Estrogen and SERMs

Cardiovascular diseases (CD) are among the major causes of mortality and morbidity in women after menopause (Cetinkaya Demir, Uyar et al. 2013). A woman over 50 years of age faces a 46 percent lifetime risk of having a cardiovascular disease and a 31 percent risk of dying from a cardiovascular event (Cetinkaya Demir, Uyar et al. 2013). Thus, menopause is a significant risk factor for cardiovascular diseases (Cetinkaya Demir, Uyar et al. 2013). Loss of endogenous estrogen production after menopause contributes not only to impaired reproductive functions, but also increases the risk of metabolic syndrome and type 2 diabetes (Mauvais-Jarvis 2011). Estrogen protects women against cardiovascular diseases and seems to play amajor role in sex-related differences in hypertension in experimental models (Posa, Kupai et al. 2013). The benefits of estrogen in cardiovascular system include a reduction in low-density lipoprotein cholesterol (LDL-C), decrease in LDL oxidation, and enhancement of vascular function (Mendelsohn and Karas 1999). Ovariectomy (OVX) in rodents is one approach for modeling human menopause, and studying the metabolic consequences of losing ovarian function. Previous studies in rodents have consistently demonstrated that OVX promotes obesity and its metabolic complications, in particular insulin resistance (Liu, Xu et al. 2004, Riant, Waget et al. 2009, Saengsirisuwan, Pongseeda et al. 2009)  Ovariectomy  in rats led to increases in energy intake, body weight and fat mass, whereas estrogen substitution antagonized these effects in a positive way (Richard, Rochon et al. 1987, Simpson, Jones et al. 2005, Hertrampf, Gruca et al. 2007). Some studies have also observed an overall insulin resistance in animals after ovariectomy,and these effects could be prevented byhormonereplacement therapy (HRT) too (Bailey and Ahmed-Sorour 1980). Estrogen replacement therapy, however, has been linked to an increased risk of tissue-specific side effects including breast cancer and uterine cancer (Cauley, Lucas et al. 1996). Some alterations in ET have also been tried, such as lowering the dose and shortening the duration of ET, combining estrogen and progesterone, or using the transdermal route instead of the oral route (Ettinger, Wang et al. 2012, Steinkellner, Denison et al. 2012). To avoid an abnormal proliferative response in the breast or uterus tissue, the combination of progestin or selective estrogen receptor modulators (SERMs) with estrogens has been strongly suggested (Weiderpass, Adami et al. 1999, Bolognese 2010). Despite these measures, reports have shown a decline in ET recommendations in the last decade and use of ET only in cases that are untreatable with other options (Beral, Banks et al. 2002).  Therapies that involve selective estrogen receptor modulators (SERMs) have shown protective effects in the treatment of menopausal symptoms in clinical situations(Wong, Yung et al. 2008, Wang, Gou et al. 2014). SERMs are a class of synthetic compounds that act as estrogen receptor antagonists or agonists, depending on the target tissues (Sato, Rippy et al. 1996, Osborne 1998). The development of SERMs allows the clinical use of estrogen substitutes that are devoid of side effects of hormone replacement therapy. Due to their tissue-specific and mixed estrogen-agonist/antagonist properties, SERMs such as raloxifene exhibit estrogenic activities in bone, cardiovascular and central nervous systems, while avoiding harmful effects in breast and uterus. Raloxifene exerts estrogen-like cardioprotective actions and improves coronary blood flow partly by releasing endothelial nitric oxide (NO) in animal studies (Zoma, Baker et al. 2000, Ogita, Node et al. 2002). Chronic treatment with raloxifene may reduce cardiovascular risks in women (Barrett-Connor, Grady et al. 2002). and prevents endothelial dysfunction in young band aging Ovx rats by increasing NO bioavailability (Rahimian, Dube et al. 2002, Wong, Yao et al. 2006). Ovariectomized (Ovx) sheep taking raloxifene have greater diameters in their coronary arteries compared with those receiving estrogen or no treatment (Gaynor, Monnet et al. 2000). Raloxifene hydrochloride (RLX), a benzothiophene SERM, confers estrogen-like effects on lipids but anti-estrogenic effects on breast tissue and uterine endometrium (Zeng, Yang et al. 2014). Metabolic syndrome is characterized by a constellation of obesity, insulin resistance, inflammation, dyslipidemia, and hypertension. In particular, obesity and insulin resistance are more prevalent in postmenopausal women than in premenopausal women, and increase the risk of cardiovascular disease and type 2 diabetes (Dandona, Aljada et al. 2005, Bonora 2006, Innes, Selfe et al. 2008, Lobo 2008). We and others have shown previously that administration of both oestradiol and raloxifene, a selective oestrogen receptor modulator (SERM) approved for the treatment of postmenopausal osteoporosis, can ameliorate collagen-induced arthritis (CIA), a murine model of human RA (Jochems, Islander et al. 2007, Jochems, Lagerquist et al. 2008). Raloxifene and tamoxifen provide protective effects on cardiovascular function by increasing the synthesis and bioactivity of endothelial factors (Colacurci, Manzella et al. 2003, Takahashi, Mori-Abe et al. 2007, Borgo, Lopes et al. 2011), thereby reducing oxidative stress or inflammatory markers (Christodoulakos, Lambrinoudaki et al. 2006, Pinna, Bolego et al. 2006, Leung, Tsang et al. 2007, Cummings, Ettinger et al. 2008).

Estrogen exerts genomic and nongenomic effects via estrogen receptor-dependent and independent mechanisms, to confer protective effects on the cardiovascular system. Alterations in plasma concentrations of lipoproteins levels, hemostatic[DVC1]  factors, glucose, insulin, and endothelium-derived factors (decreases in endothelin, increases in NO and prostaglandins) and the inhibition of smooth muscle cell migration and proliferation induced by various mitogens are thought to contribute to the vasoprotective effects of estrogen (Dubey and Jackson 2001, Grover-Paez, Zavalza-Gomez et al. 2013, Xu, Niu et al. 2013). Of these biological effects, the antioxidant effects of estrogen may play a critical role in eliciting vasoprotective effects. Estrogen inhibits the proliferation of intimal and medial vascular smooth muscle, suggesting a direct protective effect of estrogen on endothelium and vascular smooth muscle cells(Sullivan, Karas et al. 1995). The cardioprotective role of estrogen seems to be partially mediated by the induction of PGI2 biosynthesis. The COX-2-dependent prostaglandin I₂ activates its plasma membrane receptor IP which causes the induction of the antioxidant HO-1 in the vasculature (Egan, Lawson et al. 2004). Furthermore, both estradiol and raloxifene increased COX-2 levels in ovariectomized mice (Abu-Fanne, Brzezinski et al. 2008) Our results strengthen the likelihood of the theory that cardioprotective effects of estrogen and raloxifene could be mediated by HO pathway.

A few investigators have reported that systolic blood pressure is changed by HO substrates or HO inhibitors[DVC2] , but they did not investigate the role of estrogen and raloxifene replacement on the HO enzyme regulation. There are compelling evidences to support the emerging paradigm that CO, similar to NO, elicits vasodilation. Chromium mesoporphyrin (CrMP), a selective HO inhibitor[DVC3] , has been shown to increase the myogenic tone of the small muscular branch of rat femoral arteries, but not of large arterial vessels such as the aorta or the femoral arokstery[DVC4] , in organ bath experiments (Kozma, Johnson et al. 1999). Estrogens have been shown to be protective in shocklike states(Mizushima, Wang et al. 2000), they can modulate HO-1[DVC5]  induction (Lu, Peng et al. 2002). Estradiol treatment also induced increased HO-1 mRNA expression, elevated HO-1 protein levels, and HO enzyme activity in cardiac and hepatic tissue in the trauma-hemorrhage rats. Administration of the HO inhibitor[DVC6]  CrMP prevented the estradiol-induced attenuation of shock-induced organ dysfunction and damage. Thus, the salutary effects of estradiol administration on organ function after trauma-hemorrhage [DVC7] are mediated partly via the upregulation of HO-1 expression and HO-1 activity (Szalay, Shimizu et al. 2005). Estrogen treated endothelial cells of different human origins (HUVEC and HAUEC) released more CO to the culture medium than control cells (Tschugguel, Stonek et al. 2001). The results of several recent studies support the potential cardiovascular benefits of raloxifene use (Barrett-Connor, Grady et al. 2002, Leung, Tsang et al. 2007). Raloxifene improves endothelial function in ovariectomized aging or hypertensive rats (Wong, Yao et al. 2006, Chan, Leung et al. 2007) ameliorates hypertension-induced endothelial dysfunction by reducing ROS production  and enhances endothelial NO-dependent vasodilatation in vitro (Leung, Yung et al. 2007). In addition, raloxifene causes direct vasodilatation. Raloxifene supposed to reduce surrogates of  increased cardiovascular risk in patients with osteoporosis (Barrett-Connor, Grady et al. 2002) although the outcomes of the RUTH trial showed that raloxifene did not affect the overall risk of coronary heart disease in elderly women (Barrett-Connor, Mosca et al. 2006). Even though hormone replacement therapy in postmenopausal women results in an increased risk of coronary artery disease, both epidemiological in premenopausal women and experimental studies have suggested or clearly demonstrated a major atheroprotective action of oestradiol and SERMs.

According to our results, in the lack of estrogen, besides the decreased NOS level (Pavo, Laszlo et al. 2000), the diminished function of HO enzymes also plays an important role in the development of cardiovascular disorders. Estrogen or raloxifene replacement following bilateral ovariectomy increase the level and activity of HO enzymes, which could lead to the reduction of the incidence of the development of cardiovascular disorders due to the lack of estrogen.

Previous studies have demonstrated that either acute or chronic administration of various inducers of HO-1 to spontaneously hypertensive rats led to a normalization of blood pressure.(Chen, Yet et al. 2003). In another model, overexpression of HO-1 was associated with an increase in HO enzyme activity and a decrease in blood pressure in spontaneously hypertensive rats (Sabaawy, Zhang et al. 2001)

Arginine vasopressin a neurohypophyseal nonapeptide hormone, also known as antidiuretic hormone, is synthesized in the magnocellular cells. In the heart, vasopressin causes dose-dependent coronary constriction, myocardial depression and coronary smooth muscle cell proliferation at physiologic concentration, actions reversed by V1 receptor antagonists (Oghlakian and Klapholz 2009). In present study, OVX increased the contractile response to AVP but estrogen or raloxifene completely restored the tension to the level observed in ovary-intact females. The greater contractile responses of the aorta to AVP also may be due to the downregulation of vasopressin receptors in vascular smooth muscle cells. It is possible that OvX[DVC8]  not only decreased constrictor prostanoid function, but also reduced the expression of vasopressin binding sites in aortic vascular smooth muscle cells (Zhang and Davidge 1999)

Although most studies reveal that long-term treatment with estrogen enhances reactivity to AVP not all studies agree with these findings. For example, long term estrogen replacement of OvX[DVC9]  rats has been reported to be without effect on reactivity of resistance-sized mesenteric arteries to AVP (Zhang and Davidge 1999). The reason(s) for these differences are uncertain, but may involve differences in the blood vessels studied and/or the dose of estrogen replacement therapy employed.

The female cardiovascular system is influenced by changes in the endocrine system. During the menopausal transition, dramatic hormonal changes such as declining levels of estrogen and rising levels of gonadotropins may affect the cardiovascular system. Estrogen deficiency induces an imbalance between enhanced ROS production and inadequate antioxidant activity. The decline in ovarian function accompanying the menopause, OVX and POVX contributes to the induction of pro-inflammatory cytokines such as TNF-α and IL-6. Estrogen is a well known regulator of inflammation. Cardiovascular and immune system abnormalities have been reported in females with estrogen deficieny. exerting a number of known antiinflammatory effects through a variety of different mechanisms, both genomic and nongenomic. The antiinflammatory role ranges from generating NO and regulating leukocyte recruitment to reducing oxidative stress and promoting cell survival. These effects contribute to dampening inflammation in the vascular system. Estrogen deficiency has been shown to upregulate TNF-α levels in aged animals with deleterious effects on vascular function. Such effects are largely mediated through increased oxidative stress and can be reversed through exogenous estrogen, a TNF-α inhibitor or antioxidants(Arenas, Armstrong et al. 2005) . Sex hormones are known modifiers of the inflammatory response to injury, an important aspect of myocardial dysfunction and cardiomyocyte death following ischemia. Hamilton et al. in their experiments showed that the overall effect of OVX on myocardial gene expression was increased expression of genes involved in the inflammatory response. OVX increased IL-6 receptor, TNF-α, complement 8, and SOCS2 and 3 expression(Hamilton, Lin et al. 2008). During aging and after ovariectomy, estrogen deficiency is able to stimulate the spontaneous secretion of such pro-inflammatory cytokines. Along with the effects of cytokines, the MPO activity is determined as an inflammatory biomarker. Inflammation and oxidative stress are associated with atherosclerosis and cardiovascular disease. Conversely, inflammation also triggers vascular remodeling, aggravates vessel injury and exacerbates the processes of hypertension and atherosclerosis (Celik, Koc et al. 2012). MPO plays a significant role in the development of the atherosclerotic lesion and renders the plaques unstable, which is associated with the aging mechanism and cardiovascular disease (Schindhelm, van der Zwan et al. 2009). Similar results demonstrate the role of HO-1 in HO-1 knockout mice, in which the HO-1 deficiency leads to an increased production of pro-inflammatory cytokines (Marcantoni, Di Francesco et al. 2012) while HO-1 upregulation successfully slows the processes of hypertension and myocardial infarction(Chen, Li et al. 2013). Preclinical and clinical evidence clearly suggests that the progression of atherosclerosis is associated with inflammation. The CO derived from HO has been demonstrated to protection against TNF-α- induced apoptosis(Choi 2001). Aging leads to significantly increased levels of the pro-inflammatory cytokines in the liver of aged female rats as compared with young controls. Hence, the diminished activity of the HO system contributes to increased oxidative stress(Kireev, Tresguerres et al. 2010). Ross et al. demonstrated that the ventricular myocytes from young female rats were more resistant to ischemia and reperfusion injury than were cells from males, and than ischemia and reperfusion injury in female myocytes was exacerbated by aging or by OVX. Advancing age abolishes the beneficial effects of the female sex on the cell viability and the contractile function(Ross and Howlett 2012). Our data indicated that aging and estrogen- depletion process abolished the beneficial effects of the female sex in the myocardium. This suggests that the cardioprotection of the female sex may decline with age in response to a reduction in circulating estradiol levels. Indeed, we found that the removal of estrogen through OVX or POVX exacerbated the adverse effects of ischemia, which can be augmented via HO activity inhibition. Our results indicate that decreases in the expression of HO-1/HO-2 and in the activity of the HO system are important factors that contribute to the enhanced sensitivity of the OVX or POVX heart to ischemia. Moreover, we found that aging and OVX induced diminished HO activity and expression. Direct vascular effects of estradiol are believed to play a significant role in the cardioprotectivity of estrogens. These findings on OVX and POVX female rats may have important clinical implications. Extensive epidemiological studies of the morbidity and mortality rates of postmenopausal women have revealed a significant increase in cardiovascular mortality as compared with that in women who are still menstruating. After the menopause, women lose the relative cardiovascular protection that they had previously enjoyed over men(Brown, Anthony et al. 2001, Posa, Kupai et al. 2013). Although estrogen has been reported to protect the cardiovascular system, the mechanisms involved remain unclear. Estrogen exerts genomic and non-genomic effects via estrogen receptor-dependent and -independent mechanisms, to confer protective effects on the cardiovascular system. Alterations in plasma concentrations of lipoproteins (decreases in low-density lipoprotein levels and in oxidized low density lipoprotein formation, and increases in high-density lipoprotein levels), hemostatic factors, glucose, insulin, and endothelium-derived factors (decreases in endothelin and increases in NO and prostaglandins) and the inhibition of smooth muscle cell migration and proliferation induced by various mitogens are thought to contribute to the vasoprotective effects of estrogen(Lee, Cheng et al. 2005). Of these biological effects, the antioxidant effects of estrogen may play a critical role in eliciting vasoprotective effects(Dantas, Tostes et al. 2002). Endogenous and exogenous estrogens have antioxidant potential both in vitro (Dubey, Tyurina et al. 1999) and ex vivo(Wassmann, Baumer et al. 2001). An estrogen deficiency in the menopausal state therefore leads to changes in the homeostatic environment of the body, e.g. a gradual increase in oxidative stress. A previous report from our laboratory supports this concept, demonstrating the sexual dimorphism in HO activity and expression. The present study demonstrated similar results in that the HO level in the hearts of OVX rats were markedly lower than that in the sham-operated controls. HO has potent cytoprotective effects that are likely to be mediated by its products, CO, biliverdin/bilirubin, and free iron, and we were interested in the role of HO in an estrogen-deficient environment. The HO levels proved to be reduced in the aged and ovariectomized rats, which might result from the down-regulation of HO in the heart tissues. After OVX, a marked reduction in estrogen synthesis led to decreases in the activity and expression of HO.

However, only a small number of data are available concerning the effects of HO expression in vivo. It emerged from the current study that an HO activity inhibitor caused an ST depression in control female rats and augmented the ST depression in the estrogen-withdrawal model. The data suggest that the HO activity protects the heart at a fertile age.

Epidemiological, clinical, and molecular studies have shown that estrogen plays an important role on metabolic homeostasis(Barros and Gustafsson 2011), and the loss of estrogen may have profound effects on glucose homeostasis and body composition in both menopause women(Lovejoy, Champagne et al. 2008) and rodents(Jones, Thorburn et al. 2000, Rogers, Perfield et al. 2009).

It is widely accepted that the prevalent lifestyle model of Western societies characterized by limited physical activity, excessive caloric intake, and repetitive behavioral patterns contributes to the dis-regulation of the otherwise homeostatic control of body weight. Our experimental findings are agreement with those from other experiments that the combination of exercise and diet modification had greater effects on metabolic parameters(Paulino, Ferreira et al. 2010, Kang, Kim et al. 2013). Because the augmented metabolic parameters are positively correlated with cardiovascular disease-induced metabolic disorders(Paulino, Ferreira et al. 2010, Meziri, Binda et al. 2011, Touati, Meziri et al. 2011, Claudio, Endlich et al. 2013, Endlich, Claudio et al. 2013, Leite, Durigan Rde et al. 2013), our experimental findings strongly support the benefit of exercise as a means of reducing metabolic parameters and thus cardiovascular risk.

Improved insulin sensitivity is a hallmark outcome of exercise training: importantly, endurance training can restore insulin response in obese, insulin-resistant rodents and humans(Bruce, Thrush et al. 2006, Lessard, Rivas et al. 2007). Insulin resistance is a common occurrence in obesity, type 2 diabetes, dyslipidemia and hypertension and there is some experimental evidence indicating that insulin resistance and hyperinsulinemia precede the development of obesity and other MS factors(Camporez, Jornayvaz et al. 2013). Exercise training may be one of the preventive and therapeutic strategy against impaired leptin and insulin signal transduction in the hypothalamus of obese individuals and associated with a markedly increased phosphorylation or activity of several proteins involved in leptin and insulin signal transduction(Flores, Fernandes et al. 2006, Friedman 2009). Riant et al observed impaired glucose tolerance and insulin resistance in OVX mice fed with high fat diet(Riant, Waget et al. 2009). We have shown that rats fed with HT diet have hyperinsulinemia and hyperglycemia, which can be improved via recreative physical exercise combined with dietary restriction.

The HT diet does seem to influence glucose metabolism in the exposed animals. The serum levels of insulin showed significant changes in OVX rats compared to SO rats, which could be taken as an indication that they were progressively developing an insulin-resistant condition.

It is well established that an increase in plasma triglyceride elevates the risk of developing atherosclerosis and dramatic clinical events such as acute myocardial infarction and stroke. Furthermore, lowering plasma triglyceride can reverse the progression of atherosclerosis and prevent cardiovascular events(Gami, Witt et al. 2007). As an essential component of the metabolic syndrome blood triglyceride level was also investigated in this study. OVX resulted in an increase in serum triglyceride level. Exercise training and caloric restriction resulted in a reduction of the OVX increased levels of these factors. The beneficial effects of estrogens on blood lipid profile have been known for a long period of time(Zoth, Weigt et al. 2010). Also physical activity is well known to effect blood lipid profiles in a positive manner(Mestek, Plaisance et al. 2008). Even we observed significant combinatory effects in our study; we believe that the combination of both may also result in additive effects with respect to the prevention of arteriosclerosis. In our earlier experiments was demonstrated significant difference in heme oxigenase (HO) and nitrogen monoxide synthase (NOS) enzymes and cardiovascular parameters between male and female rats and we found anti-inflammatory effects of recreative exercise training so we prove this hypothesis future investigations in the cardiovascular tissue of these animals are ongoing(Posa, Kupai et al. 2013, Szalai, Szasz et al. 2014).

The leptin plays an important role in the endocrine system, a hormone secreted in the periphery by fat cells, which signals the status of body energy stores, down-regulates feeding behavior, regulating appetite and energy expenditure(Friedman and Halaas 1998) Leptin is required for energy stores to be sensed in the central nervous system and is thus essential for the functions such as normal energy homeostasis and reproduction(Zhang, Proenca et al. 1994). Leptin is characterized as an adipocytokine that is mainly expressed in adipose tissue(Zhang, Proenca et al. 1994). It is able to resist insulin secretion and features a positive correlation with body fat content(El-Haschimi, Pierroz et al. 2000).

Kang at al reported that plasma leptin level was significantly increased in high fatty diet group as compared high fatty diet combined training group and suggest the effect of leptin sensitivity in peripherial may primarily the more relate to combined dietary control and exercise training more than effect of dietary control(Kang, Kim et al. 2013). Short and long term caloric restriction without exercise training have been shown to dramatically reduce plasma leptin levels in obese human and rats(Shimokawa and Higami 2001, Shimokawa and Higami 2001, Han, Joe et al. 2010). Thus, components environment enrichment other than physical exercise could play a role in regulating feeding behavior and the related mechanisms in developing mice(Mainardi, Scabia et al. 2010). The reduction in fat deposition with training in OVX rats raises the question of whether the level of leptin is improved in these rats.

Non-alcoholic fatty liver disease (NAFLD) is emerging as an acknowledged component of metabolic syndrome. Markers of this condition, such as elevations in serum concentrations of aspartate aminotransferase transaminase (AST), alanine aminotransferase (ALT) may be considered reliable predictors of the development of the syndrome(Hanley, Williams et al. 2005). OVX resulted higher body weight, augmented AST, ALT levels as compared to those of SO rats. Cameron et al demonstrated that the endurance exercise normalised plasma AST ALT levels in diet induced MS rat model(Cameron, Alam et al. 2012).  In addition, other studies demonstrated the presence of NAFLD in rats that consumed a high-fat diet (71% of energy from fat) for three weeks. Similarly, our data showed high plasma levels of ALT and AST(Lieber, Leo et al. 2004, de Castro, dos Santos et al. 2013). 

After a 12- week HT diet OVX rats of the present study exhibited some symptoms of the metabolic syndrome. OVX non-trained rats possessed a significant higher final body weight, augmented AST, ALT level, higher triglyceride level, increased in leptin level. This effect was prevented, in part, by exercise or caloric restriction, but most effectively by combined therapy. This observation indicates that the prevention of weight gain cannot only be the result of exercise training but also of metabolic changes under control of estradiol. This indicates that exercise training has a strong influence on lowering body fat accumulation following a decrease in estrogen levels.

In summary, the results of the present short experiment indicated that a combined therapy of caloric restriction and exercise training is able to influence parameters related to lipid metabolism in OVX rats positively. As a conclusion we believe that our data provides some evidence that CR combined with physical activity could be a very effective strategy to prevent the development of a metabolic syndrome induced by overnutrition.