The AT1-Type Angiotensin Receptor in Oxidative Stress and Atherogenesis
Part II: AT1 Receptor Regulation
Despite intense investigation in the last decade, many questions concerning the etiology of atherosclerosis remain unanswered. Hypercholesterolemia and hypertension are clearly risk factors for the development of vascular lesions, although the precise molecular steps between elevated lipid or blood pressure levels and the development of atherosclerotic lesions are not completely defined.1,2⇓ Hypertension is frequently associated with additional risk factors such as hypercholesterolemia, estrogen deficiency, or hyperinsulinemia. This clustering of risk factors greatly enhances the probability to develop atherosclerosis.3 Nevertheless, the cellular events responsible for the mutual appearance of several risk factors are poorly understood. A series of recent studies have addressed the hypothesis that enhanced AT1 receptor activation could explain the association of various hormonal and metabolic disorders with hypertension, and ultimately, with accelerated progression of vascular lesions.
Regulation of AT1 Receptor Expression and Function
As early as 1980, Alexander and colleagues discovered that the vasoconstriction caused by angiotensin II in resistance vessels was variable.4 Further investigations revealed that AT1 receptor activation is subject to a negative feedback, in that increased levels of angiotensin II diminish and decreased angiotensin II concentrations enhance AT1 receptor activation.5–8⇓⇓⇓ More recently, it has been shown that multiple agonists other than angiotensin II modulate AT1 receptor expression. This phenomenon, referred to as heterologous AT1 receptor regulation, is induced by various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or fibroblast growth factor (FGF), all which downregulate AT1 receptor expression.9 Numerous other factors, including glucocorticoids, aldosterone, forskolin, TNFα, cytokines, nitric oxide, insulin, LDL, estrogen, progesterone, sodium, free radicals, IGF-1, and isoprenaline are known to influence AT1 receptor expression (Table). 10–38⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓
Hypercholesterolemia-Associated AT1 Receptor Overexpression
Hypercholesterolemia and in particular elevated LDL plasma concentrations play a fundamental role in the pathogenesis of atherosclerosis, as shown by numerous epidemiological and interventional studies.1,2⇓ Moreover, hypercholesterolemia is frequently associated with hypertension, another potent cardiovascular risk factor.3 Despite this large body of epidemiological evidence, the molecular events leading from hypercholesterolemia to hypertension and atherosclerosis are only partially understood. Recent studies have provided insight into these interactions. For example, it has been shown that exposure of vascular smooth muscle cells to LDL markedly augments AT1 receptor mRNA and protein expression.39 Consistent with these findings, the functional response of these cells on angiotensin II stimulation (eg, calcium release, cell proliferation) is also enhanced. The underlying mechanism for this upregulation of the AT1 receptor was found to be stabilization of its mRNA rather than an alteration of its transcription rate. These studies in cultured cells provided a mechanism that could link hypercholesterolemia to enhanced sensitivity of the vessel wall to angiotensin II stimulation.
Subsequent studies have extended these observations to the in vivo situation. In rabbits with diet-induced atherosclerosis and in rabbits with heritable hyperlipidemia, aortic AT1 receptor expression is increased by 2-fold.40,41⇓ This has functional significance, in that angiotensin II–induced vasoconstriction is markedly increased in these animals. Interestingly, vascular superoxide production is also increased in hypercholesterolemia and this is associated with profound alteration of endothelium-dependent vasodilation (Figure 1). These abnormalities are normalized by blockade of the AT1 receptor, despite the fact that blood pressure and lipoprotein levels are not changed by this treatment. Most importantly, development of atherosclerotic lesions can be greatly inhibited by AT1 receptor antagonism. These findings strongly support the concept that hypercholesterolemia increases AT1 receptor expression and illustrate how this phenomenon can contribute to atherosclerosis.
Subsequent work has provided evidence that hypercholesterolemia increases AT1 receptor expression in humans.42 In hypercholesterolemic subjects, angiotensin II infusion produced more than twice the increase in blood pressure as that observed in normocholesterolemic subjects. In keeping with this finding, the expression of AT1 receptors was increased by 2- to 3-fold in hypercholesterolemic subjects. Of profound importance, treatment with statins for a 4-week period normalized the pressor response to angiotensin II infusion and completely normalized AT1 receptor expression (Figure 1). These results have been confirmed in another study of normocholesterolemic individuals43 and provide a molecular link between hyperlipidemia and hypertension. Further, these studies provide insight into why lipid lowering may have antihypertensive effects.
Because statin treatment is capable of reducing expression of AT1 receptors in hypercholesterolemic individuals without normalizing LDL levels, further studies have been performed in normocholesterolemic spontaneously hypertensive rats and cultured vascular smooth muscle cells. These experiments demonstrated that statins directly downregulate AT1 receptor expression in vitro as well as in vivo.44,42,45⇓⇓
These findings likely have important clinical implications, in that they help to explain some of the molecular mechanisms leading to the development and progression of hypertension and atherosclerosis in hypercholesterolemia. Further, they demonstrate why statins, which primarily inhibit cholesterol biosynthesis, may have favorable effects on blood pressure regulation, and why AT1 receptor antagonists have a beneficial effect on the atherosclerotic process. Obviously, statins exert many pleiotropic, potentially blood pressure–lowering effects such as enhancement of NO bioavailability. The latter is governed by inhibition of small G-proteins and the reduced expression of caveolin-1.46,47⇓
Importantly, other components of the renin-angiotensin system such as ACE (angiotensin converting enzyme) are also upregulated during atherosclerosis as well as hypercholesterolemia.48 In this context, high abundance of angiotensin II has also been shown in atherosclerotic plaques.49 Therefore, an enhanced production of angiotensin II would coincide with AT1 receptor upregulation leading to a increased efficacy of the renin-angiotensin system and its deleterious consequences. Of note, ACE and AT1 receptors are components of the local renin-angiotensin system in the vessel wall. Regulation of these is likely completely independent of the circulating system, which appears not to be regulated during atherosclerosis.
Estrogen Deficiency–Associated AT1 Receptor Overexpression
Coronary heart disease is the leading cause of death among women in the United States and Europe.50 Many epidemiological studies indicate that premenopausal women have a low incidence of vascular disease, but that the risk of cardiovascular events increases rapidly after menopause. Of note, the incidence of hypertension increases dramatically at the same time.51 Numerous observational studies have shown that estrogen replacement therapy may exert beneficial effects on cardiovascular morbidity and mortality, suggesting an important role of estrogens in the pathogenesis of vascular diseases.52 One accepted effect of estrogen is its beneficial influence on lipid homeostasis. Beyond this, however, estrogen has been shown to have beneficial effects on vascular and myocardial cells. Recently, the effect of estrogen was examined on AT1 receptor gene expression in oophorectomized rats. Angiotensin II–induced vasoconstriction was significantly increased in aortas from ovariectomized rats. In keeping with this finding, AT1 receptor density and mRNA levels were increased by approximately 180% as a result of ovariectomy. Estrogen replacement therapy in these animals normalized AT1 receptor expression.53 Interestingly, overexpression of AT1 receptors induced by estrogen deficiency was associated with endothelial dysfunction and increased vascular production of superoxide, and these abnormalities were normalized by either estrogen replacement or AT1 receptor blockade.54 These data strongly suggest that abnormal vascular function observed in the estrogen-deficient state is at least in part due to angiotensin II and AT1 receptor activation. Parallel studies in cultured vascular smooth muscle cells revealed that estradiol causes a time-dependent decrease in AT1 receptor gene expression.55 This effect was mediated via nitric oxide release and dependent on posttranscriptional modulation of the AT1 receptor mRNA. Estrogen-induced downregulation of AT1 receptor expression could help to explain the association between estrogen-deficiency, hypertension, and atherosclerosis observed in many clinical studies. However, gender differences in risk factor susceptibility could be also explained by upregulation of AT1 receptor expression and activation of the renin angiotensin system via androgens.56,57⇓ These findings suggest that either ACE inhibitors or AT1 receptor antagonists may be beneficial in the postmenopausal state to antagonize the effect of increased AT1 receptor expression.
Mechanisms of AT1 Receptor Regulation
Most angiotensin II effects are mediated via AT1 receptors. Thus, the number of AT1 receptors defines the biological efficacy of angiotensin II. There are at least 4 different aspects involved in AT1 receptor regulation. First, activation of AT1 receptors with angiotensin II evokes internalization of the receptor protein and reduces receptor numbers on the cell surface.58 Second, prolonged angiotensin II stimulation reduces angiotensin II signaling via protein kinase C–dependent pathways, an event termed desensitization.59 Third, alternative splicing of the AT1 receptor pre-mRNA can alter AT1 receptor protein translation. Fourth, and likely the most important mechanism regulating AT1 receptors is modulation of its gene expression.8,9⇓ These factors are summarized in Figure 2.
Alterations of AT1 receptor gene expression are generally obvious several hours after agonist stimulation and are sustained for variable periods of time thereafter. Recently, it has been shown that internalization of the AT1 receptor seems to regulate downregulation of AT1 receptor mRNA.60
Gene expression is predominantly modulated by transcriptional and posttranscriptional mechanisms. Among other stimuli, growth factors and angiotensin II are known to reduce the rate of AT1 receptor mRNA transcription.8,9⇓ Although, numerous consensus sequences were discovered within the promotor region of the AT1 receptor (eg, Ap-1, Sp1, estrogen-response element, cAMP-response element), the exact mechanisms of transcriptional control of AT1 receptor mRNA synthesis are poorly understood.61
Posttranscriptional AT1 Receptor Regulation
In general, the abundance of a particular mRNA transcript and its resulting protein product is not only governed by its transcription rate, but also by its half-life (otherwise referred to as mRNA stability). Of relevance to the AT1 receptor has been the finding that numerous agonists that regulate AT1 receptor expression also affect posttranscriptional processing of its mRNA. Estrogens, angiotensin II, and cAMP-stimulating agents decrease AT1 receptor expression by stimulating degradation of its mRNA.55,62,63⇓⇓ In contrast, progesterone, LDL, and insulin upregulate AT1 receptor expression by decreasing its mRNA decay.13,20,21,40,55⇓⇓⇓⇓ The data concerning growth factors such as PDGF are inconsistent. It is well established that these factors reduce AT1 receptor mRNA transcription rate.9 Experiments following mRNA degradation after blockade of transcription with actinomycin D revealed that growth factors induce degradation of the AT1 receptor mRNA.9 Other data with recombinant, retroviral approaches have suggested that PDGF may have no effect on AT1 receptor mRNA stability.64,65⇓ These discrepancies are likely because of difficulties in obtaining unambiguous measures of inducible mRNA turnover. Despite these inconsistencies, there is strong evidence that posttranscriptional mechanisms predominate AT1 receptor regulation. As is the case for many other mRNAs, this process involves binding of proteins to both the 5′ and 3′ untranslated region of the AT1 receptor mRNA. Data from recombinant retroviral AT1 receptor mRNA species and experiments performed in brain tissue suggest that proteins interacting with the 5′ untranslated region of the AT1 receptor mRNA are involved in both cAMP- and estrogen-induced modulation of AT1 receptor regulation.19,64–66⇓⇓⇓ To date, however, the identity of these proteins remains undefined, as do the precise regions of the AT1 receptor mRNA involved in binding these proteins. Our own data show that a family of proteins residing in the polysomal compartment bind to the 3′ untranslated region of the AT1 receptor mRNA (Figure 3). Detailed analysis revealed that a region between bases 2175 to 2195 within the AT1 receptor mRNA, just adjacent to the poly-A-tail, is responsible for the protein-mRNA interaction. Interestingly, this AU-rich region forms a stem loop typical for a mRNA region prone to protein binding (Figure 3). Transfection experiments in vascular smooth muscle cells and in vitro decay assays within the polysomal compartment derived from vascular smooth muscle cells demonstrated that a protein binding to the bases 2175 to 2195 of the AT1 receptor mRNA mediates AT1 receptor regulation. However, the participating proteins remain unknown and need further clarification (Figure 3).67
Signal Transduction of AT1 Receptor Regulation
No uniform signal transduction pathway has been defined that inevitably results in modulation of AT1 receptor expression for all agonists in all cell types. In vascular smooth muscle cells, cAMP participates in isoprenaline and possibly in angiotensin II–induced AT1 receptor downregulation.8,63⇓ Also, superoxide radicals and hydrogen peroxide are involved in angiotensin II–induced AT1 receptor regulation. The p38 MAP kinase mediates radical-induced AT1 receptor regulation, whereas p42/44 MAP kinase is presumably involved in insulin-driven AT1 receptor overexpression.12,14⇓ In vascular smooth muscle cells, superoxide as well as hydrogen peroxide downregulate AT1 receptor mRNA expression mediated through posttranscriptional mechanisms.14 Again, transcriptional regulation seems to not be of importance. Furthermore, nitric oxide mediates estrogen-dependent AT1 receptor downregulation, PI-3 kinase has been implicated in progesterone-caused AT1 receptor upregulation (Table).55 Thus, various signal transduction pathways have been implicated; however, the detailed cascade between a cell surface impulse and the ultimate modulation of gene expression are only partially understood. Particularly, the steps immediately upstream of the described AT1 receptor mRNA binding proteins are not known.
AT1 receptor regulation very likely represents, among others, a molecular switch connecting traditional risk factors such as hypercholesterolemia, estrogen deficiency, and hyperinsulinemia with hypertension and atherosclerosis (Figure 4). From this point of view, AT1 receptor overexpression is one potential molecular mechanism that links a variety of exogenous risk factors to cellular events in the vascular disease. Presently, it is not certain if increased activity of other local components of the renin-angiotensin system such as ACE and angiotensin II is concomitantly required, or whether AT1 receptor overexpression itself is sufficient to propagate vascular lesion formation.
The genetic factors that predispose to atherosclerosis are still not precisely defined. It is tempting to speculate that AT1 receptor overexpression putatively in concert with other upregulated components of the renin-angiotensin system in response to risk factors could be one of the genetically defined predispositions. Overexpression of the AT1 receptor would likely increase the risk of atherosclerosis regardless of the underlying risk factor profile. Whether variations in the AT1 receptor regulation occur in the population and whether they are a predictor of vascular risk needs to be evaluated in large-scale clinical studies. Inducible polysomal proteins, which interact with the AT1 receptor mRNA in its 3′ untranslated region, could be key players in this scenario. We propose that these factors initiate upregulation or downregulation of AT1 receptor expression via both increased or decreased AT1 receptor mRNA decay. In this context, differences in activities or expression of the relevant RNA-binding proteins based on polymorphisms of the encoding genes could be very important as a mechanism predisposing to vascular disease. Further characterization of the molecular events related to posttranscriptional AT1 receptor regulation and identification of these relevant factors will help to determine if these pathways contribute to the genetic susceptibility to atherosclerosis.
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