Abstract-Estrogen has antiinflammatory and vasoprotective effects when administered to young women or experimental animals that appear to be converted to proinflammatory and vasotoxic effects in older subjects, particularly those that have been hormone free for long periods. Clinical studies have raised many important questions about the vascular effects of estrogen that cannot easily be answered in human subjects. Here we review cellular/molecular mechanisms by which estrogen modulates injury-induced inflammation, growth factor expression, and oxidative stress in arteries and isolated vascular smooth muscle cells, with emphasis on the role of estrogen receptors and the nuclear factor-B (NFB) signaling pathway, as well as evidence that these protective mechanisms are lost in aging subjects. Ovarian Hormones and Cardiovascular Disease in WomenCardiovascular disease is the leading cause of death among women in the United States, and coronary heart disease (CHD) develops in women on average 10 years later than in men. This lag has been attributed, at least in part, to the protective effects of female sex hormones, particularly estrogens (defined as naturally occurring activators of estrogen receptors) before menopause. 1-3 Mechanistic studies carried out in in vitro preparations and in laboratory animals have shown that both natural and synthetic estrogens have antiinflammatory and vasoprotective effects. 4 -18 Further, the natural endogenous estrogen 17␤-estradiol has been shown to cause rapid endotheliumindependent dilation of coronary arteries of men and women, to augment endothelium-dependent relaxation of human coronary arteries ex vivo, and to improve endothelial function as assessed by the brachial artery flow-mediated dilation response in postmenopausal women. 19 Importantly, the latter vasoprotective effects of estrogen have been observed in the early postmenopausal years in both healthy women and those with CHD, but not in older (Ն60 years) postmenopausal women, regardless of the presence or absence of CHD. 19,20 See accompanying article on page 277Observational studies have shown substantial benefit (Ϸ50% reduction in CHD) of hormone therapy in women who choose to use menopausal hormones (and usually begin taking them in the perimenopausal or early postmenopausal period). 21 Randomized controlled trials of menopausal hormone therapy, which typically enroll women 10 years or longer after menopause, after many years of estrogen deprivation, have shown increases in CHD events with hormone treatment (usually conjugated equine estrogenϮa progestin) in this older (60 to 79 years) age group. [22][23][24] In contrast, subgroup analyses of the Women's Health Initiative have shown that women in whom hormone therapy was initiated at a younger age (50 to 59 years), and earlier post menopause tended to have reduced risk of CHD and total mortality. 25,26 Use of unopposed conjugated estrogen was associated with lower risk of CHD than combined estrogenϩprogestin (medroxyprogesterone acetate), and an ancillary study sh...

Our previous studies demonstrated that the sexually dimorphic pattern of hypertension in the spontaneously hypertensive rat is androgen dependent Gonadectomy retards the development of hypertension in young males, but not in females, and administration of testosterone propionate to gonadectomized spontaneously hypertensive rats of both sexes confers a male pattern of blood pressure development The current study tested the hypothesis that renal and hepatic renin and angiotensinogen gene expression are also androgen dependent in the spontaneously hypertensive rat Male and female spontaneously hypertensive rats underwent gonadectomy or a sham operation at 4 weeks of age. Subgroups of gonadectomized rats of both sexes were implanted with a 15-mm or 30 -mm Silastic capsule filled with testosterone at the same time the gonadectomy was performed; a third group received an empty Silastic capsule. Northern and slot blot analyses were used to characterize and quantitate renin and angiotensinogen messenger RNA (mRNA) in the kidney and liver 18 weeks after the gonadectomy. Blood pressure, plasma renin activity, and hepatic angiotensinogen mRNA levels were higher in intact males than in females. Orchidectomy retarded the development of hypertension and lowered plasma renin and renal and hepatic angiotensinogen mRNA levels, and testosterone replacement restored the male pattern of hypertension and plasma renin and increased renal and hepatic angiotensinogen mRNA. Ovariectomy did not alter blood pressure or plasma renin but did lower renal renin and renal and hepatic angiotensinogen mRNA; testosterone increased blood pressure, plasma renin, renal renin and angiotensinogen mRNA, and hepatic angiotensinogen mRNA levels in ovariectomized females. These data suggest that the androgendependent development of hypertension in spontaneously hypertensive rats may be related to androgeninduced activation of the renin-angiotensin system. 1 In the DEA/2J and CBA strains of mice, the male develops higher blood pressure than the age-matched female.2 In the Dahl salt-sensitive rat, the spontaneously hypertensive rat of the Okamoto strain (SHR), and the desoxycorticosterone acetate-NaCI hy- pertensive rat, hypertension develops more rapidly and becomes more severe in the male than in the female.3 " 8 Recent studies from our laboratory have demonstrated that the sexually dimorphic pattern of blood pressure development in SHR is androgen dependent. 9 However, the mechanism by which androgen mediates the sexual dimorphism of blood pressure in SHR is unknown.Several studies have suggested that androgenic regulation of renin-angiotensin system activity may contribute to the development of androgen-dependent hypertension. Submandibular gland (SMG) renin activity has been shown to decrease after castration and to increase after testosterone treatment, as well as during puberty, in male mice. 10

These data indicate that the sex difference in myointimal proliferation after vascular injury is estrogen dependent. C-myc gene expression is greater in the undamaged carotid artery of the male than in that of the female, and the responsiveness of this gene to balloon injury of the artery is more rapid and more robust in the male than in the female rat. These findings have direct implications for the prevention and treatment of vascular disease in humans.

The posttranslational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ␤-N-acetylglucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification. Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are both major hallmarks of diabetes mellitus and diabetes-related cardiovascular complications. Conversely, there is a growing body of data demonstrating that the acute activation of O-GlcNAc levels is an endogenous stress response designed to enhance cell survival. Reports on the effect of altered O-GlcNAc levels on the heart and cardiovascular system have been growing rapidly over the past few years and have implicated a role for O-GlcNAc in contributing to the adverse effects of diabetes on cardiovascular function as well as mediating the response to ischemic injury. Here, we summarize our present understanding of protein O-GlcNAcylation and its effect on the regulation of cardiovascular function. We examine the pathways regulating protein O-GlcNAcylation and discuss, in more detail, our understanding of the role of O-GlcNAc in both mediating the adverse effects of diabetes as well as its role in mediating cellular protective mechanisms in the cardiovascular system. In addition, we also explore the parallels between O-GlcNAc signaling and redox signaling, as an alternative paradigm for understanding the role of O-GlcNAcylation in regulating cell function. hexosamine biosynthesis; protein O-glycosylation; ␤-N-acetylglucosamine transferase; diabetes mellitus POSTTRANSLATIONAL MODIFICATION (PTM) of proteins is a common mechanism for the modulation of protein function, location, and turnover. Although protein phosphorylation is probably the most widely studied form of PTM, there are many other PTMs, including acylation, ubiquitylation, methylation, acetylation, thiolation, nitration, and glycosylation (107). The focus of this review is protein glycosylation, specifically, O-glycosylation of nuclear and cytoplasmic proteins. Classical protein glycosylation occurs in the endoplasmic reticulum and Golgi, leading to the formation of stable and complex elongated oligosaccharide structures via both N-linkage on asparagine and O-linkage on the hydroxy amino acids serine and threonine in addition to hydroxyproline, hydroxylysine, and tyrosine residues of proteins that become secreted or membrane component glycoproteins (189). In contrast, glycosylation of nuclear and cytoplasmic proteins is a rapid and dynamic modification of serine or threonine residues by the O-linked attachment of the monosaccharide ␤-N-acetylglucosamine (OGlcNAc) (97,195); this process is referred to as protein O-GlcNAcylation to contrast it wi...

The current study examined the effects of bosentan, an orally active antagonist of endothelin-A and -B receptors, on the development and maintenance of hypoxia (10% O2)-induced pulmonary hypertension and vascular remodeling in the rat. Pretreatment with bosentan (100 mg.kg-1.day-1, 1 gavage/day for 2 days) completely blocked the pulmonary vasoconstrictor response to acute hypoxia. Chronic bosentan treatment (100 mg.kg-1.day-1 po in the food) instituted 48 h before hypoxic exposure prevented the subsequent development of pulmonary hypertension, attenuated the associated right heart hypertrophy, and prevented the remodeling of small (50-100 microns) pulmonary arteries without altering systemic arterial pressure. Institution of bosentan treatment (for 4 wk) after 2 wk of hypoxia produced a significant reversal of established hypoxia-induced pulmonary hypertension (from 36 +/- 1 to 25 +/- 1 mmHg), right heart hypertrophy, and pulmonary vascular remodeling despite continuing hypoxic exposure. These findings support the hypothesis that endogenous endothelin-1 plays a major role in hypoxic pulmonary vasoconstriction and/or hypertension, right heart hypertrophy, and pulmonary vascular remodeling and suggest that endothelin-receptor blockade may be useful in the treatment of hypoxic pulmonary hypertension humans.

Abstract-This study tested the hypothesis that atrial natriuretic peptide has direct antihypertrophic actions on the heart by modulating expression of genes involved in cardiac hypertrophy and extracellular matrix production. Hearts of male, atrial natriuretic peptide-null and control wild-type mice that had been subjected to pressure overload after transverse aortic constriction and control unoperated hearts were weighed and subjected to microarray, Northern blot, and immunohistochemical analyses. Microarray and Northern blot analyses were used to identify genes that are regulated differentially in response to stress in the presence and absence of atrial natriuretic peptide. Immunohistochemical analysis was used to identify and localize expression of the protein products of these genes. Atrial natriuretic peptide-null mice demonstrated cardiac hypertrophy at baseline and an exaggerated hypertrophic response to transverse aortic constriction associated with increased expression of the extracellular matrix molecules periostin, osteopontin, collagen I and III, and thrombospondin, as well as the extracellular matrix regulatory proteins, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3, and the novel growth factor pleiotrophin compared with wild-type controls. These results support the hypothesis that atrial natriuretic peptide protects against pressure overload-induced cardiac hypertrophy and remodeling by negative modulation of genes involved in extracellular matrix deposition. Key Words: atrial natriuretic factor Ⅲ constriction Ⅲ aorta Ⅲ pressure overload Ⅲ hypertrophy, cardiac Ⅲ extracellular matrix Ⅲ growth substances A trial natriuretic peptide (ANP) inhibits cell growth and proliferation and induces apoptosis in a variety of cell types, including vascular smooth muscle cells (VSMCs) and cardiomyocytes. 1-3 Intracardiac ANP might also play an important autocrine/paracrine role in modulating cardiac remodeling under stress conditions and might protect against the development of pathologic cardiac hypertrophy. 4 -7 Synthesis and release of ANP in the heart are increased under stressful conditions such as pressure and volume overloadinduced pathologic cardiac hypertrophy, exercise-induced physiologic cardiac hypertrophy, heart failure, and hypoxic pulmonary hypertension. 1,2 Expression of ANP is inversely related to cardiac growth/hypertrophy. 5,8 -11 Transgenic mice overexpressing ANP have smaller hearts than do wild-type mice, and ANP gene delivery attenuates cardiac hypertrophy in spontaneously hypertensive rats. 5,7 Conversely, transgenic mice with homozygous disruption of the pro-ANP gene (Nppa -/-mice) or the natriuretic peptide receptor-A (NPR-A) gene (Npr1 -/-mice) exhibit cardiac hypertrophy at baseline that is out of proportion to the modest elevations in blood pressure (BP) observed in these models. 8 -11 Furthermore, in Npr1 -/-mice, pressure overload induced by transverse aortic constriction (TAC) results in a greater (55%) increase in left ventricular (LV) weight than in Np...

Background-We have previously shown that estrogen (17␤-estradiol; E2) inhibits neointima formation and migration of leukocytes, particularly neutrophils, into rat carotid arteries after acute endoluminal injury. This study tested the hypothesis that E2 inhibits expression of adhesion molecules, chemokines, and proinflammatory cytokines in rat carotid arteries in the early hours after balloon injury, thus attenuating the stimulus for leukocyte entry and negatively modulating the injury response. Methods and Results-Ovariectomized (OVX) rats were randomly assigned to treatment with E2 or vehicle (V) and subjected to balloon injury of the right carotid artery. After 2, 6, and 24 hours, rats were euthanized, and both carotid arteries were processed for real-time reverse transcription-polymerase chain reaction (2 and 24 hours), ELISA (6 hours), or neutrophil chemotaxis assay (24 hours). Expression of mRNA for adhesion molecules (P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1), chemoattractants (cytokine-induced neutrophil chemoattractant [CINC]-2␤ and monocyte chemoattractant protein [MCP]-1), and proinflammatory cytokines (interleukin [IL]-1 and IL-6) was markedly increased (2 to 5000 times) in injured arteries of OVXϩV rats at 2 hours and was reduced by 24 hours. E2 significantly attenuated expression of the proinflammatory mediators (by 60% to 80%) at 2 hours. ELISA confirmed injury-induced upregulation of neutrophil and monocyte/macrophage chemoattractants (CINC-2␣, MCP-1) in OVXϩV arteries and E2-induced inhibition of CINC-2␣ expression. E2 significantly (by 65%) inhibited neutrophil chemotactic activity of arterial homogenates. Conclusions-E2 attenuates the early vascular injury response, at least in part, by negatively modulating proinflammatory mediator expression and the resultant chemotactic activity of injured vessels for neutrophils.

To test the hypothesis that endothelin (ET)-1 synthesis and ET receptor levels are increased selectively in the lung of rats with chronic hypoxic pulmonary hypertension, the current study examined the effects of exposure to chronic hypoxia (10% O2, 1 atm, 4 wk) on pulmonary arterial pressure, ET-1 levels in plasma and lung, and ET-1 and ETA and ETB receptor mRNA levels in lung, heart, pulmonary artery, aorta, kidney, spleen, and liver. Hypoxic exposure was associated with increases in pulmonary arterial pressure, plasma ET-1 levels, ET-1 mRNA in lung and pulmonary artery, and ET-1 stores and ETA and ETB receptor mRNA levels in lung. In thoracic aorta and the four heart chambers, ETA and ETB receptor mRNA levels were increased, but ET-1 mRNA levels were unchanged from air control levels. No change in ET-1 or ET receptor mRNA levels was seen in organs perfused by the systemic vascular bed, except in liver, where ETA receptor mRNA levels were decreased. The findings of concomitant increases in gene transcript levels for ET-1 and the ETA and ETB receptors in lung, but not in the great vessels or any other organ examined, are consistent with the hypothesis that increased ET-1 synthesis in the lung contributes to pulmonary vascular remodeling and the maintenance of chronic hypoxic pulmonary hypertension.