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Phosphodiesterase 5 inhibition (PDE5i) activates cGMP-dependent protein kinase (PKG) and ameliorates heart failure; however, its impact on cardiac mitochondrial regulation has not been fully determined. Here, we investigated the role of the mitochondrial regulator peroxisome proliferator-activated receptor γ co-activator-1α (PGC1α) in the PDE5i-conferred cardioprotection, utilizing PGC1α null mice. In PGC1α(+/+) hearts exposed to 7 weeks of pressure overload by transverse aortic constriction, chronic treatment with the PDE5 inhibitor sildenafil improved cardiac function and remodeling, with improved mitochondrial respiration and upregulation of PGC1α mRNA in the myocardium. By contrast, PDE5i-elicited benefits were abrogated in PGC1α(−/−) hearts. In cultured cardiomyocytes, PKG overexpression induced PGC1α, while inhibition of the transcription factor CREB abrogated the PGC1α induction. Together, these results suggest that the PKG–PGC1α axis plays a pivotal role in the therapeutic efficacy of PDE5i in heart failure.

Background: Augmentation of NP (natriuretic peptide) receptor and cyclic guanosine monophosphate (cGMP) signaling has emerged as a therapeutic strategy in heart failure (HF). cGMP-specific PDE9 (phosphodiesterase 9) inhibition increases cGMP signaling and attenuates stress-induced hypertrophic heart disease in preclinical studies. A novel cGMP-specific PDE9 inhibitor, CRD-733, is currently being advanced in human clinical studies. Here, we explore the effects of chronic PDE9 inhibition with CRD-733 in the mouse transverse aortic constriction pressure overload HF model. Methods: Adult male C57BL/6J mice were subjected to transverse aortic constriction and developed significant left ventricular (LV) hypertrophy after 7 days ( P Results: CRD-733 treatment reversed existing LV hypertrophy compared with vehicle ( P P =0.009), and attenuated left atrial dilation ( P P =0.037) compared with vehicle, while lung weights, a surrogate for pulmonary edema, were reduced to sham levels. Chronic CRD-733 treatment increased plasma cGMP levels compared with vehicle ( P 273 of cardiac myosin binding protein-C, a cGMP-dependent protein kinase I phosphorylation site. Conclusions: The PDE9 inhibitor, CRD-733, improves key hallmarks of HF including LV hypertrophy, LV dysfunction, left atrial dilation, and pulmonary edema after pressure overload in the mouse transverse aortic constriction HF model. Additionally, elevated plasma cGMP may be used as a biomarker of target engagement. These findings support future investigation into the therapeutic potential of CRD-733 in human HF.

Introduction: Regulator of G protein signaling 2 (RGS2) is a negative regulator of G protein-coupled signaling, and is potentiated by cGMP-PKG. Our prior work using a global deletion model demonstrated the protective role for RGS2 against mal-adaptive cardiac remodeling, which is essential to the cGMP-PDE5 inhibitor efficacy; however, cell type-specific contribution remains to be determined, given the expression of RGS2 in myocytes, fibroblasts and vascular smooth muscle cells. We hypothesized that myocyte RGS2 might serve as a primary contributor to the cardio-protection and mediates cGMP-PKG benefits. Method: We generated cardiomyocyte-specific RGS2 knockout mice (cKO) on a C57BL/6 background. Pressure overload was produced by transverse aortic constriction (TAC) in male RGS2-cKO mice (RGS2cKO) and their littermate controls (WT). To investigate cGMP-PKG enhancing effects, a soluble guanylyl cyclase stimulator (riociguat) was orally administrated daily (3mg/kg/day). Invasive hemodynamics, anatomy, histology and molecular signaling were assessed at 1wk after TAC. Results: RGS2-cKO revealed exacerbated dilative cardiac remodeling (LVDD: cKO 3.57±0.36 vs WT 2.90±0.16) and severely impaired cardiac function (dPdt/IP: cKO 166±3.49 vs WT 195±7.40), while their basal phenotype was undistinguishable from WT. cKO-TAC hearts showed larger cardiomyocyte size and more fibrosis than WT-TAC hearts, associated with aberrant gene expression induction including Myh6, Myh7, Col1a2, and TGFβ . Intriguingly, concomitant riociguat treatment reduced LV chamber size and fibrosis but failed to improve cardiac performance in cKO-TAC hearts, while riociguat ameliorated all these in WT-TAC hearts. Conlusion: These results suggest that RGS2-PKG axis in cardiac myocytes critically contributes to cardiac functional performance and that cGMP-PKG exerts anti-fibrotic effects independently of myocyte conditions.

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone’s involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS–dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

Hypertensive heart disease causes significant mortality in older patients, yet there is an incomplete understanding of molecular mechanisms that regulate age-dependent hypertensive left ventricular hypertrophy (LVH). Therefore, we tested the hypothesis that the cGMP-dependent protein kinase G I alpha (PKGIα) attenuates hypertensive LVH by evaluating the cardiac phenotype in mice with selective mutations of the PKGIα leucine zipper domain. These leucine zipper mutant (LZM) mice develop basal hypertension. Compared with wild-type controls, 8-month-old adult LZM mice developed increased left ventricular end-diastolic pressure but without frank LVH. In advanced age (15 months), the LZM mice developed overt pathological LVH. These findings reveal a role of PKGIα in normally attenuating hypertensive LVH. Therefore, mutation of the PKGIα LZ domain produces a clinically relevant model for hypertensive heart disease of aging.

Hypertension is a cardiovascular risk factor present in over two-thirds of people over age 60 in North America; elevated blood pressure correlates with increased risk of heart attack, stroke and progression to heart and kidney failure. Current therapies are insufficient to control blood pressure in almost half of these patients. The mineralocorticoid receptor (MR), acting in the kidney, is known to regulate blood pressure through aldosterone binding and stimulation of sodium retention. However, recent studies support the concept that the MR also has extrarenal actions and that defects in sodium handling alone do not fully explain the development of hypertension and associated cardiovascular mortality. We and others have identified functional MR in human vascular smooth muscle cells (SMCs), suggesting that vascular MR might directly regulate blood pressure. Here we show that mice with SMC-specific deficiency of the MR have decreased blood pressure as they age without defects in renal sodium handling or vascular structure. Aged mice lacking MR in SMCs (SMC-MR) have reduced vascular myogenic tone, agonist-dependent contraction and expression and activity of L-type calcium channels. Moreover, SMC-MR contributes to angiotensin II–induced vascular oxidative stress, vascular contraction and hypertension. This study identifies a new role for vascular MR in blood pressure control and in vascular aging and supports the emerging hypothesis that vascular tone contributes directly to systemic blood pressure.

Objective— Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease. Methods and Results— Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. Quantitative reverse transcription–polymerase chain reaction was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared with the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis. Conclusion— This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress–dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease, and these pathways may provide novel drug targets to prevent atherosclerosis in humans.

Objective— Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action. Methods and Results— We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by ≈75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by ≈45%. TLR4 deficiency prevented the increased interleukin-1α (IL-1α) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1α levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner. Conclusion— TLR4 contributes to early-stage intimal foam cell accumulation at lesion-prone aortic sites in ApoE KO mice, as does TLR2 to a lesser extent. Intimal SMC surround and penetrate early lesions, where TLR4 signaling within them may influence lesion progression.

G protein-coupled receptors (GPCRs) have key roles in cardiovascular regulation and are important targets for the treatment of hypertension. GTPase-activating proteins, such as RGS2, modulate downstream signaling by GPCRs. RGS2 displays regulatory selectivity for the Gαq subclass of G proteins, and mice lacking RGS2 develop hypertension through incompletely understood mechanisms. Using total body RGS2-deficient mice, we used a kidney crosstransplantation strategy to examine separately the contributions of RGS2 actions in the kidney from those in extrarenal tissues with regard to BP regulation. Loss of renal RGS2 was sufficient to cause hypertension, whereas the absence of RGS2 from all extrarenal tissues including the peripheral vasculature did not significantly alter BP. Accordingly, these results suggest that RGS2 acts within the kidney to modulate BP and prevent hypertension. These data support a critical role for the renal epithelium and/or vasculature as the final determinants of the intra-arterial pressure in hypertension.

The higher incidence of cardiovascular disease in men than in women of similar age, and the menopause-associated increase in cardiovascular disease in women, has led to speculation that gender-related differences in sex hormones have a key role in the development and evolution of cardiovascular disease. Compelling data have indicated that sex differences in vascular biology are determined not only by gender-related differences in sex steroid levels, but also by gender-specific tissue and cellular differences that mediate sex-specific responses. In this Review, we describe the sex-specific effects of estrogen and testosterone on cardiovascular risk, direct vascular effects of these sex hormones, and how these effects influence development of atherosclerosis. Cardiovascular effects of exogenous hormone administration are also discussed. Importantly, evidence has indicated that estrogens alone or in combination with progestins in postmenopausal women increase cardiovascular risk if started late after menopause, but that it possibly has beneficial cardiovascular effects in younger postmenopausal women, although data on long-term testosterone therapy are lacking. Hormone therapy should not be considered solely for primary prevention or treatment of cardiovascular disease at this time.

available in PMC 2010 April 1., Increased arterial stiffness and wave reflection have been identified as cardiovascular disease risk factors. In light of significant sex differences and the moderate heritability of vascular function measures, we hypothesized that variation in the genes coding for oestrogen receptors α (ESR1) and β (ESR2) and aromatase (CYP19A1) is associated with aortic stiffness and pressure wave reflection as measured by non-invasive arterial tonometry. In all, 1261 unrelated Framingham Offspring Study participants who attended the seventh examination cycle (mean age 62±10 years, 52% women) and had arterial tonometry and genotyping data were included in the study. Analysis of covariance was used to assess the association of polymorphisms with forward wave amplitude, augmented pressure, augmentation index (AI), carotid–femoral pulse wave velocity and mean arterial pressure with adjustment for potential confounders. In the sex-pooled analysis, those homozygous for the minor allele at any of four ESR1 variants that were in strong linkage disequilibrium ((TA)n, rs2077647, rs2234693 and rs9340799) had on an average 18% higher augmented pressure and 16% greater AI compared with carriers of one or two major alleles (P=0.0002–0.01). A similar magnitude of association was detected in those homozygous for the common allele at two ESR2 single-nucleotide polymorphisms (P=0.007–0.02). Our results are consistent with the hypothesis that variation in ESR1 and ESR2, but not CYP19A1, is associated with an increased wave reflection that may contribute to associations between these variants and adverse clinical events demonstrated earlier. Our findings will need to be replicated in additional cohorts.

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.

Cardiovascular disease is the leading cause of mortality for both men and women in developed countries. The sex steroid hormone estrogen is required for normal vascular physiology. Estrogen functions by binding to intracellular estrogen receptors (ER), ERalpha and ERbeta, ligand-activated transcription factors that are expressed in both vascular endothelial and smooth muscle cells. We recently demonstrated that long-term (8 d) estrogen treatment in vivo in mice recruits distinct vascular gene sets mediated by ERalpha and ERbeta and that the promoters from these gene sets are enriched for binding sites of specific transcription factors, leading to the hypothesis that estrogen initiates a cascade of early transcriptional events that modulate gene expression in the vasculature. Here we test this hypothesis using gene expression profiling to examine initial transcriptional events (2-8 h) mediated by estrogen in blood vessels. Our data reveal that 1) estrogen regulates temporally distinct cascades of vascular gene expression, 2) initially, estrogen-mediated vascular gene repression predominates, 3) the earliest estrogen-recruited gene program is enriched in vascular transcription factors that can interact with binding sites present in estrogen-regulated vascular genes recruited subsequently, and 4) estrogen-regulated genes recruited next have specific functions, including lipid metabolism and cellular growth and proliferation that are potentially important for estrogen's known vascular functions. In summary, estrogen directly and rapidly recruits specific transcriptional factors that then propagate distinct cascades of gene expression. These data define the temporal recruitment of specific vascular genes by estrogen and enable further analysis of the mechanisms by which estrogen directly regulates vascular function.

Estrogen has both rapid and longer term direct effects on cardiovascular tissues mediated by the two estrogen receptors, ESR1 and ESR2. Previous work identified that estrogen regulates the expression of inducible nitric oxide synthase (NOS2A) in vascular smooth muscle cells (VSMC). ESR2 knockout mice have vascular dysfunction due to dysregulation of NOS2A expression and these mice are hypertensive (Zhu et al. Science 2002 295 505–508). Here, we report studies to examine the differential regulation of NOS2A gene expression by ESR1 and 2. Immunoblotting and RT-PCR studies revealed that different VSMC lines expressed different levels of ESR1 and ESR2 protein and mRNA. VSMC from different vascular beds were studied, including aortic VSMC expressing ESR1 and radial (Rad) VSMC expressing ESR2. E2 inhibited NO production and NOS2A protein expression in aortic VSMC. Human NOS2A promoter–reporter studies revealed suppression of NOS2A reporter activity by E2 in aortic VSMC, and stimulation of NOS2A reporter activity by E2 in Rad arterial VSMC. In heterologous expression studies of COS-7 cells lacking endogenous ER, E2 treatment of COS-7 cells did not alter NOS2A reporter activity in the presence of ESR1, while reporter activity increased 2.3-fold in the presence of ESR2. Similar experiments in COS-7 cells using the selective estrogen receptor modulator raloxifene showed that raloxifene caused a reduction in NOS2A reporter activity with ESR1 coexpression and an increase with ESR2 coexpression. Rat VSMC expressing ESR2 but not ESR1 also showed increased NOS2A reporter activity with E2 treatment, an effect lost when ESR1 was introduced into the cells. Taken together, these data support that hNOS2A transcription is regulated positively by ESR2 and negatively by ESR1 in VSMC, supporting differential actions of these two estrogen receptors on a physiologically relevant gene in VSMC.

Younger age at the onset of menopause and lower circulating levels of estrogen are risk factors for cardiovascular disease. Several studies have detected associations between variations in genes encoding estrogen receptors alpha (ESR1) and beta (ESR2), and enzyme aromatase (CYP19A1), which regulates the estrogen to testosterone ratio, and cardiovascular phenotypes in the Framingham Heart Study. To explore potential mechanisms by which these gene variants may contribute to cardiovascular disease, we tested the hypothesis that the polymorphisms were associated with endogenous steroid hormone levels.Multiple regression analysis was used to assess the relation between reported polymorphisms and total serum estradiol, testosterone, and dehydroepiandrosterone sulfate levels in 834 men and 687 women who attended the third and fourth Framingham Heart Study examination cycles.In men, significant associations were detected between CYP19A1 polymorphisms and estradiol and testosterone levels, and the estradiol to testosterone ratio (P ranges 0.0005-0.01). Specifically, carriers of common haplotype rs700518[G]-(TTTA)(n) [L]-rs726547[C] had higher estradiol levels (5% per copy; P = 0.0004), lower testosterone levels (17% per copy; P = 0.036), and a higher estradiol to testosterone ratio (24% per copy; P0.0001) compared with the rs700518[A]-(TTTA)(n) [S]-rs726547[C] carriers. In addition, postmenopausal carriers of the ESR2 (CA)(n) long allele and rs1256031 [C] allele had moderately higher estradiol levels (Por = 0.03). No significant associations with the ESR1 variants were detected.Our findings suggest that variations in CYP19A1 correlate with steroid hormone levels in men. Knowledge that a specific carrier status may predispose to altered steroid hormone levels may lead to targeted intervention strategies to reduce health risks in genetically susceptible individuals.

Hypertension, a major cardiovascular risk factor and cause of mortality worldwide, is thought to arise from primary renal abnormalities. However, the etiology of most cases of hypertension remains unexplained. Vascular tone, an important determinant of blood pressure, is regulated by nitric oxide, which causes vascular relaxation by increasing intracellular cGMP and activating cGMP-dependent protein kinase I (PKGI). Here we show that mice with a selective mutation in the N-terminal protein interaction domain of PKGIα display inherited vascular smooth muscle cell abnormalities of contraction, abnormal relaxation of large and resistance blood vessels, and increased systemic blood pressure. Renal function studies and responses to changes in dietary sodium in the PKGIα mutant mice are normal. These data reveal that PKGIα is required for normal VSMC physiology and support the idea that high blood pressure can arise from a primary abnormality of vascular smooth muscle cell contractile regulation, suggesting a new approach to the diagnosis and therapy of hypertension and cardiovascular diseases.

Genetic polymorphisms that influence smooth muscle cell contraction and relaxation may affect the response to anti-hypertensive therapy. Our finding of lower blood pressure (BP) in the setting of treatment for hypertension, particularly with beta-blockade, in Caucasian KCNMB1-E65K-carriers in two community cohorts has implications for personalization of therapy in hypertension.

Background We have shown previously that 17β-estradiol (E2) increases left ventricular (LV) and cardiomyocyte hypertrophy after myocardial infarction (MI). However, E2 decreases hypertrophy in pressure overload models. We hypothesized that the effect of estrogen on cardiac hypertrophy was dependent on the type of hypertrophic stimulus. Methods and Results Ovariectomized wild-type female mice (n = 192) were given vehicle or E2 treatment followed by coronary ligation (MI), transverse aortic constriction (TAC), or sham operation. Signaling pathway activation was studied at 3, 24, and 48 hours, whereas echocardiography and hemodynamic studies were performed at 14 days. MI induced early but transient activation of p38 and p42/44 MAPK pathways, whereas TAC induced sustained activation of both pathways. E2 had no effect on these pathways, but increased Stat3 activation after MI while decreasing Stat3 activation after TAC. MI caused LV dilation and decreased fractional shortening (FS) that were unaltered by E2. TAC caused LV dilation, reduced FS, and increased LV mass, but in this model, E2 improved these parameters. After MI, E2 led to increases in myocyte cross-sectional area, atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) gene expression, but E2 diminished TAC-induced increases ANP and β-MHC gene expression. Conclusions These data demonstrate that the effects of E2 on LV and myocyte remodeling depend on the nature of the hypertrophic stimulus. The opposing influence of E2 on hypertrophy in these models may, in part, result from differential effects of E2 on Stat3 activation. Further work will be necessary to explore this and other potential mechanisms by which estrogen affects hypertrophy in these models.

Estrogen plays an important role in the regulation of vascular tone and in the pathophysiology of cardiovascular disease. Physiological effects of estrogen are mediated through estrogen receptors alpha (ERalpha) and beta (ERbeta), which are both expressed in vascular smooth muscle and endothelial cells. However, the molecular pathways mediating estrogen effects in blood vessels are not well defined. We have performed gene expression profiling in the mouse aorta to identify comprehensive gene sets the expression of which is regulated by long-term (1 wk) estrogen treatment. The ER subtype dependence of the alterations in gene expression was characterized by parallel gene expression profiling experiments in ERalpha-deficient [ERalpha knockout (ERalphaKO)] and ERbeta-deficient (ERbetaKO) mice. Importantly, these data revealed that ERalpha- and ERbeta-dependent pathways regulate distinct and largely nonoverlapping sets of genes. Whereas ERalpha is essential for most of the estrogen-mediated increase in gene expression in wild-type aortas, ERbeta mediates the large majority (nearly 90%) of estrogen-mediated decreases in gene expression. Biological functions of the estrogen-regulated genes include extracellular matrix synthesis, in addition to electron transport in the mitochondrion and reactive oxygen species pathways. Of note, the estrogen/ERbeta pathway mediates down-regulation of mRNAs for nuclear-encoded subunits in each of the major complexes of the mitochondrial respiratory chain. Several estrogen-regulated genes also encode transcription factors. Computational analysis of promoters from coexpressed genes revealed overrepresentation of binding sites for such factors, lending support for an estrogen-regulatory transcriptional network in the vasculature. Overall, these findings provide a foundation for understanding the molecular basis for estrogen effects on vasculature gene expression.

Left ventricular (LV) mass and other LV measures have been shown to be heritable. In this study we hypothesized that functional variation in the gene coding for estrogen receptor-α ( ESR1 ), known for mediating the effect of estrogens on myocardium, is associated with age-related changes in LV structure. Four genetic markers ( ESR1 TA repeat; rs2077647 , or + 30T > C ; rs2234693 , or Pvu II; and rs9340799 , or Xba I) were genotyped in 847 unrelated individuals (488 women) from the Framingham Offspring Study, who attended 2 examination cycles 16 years apart (mean ages at first examination: 43±9 years; at follow-up: 59±9 years). ANCOVA was used to assess the association of polymorphisms and their haplotypes with cross-sectional measurements and longitudinal changes in LV mass, wall thickness, end-diastolic and end-systolic internal diameter, and fractional shortening after adjustment for factors known to influence these variables. Changes over time were detected for all of the LV measurements ( P ranging from SS genotype of the ESR1 TA repeat polymorphism in the promoter region was associated with longitudinal changes in LV mass and LV wall thickness ( P ranging from 0.0006 to 0.01). Moreover, the TA [ S ]– +30 [ T ]– Pvu II[ T ]– Xba I[ A ] haplotype (frequency: 47.5%) was associated with greater LV changes as compared with the TA [ L ] –+30 [ C ]– Pvu II[ C ]– Xba I[ G ] haplotype (frequency: 31.8%). Our results are consistent with the hypothesis that common ESR1 polymorphisms are significantly associated with age-related changes in LV structure. Understanding the mechanisms predisposing to unfavorable LV remodeling of the heart with advancing age may aid in the discovery of new therapeutic targets for the prevention of heart failure.

Objective— Clinical studies demonstrate that mineralocorticoid receptor (MR) antagonism improves outcomes in cardiovascular patients and that vascular calcification correlates with adverse cardiac events. We have recently demonstrated that human vascular smooth muscle cells (VSMCs) express functional MRs that, in response to aldosterone, modulate expression of osteogenic genes including alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP2). This study examines the effects of MR activation by aldosterone on the process of in vitro vascular calcification. Methods and Results— Using immunoblotting and adenoviral promoter-reporter assays, we demonstrated that calcifying vascular cells (CVCs), an in vitro model of vascular calcification, express MRs that mediate both aldosterone- and cortisol-stimulated gene transcription. In this model, aldosterone stimulated ALP activity, an early marker of osteoblastic differentiation, as well as mineralization. Aldosterone antagonism with spironolactone abolished both effects implicating CVC MRs in the mechanism of aldosterone-stimulated vascular calcification. Inhibition of BMP2 signaling by overexpression of dominant negative BMP2 receptor did not attenuate aldosterone-induced osteoblastic differentiation. Conclusions— Aldosterone activation of MR promotes osteoblastic differentiation and mineralization of VSMCs independent of BMP2 signaling. These data provide a mechanistic link between hormone-mediated VSMC MR activation and vascular calcification, two processes associated with increased risk of cardiovascular ischemic events in humans.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) constitutes a key event in atherosclerosis, neointimal hyperplasia, and the response to vascular injury. Estrogen receptor α (ERα) mediates the protective effects of estrogen in injured blood vessels and regulates ligand-dependent gene expression in vascular cells. However, the molecular mechanisms mediating ERα-dependent VSMC gene expression and VSMC proliferation after vascular injury are not well defined. Here, we report that the ER coactivator steroid receptor coactivator 3 (SRC3) is also a coactivator for the major VSMC transcription factor myocardin, which is required for VSMC differentiation to the nonproliferative, contractile state. The N terminus of SRC3, which contains a basic helix-loop-helix/Per-ARNT-Sim protein–protein interaction domain, binds the C-terminal activation domain of myocardin and enhances myocardin-mediated transcriptional activation of VSMC-specific, CArG-containing promoters, including the VSMC-specific genes SM22 and myosin heavy chain. Suppression of endogenous SRC3 expression by specific small interfering RNA attenuates myocardin transcriptional activation in cultured cells. The SRC3–myocardin interaction identifies a site of convergence for nuclear hormone receptor-mediated and VSMC-specific gene regulation and suggests a possible mechanism for the vascular protective effects of estrogen on vascular injury.

The retinoic acid receptors (RARs) are ligand-dependent transcription factors that play critical roles in cell differentiation, embryonic development, and tumor suppression. RAR transcriptional activities are mediated by a growing family of nuclear receptor (NR) coregulators. Here we report the cloning and characterization of a novel protein RIF1 (receptor interacting factor) that interacts with RARalpha in vivo and in vitro. RIF1 encodes a novel 739 amino acid protein that is ubiquitously expressed in a variety of tissues and cell lines. GST-pull down assays show that RIF1 also interacts with a number of other NRs. The interaction domain of RIF1 for RARalpha is located at the C-terminal region of RIF1, between amino acids 512 and 674. RIF1 is localized exclusively in the cell nucleus and specifically to the nuclear matrix. Mutation of the nuclear localization signal abolishes this nuclear localization and causes RIF1 to appear in the cytoplasm. Co-transfection of RIF1 with RAR causes RAR to localize to the nuclear matrix. RIF1 contains a strong transcriptional repression domain that robustly inhibits ligand-dependent transcriptional activation by RARalpha. This domain is located to the distal C-terminal 100 amino acids, distinct from the RARalpha-interaction and nuclear matrix-targeting domains. The transcriptional repression activity of RIF1 is mediated at least in part through direct recruitment of histone deacetylases. This study identifies RIF1 as a novel nuclear matrix transcription repressor, and suggests a potential role of RIF1 that regulates NR transcriptional activity.

Background: Heart failure is a significant contributor to cardiovascular mortality. cGMP inhibits cardiac remodeling in part via cGMP-dependent protein kinase G I α (PKGIα). Knock in mice with a mu...

Plasma lipid profile is affected by endogenous estrogen levels and hormone replacement therapy (HRT). As plasma lipid concentrations have a significant heritable basis and the effects of both endogenous estrogen and use of HRT are mediated by estrogen receptors, we sought to investigate the relationships between polymorphisms in estrogen receptor-α ( ESR1 ) and plasma lipid and lipoprotein concentrations. We analyzed data from 854 women (mean age 52±10 years) from the Framingham Heart Study. A TA repeat in the promoter region, c.30T > C in exon 1, c.454-397T > C , and c.454-351A > G in intron 1, all in linkage disequilibrium (LD), were significantly associated with low-density lipoprotein (LDL) particle size and concentration of small LDL particles. Women with the c.454-397C allele had larger LDL particle size (21.09±0.02nm versus 21.01±0.03nm, p =0.021) concurrent with lower small LDL particle concentration (0.47±0.02mmol/L versus 0.58±0.03mmol/L, p =0.008). Moreover, the TA[L] – c.30C – c.454-397C – c.454-351G haplotype (frequency, 32%) was associated with lower small LDL particle concentrations (−0.06±0.03mmol/L change associated with each copy of this haplotype, p =0.011) when compared to the TA[S] – c.30T – c.454-397T – c.454-351A haplotype (frequency, 46%), where L and S are long and short TA repeats. Our results suggest that common ESR1 polymorphisms have a significant effect on lipoprotein metabolism in women.

Background Left ventricular (LV) hypertrophy is a significant risk factor for cardiovascular disease. Given sex-based differences in cardiac structure and remodeling, we hypothesized that variation in estrogen pathway genes might be associated with alteration of LV structure. Methods We studied 1249 unrelated individuals, 547 men and 702 women (mean age 59 years) from the Framingham Heart Study. Eight single nucleotide polymorphisms in the genes for estrogen receptor α and estrogen receptor β (ESR2) were tested for association with 5 LV measures: LV mass (LVM), LV wall thickness (LVWT), LV internal diameter at end-diastole and end-systole, and fractional shortening. Sex-specific multiple regression analyses were performed adjusting for age, weight, height, systolic and diastolic blood pressure, hypertension treatment, diabetes, and in women, menopausal status. Results In men, there was no evidence of association between the estrogen pathway polymorphisms tested and LV structure or function. In women, however, two polymorphisms, ESR2 rs1256031 and ESR2 rs1256059, in linkage disequilibrium with one another, were associated with LVM and LVWT (P = .0007 to .03); the association was most pronounced in those women with hypertension (P = .0006 to .01). The association did not appear to be explained by variation in blood pressure, plasma lipoprotein levels, or hyperglycemia. Conclusions The ESR2 polymorphisms are associated with LV structural differences in women with hypertension in a community-based population. These data are consistent with the hypothesis that genetic factors may mediate part of the observed sex-based differences in LV structure and remodeling.

High levels of small low density lipoprotein (LDL) particles are a major risk factor for cardiovascular morbidity and mortality. Both estrogens and smoking, with known anti-estrogenic effects, alter the atherogenic lipid profile. We tested for a role of interaction between smoking and estrogen receptor alpha gene (ESR1) variation in association with plasma concentration of atherogenic small LDL particles and LDL particle size. We studied 1727 unrelated subjects, 854 women and 873 men, mean age 51 years (SD 10), from the population-based Framingham Heart Study. After covariate adjustment, women who smoked and had the common ESR1 c.454-397 TT genotype (in 30% of women, T was present on both chromosomes at position 397 prior to the start of exon 2) had >1.7-fold higher levels of small LDL particles than women with the alternative genotypes (P-value for smoking-genotype interaction was 0.001). Similar results were obtained for three other ESR1 variants including c.454-351A > G, in the same linkage disequilibrium block. A similar substantial gender-specific result was also evident with a fifth variant, in a separate linkage disequilibrium block, in exon 4 (P = 0.003). Women who smoked and had specific, common ESR1 genotypes had a substantially higher plasma concentration of atherogenic small LDL particles. Significant results revealed a dose-dependent effect of smoking and were evident in both pre- and postmenopausal women. The reported association has the potential to explain the risks associated with estrogen use in certain women and a recent report of association between an ESR1 haplotype comprised of c.454-397 T and c.454-351 A alleles with increased myocardial infarction and ischaemic heart disease, independent of the standard, established cardiovascular risk factors.

Cardiovascular diseases (CVDs), the major cause of morbidity and mortality for both men and women, occur uncommonly in premenopausal women, but their incidence rises sharply after the menopausal transition. Cardiovascular gender differences are apparent long before CVDs appear in men and women, and improved understanding of the biology underlying these differences has the potential to advance the diagnosis and treatment of CVDs in both sexes. This review considers gender differences in the molecular and cellular physiology of the heart and blood vessels in health and disease, highlighting understudied areas that can help resolve the current controversy regarding hormone replacement therapy and improve cardiovascular health in women.

Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate gene expression. SHRs also mediate rapid, nongenomic cellular activation by steroids. In vascular endothelial cells, the SHR for estrogen, estrogen receptor (ER) alpha, is targeted by unknown mechanisms to a functional signaling module in membrane caveolae that enables estrogen to rapidly activate the mitogen-activated protein kinase and phosphatidylinositol 3-Akt kinase pathways, and endothelial NO synthase (eNOS). Here we identify the 110-kDa caveolin-binding protein striatin as the molecular anchor that localizes ERalpha to the membrane and organizes the ERalpha-eNOS membrane signaling complex. Striatin directly binds to amino acids 183-253 of ERalpha, targets ERalpha to the cell membrane, and serves as a scaffold for the formation of an ERalpha-Galphai complex. Disruption of complex formation between ERalpha and striatin blocks estrogen-induced rapid activation mitogen-activated protein kinase, Akt kinase, and eNOS, but has no effect on ER-dependent regulation of an estrogen response element-driven reporter plasmid. These findings identify striatin as a molecular scaffold required for rapid, nongenomic estrogen-mediated activation of downstream signaling pathways. Furthermore, by demonstrating independent regulation of nongenomic vs. genomic ER-dependent signaling, these findings provide conceptual support for the potential development of "pathway-specific" selective ER modulators.

The Women's Health Initiative (WHI) randomized controlled trial failed to show cardioprotection by estrogen plus progestin treatment of postmenopausal women. But by design, the WHI population was 10-fold underpowered to show cardioprotection of women starting hormone treatment during the menopausal transition. Thus, observational studies that showed cardioprotection in such women remain the only applicable clinical guide to this issue. Randomized controlled trials are urgently needed to test cardioprotection in women starting treatment during the menopausal transition.

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-alpha (PKGI-alpha), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-alpha attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-alpha binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G(q), terminating PAR-1 signaling. Disruption of the RGS-2-PKGI-alpha interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2-/- mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-alpha binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.

Endothelial nitric oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. Heat shock protein 90 (HSP90) binds directly to eNOS, augmenting NO production. We have used purified proteins to characterize further the mechanism by which HSP90 increases eNOS activity at low (100 nm) and high (10 microm) Ca(2+) levels. In the presence of calmodulin (CaM), HSP90 increased eNOS activity dose dependently at both low and high Ca(2+) concentrations. This effect was abolished by the specific HSP90 inhibitor geldanamycin (GA) at both calcium concentrations. The EC(50) values of eNOS for both Ca(2+) and CaM were decreased in the presence of HSP90. HSP90 also significantly increased the rate of NADPH-dependent cytochrome c reduction by eNOS at both low and high Ca(2+) concentrations. HSP90 bound to eNOS in a dose-dependent manner, and the amount of bound HSP90 also increased with increasing Ca(2+)/CaM. At 100 nm Ca(2+), HSP90 promoted dose-dependent CaM binding to eNOS that was fully inhibitable by GA. At high calcium, HSP90 did not affect CaM binding to eNOS, but GA inhibited HSP90 binding to eNOS. At high Ca(2+), HSP90 caused the V(max) of eNOS for l-arginine to increase by 2-fold, but the K(m) of eNOS was unchanged. HSP90 bound preferentially to CaM-prebound eNOS and significantly increased both its NO synthesis and reductase activities. These data support that HSP90 promotes eNOS activity by two mechanisms: (i) a CaM-dependent mechanism operative at low Ca(2+) concentrations, characterized by an increase in the affinity of eNOS for CaM and (ii) a CaM-independent mechanism apparent at high Ca(2+) concentrations, characterized by stimulation of eNOS reductase activity without further change in CaM binding. These studies contribute to our understanding of eNOS activation by HSP90 and provide a basis for in vitro studies of other eNOS-interacting proteins.

Left ventricular (LV) remodelling following myocardial infarction (MI) is associated with increased morbidity and mortality. Previous data suggest that angiotensin II (Ang II) plays a central role in the molecular events contributing to LV remodelling. We explored the effects of angiotensin-converting-enzyme (ACE) inhibition versus Ang II (AT1) receptor blockade on LV remodelling in mice post-MI. Mice underwent sham procedure or left coronary artery ligation, and received placebo, the AT1 receptor antagonist, losartan or the ACE inhibitor, enalapril. At 6 weeks, echocardiography and haemodynamic studies were performed. Infarct size and interstitial collagen content were determined. Expression of genes encoding atrial natriuretic peptide (ANP), collagen type I, AT1a and AT1b receptors were measured. The placebo MI group showed increased LV end-diastolic diameter, LV end-systolic diameter with depressed fractional shortening (P

Estrogen receptor (ER)α mediates many of the effects of estrogen on the vascular endothelium. The purpose of the present study was to determine whether estrogen modifies endothelial ERα expression. In experiments in cultured ovine endothelial cells, physiological concentrations of 17β-estradiol (E 2 , 10 −10 to 10 −8 mol/L) caused an increase in ERα protein abundance that was evident after 6 hours of hormone exposure. Shorter (2-hour) E 2 treatment caused ERα downregulation. In contrast to the upregulation in ERα after long-term E 2 , the expression of the other ER isoform, ERβ, was downregulated. Both nonselective ER antagonism with ICI 182,780 and the inhibition of gene transcription with actinomycin D blocked the increase in ERα with E 2 . In studies using the human ERα gene promoter P-1 coupled to luciferase, an increase in ERα gene transcription was evident in endothelial cells within 4 hours of E 2 exposure. The transcriptional activation was fully blocked by ICI 182,780, whereas the specific ERβ antagonist RR-tetrahydrochrysene yielded partial blockade. Overexpression of ERα or ERβ caused comparable 10- and 8-fold increases, respectively, in ERα promoter activation by E 2. Thus, long-term exposure to E 2 upregulates ERα expression in endothelial cells through the actions of either ERα or ERβ on ERα gene transcription; in contrast, E 2 causes ERβ downregulation in the endothelium. We postulate that E 2 -induced changes in ERα and ERβ expression modify the effects of the hormone on vascular endothelium.

Estrogen has both rapid vasodilatory effects and longer-term effects on the vasculature. The longer-term effects of estrogen are produced, at least in part, by changes in vascular cell gene and protein expression that are mediated by the ligand-activated transcription factors, estrogen receptor (ER)-alpha and ER-beta. The rapid vasodilatory effects of estrogen do not require changes in gene expression and are produced by estrogen-stimulated increases in endothelial cell nitric oxide synthase activity. This results in nitric oxide-mediated increases in cyclic guanosine monophosphate in vascular smooth muscle cells, which mediate vasodilatation. This article briefly reviews recent progress in this field of vascular biology.

C disease (CVD) is the leading cause of death among US women. Despite this fact, CVD is perceived mainly as a disease of middle-aged men. Female patients often seriously underestimate the risk of CVD faced by themselves and other women, focusing instead on other causes of morbidity and mortality, such as cancer.1 At the same time, physicians may underdiagnose heart disease in women because of a lack of awareness. Women often present differently from men, with atypical chest pain and less specificity for exercise stress testing. Perhaps, because of missed diagnoses, women tend to have more advanced disease at the time of diagnosis and less favorable outcomes after first or subsequent myocardial infarctions.2 The risk of CVD in women increases substantially after menopause. This finding is believed to be caused in part by the loss of estrogen, which has multiple protective effects on the vascular system (reviewed by Mendelsohn and Karas).3 Numerous observational studies have shown a cardioprotective benefit when estrogen replacement therapy/hormone replacement therapy (ERT/HRT) is given to healthy women after menopause. However, recent randomized trials (Heart and Estrogen/ progestin Replacement Study [HERS], Estrogen Replacement and Atherosclerosis [ERA] trial) have suggested that in women with well established CVD, ERT and HRT do not have a protective effect, and they may even lead to an increased risk of coronary events in their first year of use.4,5 It is important to emphasize that these trials were for secondary CVD prevention in older postmenopausal women with established CVD only, and that they included only a single combination HRT regimen. The results obtained in these trials are therefore not relevant to the potential effect of primary prevention of CVD in healthy women or to use of the newer HRT formulations that are now available. Unfortunately, these studies have been extensively overinterpreted as well as misinterpreted.6 Because of the misinterpretation of these studies, there is considerable confusion in the medical community about the use of HRT and its potential effects on CVD risk. Table 1 lists some of the most common misconceptions. These issues were addressed at a roundtable discussion held in Los Angeles on June 21, 2001, attended by a multidisciplinary panel of physicians with expertise in the fields of cardiology, obstetrics and gynecology, and epidemiology. The objecFrom the Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA; and Tufts University School of Medicine, Molecular Cardiology Research Institute, Tufts–New England Medical Center, Boston, Massachusetts, USA. Address for reprints: Daniel R. Mishell, Jr., MD, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Mail Code 999, Building WCH L-1009, 1240 North Mission Road, Los Angeles, California 90033.

Blood vessel cells express the 2 known estrogen receptors, α and β (ERα, ERβ), which are thought to mediate estrogen inhibition of vascular injury and atherosclerosis, but the relative role of ERα and ERβ in these events is controversial. Estrogen inhibits the vascular injury response to the same extent in ovariectomized female wild-type mice and in the original single gene knockout mice for ERα (ERαKO Chapel Hill [ERαKO CH ]) and ERβ (ERβKO Chapel Hill [ERβKO CH ]). In double gene knockout mice generated by crossing these animals (ERα,βKO CH ), estrogen no longer inhibits medial thickening after vascular injury, but still inhibits vascular smooth muscle cell proliferation and increases uterine weight. The partial retention of estrogen responsiveness in ERα,βKO CH mice could be due either to the presence of a novel, unidentified estrogen receptor or to functional expression of an estrogen receptor-α splice variant in the parental ERαKO CH mice. To distinguish between these possibilities, we studied recently generated mice fully null for estrogen receptor α (ERαKO Strasbourg [ERαKO St ]) and examined the effect of estrogen on the response to vascular injury. In the present study, we show that after vascular injury in ovariectomized ERαKO St mice, estrogen has no detectable effect on any measure of vascular injury, including medial area, proteoglycan deposition, or smooth muscle cell proliferation. These data demonstrate that estrogen receptor-α mediates the protective effects of estrogen on the response to vascular injury.

Abstract— The two known estrogen receptors, ERα and ERβ, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERα knockout (ERαKO CH ), and ERβ knockout (ERβKO CH ) mice. We generated mice harboring disruptions of both ERα and ERβ genes (ERα,βKO CH ) by breeding and studied the effect of 17β-estradiol (E2) on vascular injury responses in ovariectomized female ERα,βKO CH mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERα,βKO CH mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERα,βKO CH mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERα,βKO CH mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERα,βKO CH mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERα splice variant reported previously in the parental ERαKO CH mice. These possibilities may be resolved by studies of mice in which ERα has been fully disrupted (ERαKO St ), which are in progress.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Estrogen receptors are best known as ligand-activated transcription factors that regulate vascular cell gene expression. For many years now, a rapid signaling pathway mediated by cell membrane-associated estrogen receptors also has been recognized, but the physiological relevance of this pathway has remained unclear. In this issue of the JCI, Chambliss et al. provide new data to indicate that activation of non-nuclear estrogen receptor signaling regulates processes central to cardiovascular health and disease. These investigators show that an estrogen-dendrimer conjugate (EDC), which activates estrogen receptors but remains non-nuclear, stimulates vascular EC migration in vitro and protects against vascular injury in vivo. They show further that the vascular benefits of EDC in vivo occur selectively in the vasculature, without stimulating the uterus or enhancing growth of breast cancer xenografts. Taken together, these findings indicate that activation of non-nuclear estrogen receptor signaling regulates vascular events of physiological relevance and suggest that translation of these findings into clinically relevant therapeutic interventions is a logical next goal.

Estrogen receptors (ERs) are ligand-activated transcription factors that regulate gene expression and cell growth. Two ERs now have been identified: ERα and the more recently discovered ERβ. The physiological function of ERβ remains unclear, but evidence from vascular injury studies and from ERβ knockout mice suggests that ERβ may be involved in the regulation of cellular proliferation. Here we show a direct and specific interaction between ERβ and the cell cycle mitotic spindle assembly checkpoint protein, MAD2 (mitosis arrest-deficient 2). The ERβ-MAD2 interaction was identified by screening of a yeast two-hybrid system vascular endothelial cell library with ERβ and confirmed with glutathione S -transferase-fusion protein interaction studies. In contrast, ERα did not interact with MAD2 in either the two-hybrid system or in the protein–protein interaction experiments. Amino acids 173–208 in the hinge region of ERβ were sufficient to mediate the interaction with MAD2 in the two-hybrid system and in glutathione S -transferase-fusion protein studies. These data identify a link between ERβ and MAD2 of potential importance to regulation of the cell cycle and support a function of ERβ distinct from the established role of ERs as transcription factors.

Heart Block in a Mouse Model of Myotonic Dystrophy. Introduction: A mouse strain lacking functional myotonic dystrophy protein kinase (DMPK) has recently been developed. DMPK-/- mice exhibit muscular and conduction abnormalities consistent with the disease; however, the site of abnormal cardiac conduction is unknown. Methods and Results: Nine homozygous DMPK-/- mice and seven age matched wild-type (WT) controls underwent in vivo electrophysiologic studies using an endocardial 2-French catheter. Baseline intervals as well as Wenckebach and 2:1 cycle lengths were measured to assess AV and ventriculoatrial (VA) conduction. Effective refractory periods (ERP) and functional refractory periods were determined during atrial and ventricular premature stimulation. His-bundle recordings were obtained on all the studied animals (16/16). DMPK-/- mice bad significantly prolonged PR (48.1 ± 5.5 vs 40.9 ± 3.9 msec, P = 0.010) and AH (36.7 ± 4.0 vs 31.6 ± 4.8 msec, P = 0.037) intervals compared to WT controls. HV intervals were very significantly prolonged as well (14.7 ± 2.0 vs 10.3 ± 0.8 msec; P < 0.0001). Three of 9 DMPK-/- and I of 7 WT mice exhibited VA block. Atrial ERP was reached before AV node ERP in 2 (22%) of 9 of the knockout mice and 5 (71%) of 7 of the controls (P = 0.06). Only one mouse (DMPK-/-) exhibited infra-Hisian block on premature atrial stimulation. Conclusion: In this mouse model of myotonic dystrophy, AV conduction abnormalities were localized to the supra-Hisian and infra-Hisian conduction tissues, with a higher predilection to the latter, a finding similar to the human form of the disease.