decrease lysosomal enzyme activities and increase mitochondrial FOF1-ATPase activity. These results suggest that estrogen may play an important role in maintaining the structural integrity of cells. But these experiments have not explained the influence of estrogen on endothelium and myocardium morphologically.The paper studies the effect of long-time estrogen replacement on circulating and cardiac RAS system and on the structure of myocardium and aorta, by establishing sham, OVX and OVX＋E2 three SD female rat models. The study will offer theoretical evidence for understanding the high risk of cardiovascular diseases of postmenopausal women and the benefits and risks of estrogen replacement therapy.Chapter One: Influence of long-term estrogen treatment on circulating renin-angiotensin system and endothelial ultrastructureObjective: To establish three SD rat models of sham, ovariectomy and estrogen replacement treatment; To study the influence of ovariectomy and long-term 17β-estradiol treatment on circulating renin-angiotensin system (RAS), nitrite oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), the ultrastructural characteristics and endothelium-dependent relaxation of thoracic aorta.Methods: Rats were ovariectomized (OVX, n=17), not ovariectomized (sham, n=16) or ovariectomized and treated with subcutaneous 17β-estradiol (15μg/kg/day, OVX＋E2, n=17). Within 13 weeks after operation, the blood pressure, the serum estrogen, NO, plasma angiotensinⅡ(AngⅡ), ANP and renin activity were monitored. At the 13th week after operation, vasodilator responses to acetylcholine in the aortic rings and ultrastructural characteristics of thoracic aorta were determined.Results: By 3 weeks and, until the end of the study, OVX had significantly higher weights than other two groups (p＜0.05). E2-treatment maintained uterus weights that were significantly higher than OVX (p＜0.05) and similar with sham rats (p＞0.05). Plasma estradiol levels were significantly lower in the OVX compared with sham (P＜0.05) and OVX＋E2 rats (p＜0.05). At the 9th and 13th week, OVX had significantly higher blood pressure than the other two groups (p＜0.05). Overiectomy led to a significant decrease in plasma AngⅡconcentration and a significant increase in renin activity of OVX rats compared with sham, which could be restored by estrogen treatment (p＜0.05). At the 5th, 9th and 13th week, serum NO of OVX was significantly lower compared with sham, which could be restored by estrogen treatment (p＜0.05). Plasma ANP at the 9th and 13th week was significantly lower in OVX (p＜0.05). Ovariectomy markedly reduced endothelium-dependent relaxation to acetylcholine in the isolated rat thoracic aortic rings (p＜0.05) and chronic estrogen treatment significantly restored the vasodilator responses to acetylcholine (p＜0.05). It was found under electron microscopy that the endothelial cells of OVX rats were swollen, even necrosed, estrogen treatment can inhibit the changes.Conclusion: These results suggest that estradiol protects from the development of hypertension and posses a protective effect on the endothelium through increasing NO and ANP concentration, and decreasing renin activity. However, there existed the discordance between the effects of estradiol on angiotensinⅡ, and on blood pressure, which may be a negative feedback that ultimately results in overall suppression of the RAS.Chapter Two: Influence of ovariectomy and long-term estrogen treatment on cardiac renin-angiotensin systemObjective: To study the effect of ovariectomy and long-term estrogen treatment on the expressions of AT1, AT2 and angiotensinⅡin heart; To test whether the expressions of AT1 and AT2 modulated by estrogen-NO pathway. Methods: 13 weeks after operation, radioimmunoassay was used to analyse AngⅡconcentrations of left ventricles in three group SD rats. RT-PCR and Westernblot were used to analyse the the expressions of AT1 and AT2 in myocardium of the three group SD rats at mRNA and protein levels. To test whether the expressions of AT1 and AT2 modulated by estrogen-NO pathway, H9c2 cells were exposed to 17β-estradiol (100nM) plus L-NAME (10μM or 100μM) and SNAP(10μM or 100μM) respectively for 48 h, then the expressions of AT1 and AT2 was analysed by RT-PCR and Westernblot.Results: AngⅡconcentration of left ventricles in OVX rats was higher than sham (p＜0.05) and OVX＋E2 rats (p＜0.05). The expression of AT1 was upregulated in the OVX rats at both mRNA and protein levels (p＜0.05), which was prevented by estrogen treatment (p＜0.05). The expression of AT2 of OVX rats was lower than sham (p＜0.05) and OVX＋E2 rats (p＜0.05) at both mRNA and protein levels. AT1 expressions of H9c2 cells cultured with 1 nM or 100 nM E2 for 48h were significantly lower than 0 nM (p＜0.05) or 0.01 nM E2 group (p＜0.05). AT2 expressions of H9c2 cells cultured with 1 nM or 100 nM E2 for 48h were significantly higher than 0 nM (p＜0.05) or 0.01 nM E2 group (p＜0.05). The expressions of AT1 in H9c2 cells pretreated with 100 nM E2 plus L-NAME (10μM or 100μM) for 48hr was significantly higher than 100 nM E2 group (p＜0.05), and significantly lower than the cells pretreated with only 100μM L-NAME at mRNA and protein levels (p＜0.05). The expressions of AT1 in H9c2 cells pretreated with 100 nM E2 plus 100μM SNAP for 48hr was significantly lower than 100 nM E2 group (p＜0.05), and significantly lower than the cells pretreated with 100 nM E2 plus 10μM SNAP at mRNA and protein levels (p＜0.05). The expression of AT2 in H9c2 cells pretreated with 100 nM E2 plus L-NAME (10μM or 100μM)) or SNAP (10μM or 100μM)) had no significant difference compared with 100 nM E2 group (p＞0.05), but higher 0 nM E2 group (p＜0.05).Conclusion: Long-term estrogen replacement could protect the cardiac function by modulating the cardiac RAS system to adapt to ischemia et al badly stimuli. NOS/NO pathway involved in the decrease of AT1 expression modulated by estrogen. The study offered a new evidence for understanding the high risk of cardiovascular diseases of postmenopausal women.Chapter Three: Effect of ovariectomy and long-term estrogen treatment on uitrastructure of myocardiumObjective: To study the effect of ovariectomy and long-term estrogen treatment on the on ultrastructure of myocardium in SD female rats.Methods: At the 13th week, the rats were discected, then left ventricles were removed rapidly and fixed in 4% paraformaldehyde or 2.5% glutaraldehyde respectively. Paraffin block sections (4μm thick) or ultrasections (about 500(?)) were cut, and examined using light microscope and transmission electron microscope. Thiobarbituric acid method was used to analyse MDA concentrations of left ventricles in three group SD rats.Results: MDA concentration of left ventricles in OVX rats was higher than sham (p＜0.05) and OVX＋E2 rats (p＜0.05). By the electron microscopy examination of the left ventricles, the sham rats did not reveal abnormal findings of the cardiac myocytes, but in rats of OVX group, there was disarranged and lysis of some myofibrils. Most of mitochondria were markedly abnormal in shape and were swollen. In OVX＋E2 rats, myofibdls arranged regular. Light band and dark band of sarcomere were clearer than OVX rats. Except some mitochondrias were swollen, most were normal.Conclusion: Ovariectomy may lead to ultralstructural disorder of myocardium, long-term estrogen replacement can protect against the ultralstructural injury. Estrogen plays an important role in maintaining the normal ultralstructure maybe through modulating the cardiac RAS system.