Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

Reduced microvascular flow-mediated dilation in Syrian hamsters lacking d-sarcoglycan is caused by increased oxidative stress.

d-sarcoglycan mutation reduces mechanotransduction and induces dilated cardiomyopathy with aging. We hypothesized that in young hamsters with d-sarcoglycan mutation, which do not show cardiomyopathy, flow mechanotransduction might be affected in resistance arteries as the control of local blood flow. Flow-mediated-dilation (FMD) was measured in isolated mesenteric resistance arteries, using 3-months old hamsters carrying a mutation in the d-sarcoglycan gene (CH-147) and their control littermates. The FMD was significantly reduced in the CHF-147 group. Nevertheless, passive arterial diameter, vascular structure and endothelium-independent dilation to sodium nitroprusside were not modified. Contraction induced by KCl was not modified, whereas contraction due to phenylephrine was increased. The basal NO production and total eNOS expression levels were not altered. Nevertheless, eNOS phosphorylation, FAKs and RhoA expression were reduced in CH-147. In contrast, p47phox, COX2, iNOS and reactive oxygen species levels were higher in the endothelium of CHF-147 hamsters. Reducing ROS levels using the superoxide dismutase analog Tempol significantly restored the flow-mediated dilation (FMD) levels in CHF-147 hamsters. However, treatment with the COX-2 inhibitor NS-398 showed a non-significant improvement in FMD.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1-/- male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1-/- mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1-/- mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1-/- mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1-/- mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.

Sildenafil-Induced Revascularization of Rat Hindlimb Involves Arteriogenesis through PI3K/AKT and eNOS Activation.

Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil-treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.

Early Inactivation of Membrane Estrogen Receptor Alpha (ERα) Recapitulates the Endothelial Dysfunction of Aged Mouse Resistance Arteries.

Flow-mediated dilation (FMD) of resistance arteries is essential for tissue perfusion but it decreases with ageing. As estrogen receptor alpha (Erα encoded by Esr1), and more precisely membrane ERα, plays an important role in FMD in young mice in a ligand-independent fashion, we evaluated its influence on this arteriolar function in ageing. We first confirmed that in young (6-month-old) mice, FMD of mesenteric resistance arteries was reduced in Esr1-/- (lacking ERα) and C451A-ERα (lacking membrane ERα). In old (24-month-old) mice, FMD was reduced in WT mice compared to young mice, whereas it was not further decreased in Esr1-/- and C451A-ERα mice. Markers of oxidative stress were similarly increased in old WT and C451A-ERα mice. Reduction in oxidative stress with superoxide dismutase plus catalase or Mito-tempo, which reduces mitochondrial superoxide restored FMD to a normal control level in young C451A-ERα mice as well as in old WT mice and old C451A-ERα mice. Estradiol-mediated dilation was absent in old WT mice. We conclude that oxidative stress is a key event in the decline of FMD, and that an early defect in membrane ERα recapitulates phenotypically and functionally ageing of these resistance arteries. The loss of this function could take part in vascular ageing.

Estrogens and the Angiotensin II Type 2 Receptor Control Flow-Mediated Outward Remodeling in the Female Mouse Mesenteric Artery.

Flow-mediated outward remodeling (FMR) is involved in postischemic revascularization. Angiotensin II type 2 receptor (AT2R), through activation of T-cell-mediated IL-17 production, and estrogens are involved in FMR. Thus, we investigated the interplay between estrogens and AT2R in FMR using a model of ligation of feed arteries supplying collateral pathways in mouse mesenteric arteries in vivo. Arteries were collected after 2 (inflammatory phase), 4 (diameter expansion phase), and 7 days (remodeling completed). We used AT2R+/+ and AT2R-/- ovariectomized (OVX) female mice treated or not with 17-beta-estradiol (E2). Seven days after ligation, arterial diameter was larger in high flow (HF) compared to normal flow (NF) arteries. FMR was absent in OVX mice and restored by E2. AT2R gene expression was higher in HF than in NF arteries only in E2-treated OVX AT2R+/+ mice. CD11b and TNF alpha levels (inflammatory phase), MMP2 and TIMP1 (extracellular matrix digestion), and NOS3 (diameter expansion phase) expression levels were higher in HF than in NF arteries only in E2-treated AT2R+/+ mice, not in the other groups. Thus, E2 is necessary for AT2R-dependent diameter expansion, possibly through activation of T-cell AT2R, in arteries submitted chronically to high blood flow.

Mutation of Arginine 264 on ERα (Estrogen Receptor Alpha) Selectively Abrogates the Rapid Signaling of Estradiol in the Endothelium Without Altering Fertility.

OBJECTIVE: ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17ß-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the C451A-ERα, the R264A-ERα females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα-dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in R264A-ERα mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved. CONCLUSIONS: These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions.

Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension.

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

Testosterone Prevents Cutaneous Ischemia and Necrosis in Males Through Complementary Estrogenic and Androgenic Actions.

OBJECTIVE: Chronic nonhealing wounds are a substantial medical concern and are associated with morbidity and mortality; thus, new treatment strategies are required. The first step toward personalized/precision medicine in this field is probably in taking sex differences into account. Impaired wound healing is augmented by ischemia, and we previously demonstrated that 17ß-estradiol exerts a major preventive effect against ischemia-induced skin flap necrosis in female mice. However, the equivalent effects of testosterone in male mice have not yet been reported. We then investigated the role of steroid hormones in male mice using a skin flap ischemia model. APPROACH AND RESULTS: Castrated male mice developed skin necrosis after ischemia, whereas intact or castrated males treated with testosterone were equally protected. Testosterone can (1) activate the estrogen receptor after its aromatization into 17ß-estradiol or (2) be reduced into dihydrotestosterone, a nonaromatizable androgen that activates the androgen receptor. We found that dihydrotestosterone protected castrated wild-type mice by promoting skin revascularization, probably through a direct action on resistance arteries, as evidenced using a complementary model of flow-mediated outward remodeling. 17ß-estradiol treatment of castrated male mice also strongly protected them from ischemic necrosis through the activation of estrogen receptor-α by increasing skin revascularization and skin survival. Remarkably, 17ß-estradiol improved skin survival with a greater efficiency than dihydrotestosterone. CONCLUSIONS: Testosterone provides males with a strong protection against cutaneous necrosis and acts through both its estrogenic and androgenic derivatives, which have complementary effects on skin survival and revascularization.

Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions.

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2(0), whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

Determinants of flow-mediated outward remodeling in female rodents: respective roles of age, estrogens, and timing.

OBJECTIVE: Flow (shear stress)-mediated outward remodeling (FMR) of resistance arteries is a key adaptive process allowing collateral growth after arterial occlusion but declining with age. 17-ß-estradiol (E2) has a key role in this process through activation of estrogen receptor α (ERα). Thus, we investigated the impact of age and timing for estrogen efficacy on FMR. APPROACH AND RESULTS: Female rats, 3 to 18 months old, were submitted to surgery to increase blood flow locally in 1 mesenteric artery in vivo. High-flow and normal-flow arteries were collected 2 weeks later for in vitro analysis. Diameter increased by 27% in high-flow arteries compared with normal-flow arteries in 3-month-old rats. The amplitude of remodeling declined with age (12% in 18-month-old rats) in parallel with E2 blood level and E2 substitution failed restoring remodeling in 18-month-old rats. Ovariectomy of 3-, 9-, and 12-month-old rats abolished FMR, which was restored by immediate E2 replacement. Nevertheless, this effect of E2 was absent 9 months after ovariectomy. In this latter group, ERα and endothelial nitric oxide synthase expression were reduced by half compared with age-matched rats recently ovariectomized. FMR did not occur in ERα(-/-) mice, whereas it was decreased by 50% in ERα(+/-) mice, emphasizing the importance of gene dosage in high-flow remodeling. CONCLUSIONS: E2 deprivation, rather than age, leads to decline in FMR, which can be prevented by early exogenous E2. However, delayed E2 replacement was ineffective on FMR, underlining the importance of timing of this estrogen action.

AGEs breaking and antioxidant treatment improves endothelium-dependent dilation without effect on flow-mediated remodeling of resistance arteries in old Zucker diabetic rats.

BACKGROUND: A chronic increase in blood flow in resistance arteries is associated with increased lumen diameter (outward remodeling) and improved endothelium (NO)-mediated relaxation. Flow-mediated remodeling of resistance arteries is essential for revascularization in ischemic diseases. Nevertheless, it is impaired in 12 to 24-month old rats and in young Zucker Diabetic Fatty (ZDF) rats due to advanced glycation end products (AGEs) and oxidative stress. As type 2 diabetes occurs preferentially in older subjects we investigated flow-mediated remodeling and the effect of the AGEs breaker ALT-711 associated or not to the antioxidant TEMPOL in one-year old lean (LZ) and ZDF rats. METHODS: Mesenteric resistance arteries were exposed to high (HF) or normal blood flow (NF) in vivo. They were collected after 2 weeks for in vitro analysis. RESULTS: In LZ rats, diameter expansion did not occur despite a significant increase in blood flow in HF arteries. Nevertheless, endothelium-mediated relaxation was higher in HF than in NF arteries. ALT-711, alone or in combination with TEMPOL, restored outward remodeling in HF arteries in association with AGEs reduction. TEMPOL alone had no effect. ALT-711, TEMPOL or the combination of the 2 drugs did not significantly affect endothelium-mediated relaxation in HF and NF arteries.In ZDF rats, diameter did not increase despite the increase in blood flow and endothelium-mediated relaxation was further decreased in HF arteries in association with AGEs accumulation and excessive oxidative stress. In both NF and HF arteries, endothelium-mediated relaxation was lower in ZDF than in LZ rats. ALT-711, TEMPOL or their combination did not improve remodeling (diameter equivalent in HF and NF arteries). In parallel, they did not reduce AGEs level and did not improve MMPs activity. Nevertheless, ALT-711 and TEMPOL partly improved endothelium-mediated relaxation through a reduction of oxidative stress and the association of ALT-711 and TEMPOL fully restored relaxation to the level found in LZ rats. CONCLUSIONS: ALT-711 did not improve outward remodeling in mature ZDF rats but it reduced oxidative stress and consequently improved endothelium-dependent relaxation. In mature LZ rats, ALT-711 improved outward remodeling and reduced AGEs level. Consequently, AGEs breaking is differently useful in ageing whether it is associated with diabetes or not.

Kynurenic Acid: A Novel Player in Cardioprotection against Myocardial Ischemia/Reperfusion Injuries.

BACKGROUND: Myocardial infarction is one of the leading causes of mortality worldwide; hence, there is an urgent need to discover novel cardioprotective strategies. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway, has been previously reported to have cardioprotective effects. However, the mechanisms by which KYNA may be protective are still unclear. The current study addressed this issue by investigating KYNA's cardioprotective effect in the context of myocardial ischemia/reperfusion. METHODS: H9C2 cells and rats were exposed to hypoxia/reoxygenation or myocardial infarction, respectively, in the presence or absence of KYNA. In vitro, cell death was quantified using flow cytometry analysis of propidium iodide staining. In vivo, TTC-Evans Blue staining was performed to evaluate infarct size. Mitochondrial respiratory chain complex activities were measured using spectrophotometry. Protein expression was evaluated by Western blot, and mRNA levels by RT-qPCR. RESULTS: KYNA treatment significantly reduced H9C2-relative cell death as well as infarct size. KYNA did not exhibit any effect on the mitochondrial respiratory chain complex activity. SOD2 mRNA levels were increased by KYNA. A decrease in p62 protein levels together with a trend of increase in PARK2 may mark a stimulation of mitophagy. Additionally, ERK1/2, Akt, and FOXO3α phosphorylation levels were significantly reduced after the KYNA treatment. Altogether, KYNA significantly reduced myocardial ischemia/reperfusion injuries in both in vitro and in vivo models. CONCLUSION: Here we show that KYNA-mediated cardioprotection was associated with enhanced mitophagy and antioxidant defense. A deeper understanding of KYNA's cardioprotective mechanisms is necessary to identify promising novel therapeutic targets and their translation into the clinical arena.

Altered Mitochondrial Opa1-Related Fusion in Mouse Promotes Endothelial Cell Dysfunction and Atherosclerosis.

Flow (shear stress)-mediated dilation (FMD) of resistance arteries is a rapid endothelial response involved in tissue perfusion. FMD is reduced early in cardiovascular diseases, generating a major risk factor for atherosclerosis. As alteration of mitochondrial fusion reduces endothelial cells' (ECs) sprouting and angiogenesis, we investigated its role in ECs responses to flow. Opa1 silencing reduced ECs (HUVECs) migration and flow-mediated elongation. In isolated perfused resistance arteries, FMD was reduced in Opa1+/- mice, a model of the human disease due to Opa1 haplo-insufficiency, and in mice with an EC specific Opa1 knock-out (EC-Opa1). Reducing mitochondrial oxidative stress restored FMD in EC-Opa1 mice. In isolated perfused kidneys from EC-Opa1 mice, flow induced a greater pressure, less ATP, and more H2O2 production, compared to control mice. Opa1 expression and mitochondrial length were reduced in ECs submitted in vitro to disturbed flow and in vivo in the atheroprone zone of the mouse aortic cross. Aortic lipid deposition was greater in Ldlr-/--Opa1+/- and in Ldlr-/--EC-Opa1 mice than in control mice fed with a high-fat diet. In conclusion, we found that reduction in mitochondrial fusion in mouse ECs altered the dilator response to shear stress due to excessive superoxide production and induced greater atherosclerosis development.

Flow-mediated outward arterial remodeling in aging.

The present review focuses on the effect of aging on flow-mediated outward remodeling (FMR) via alterations in estrogen metabolism, oxidative stress and inflammation. In ischemic disorders, the ability of the vasculature to adapt or remodel determines the quality of the recovery. FMR, which has a key role in revascularization, is a complex phenomenon that recruits endothelial and smooth muscle cells as well as the immune system. FMR becomes progressively less with age as a result of an increase in inflammation and oxidative stress, in part of mitochondrial origin. The alteration in FMR is greater in older individuals with risk factors and thus the therapy cannot merely amount to exercise with or without a mild vasodilating drug. Interestingly, the reduction in FMR occurs later in females. Estrogen and its alpha receptor (ERα) play a key role in FMR through the control of dilatory pathways including the angiotensin II type 2 receptor, thus providing possible tools to activate FMR in older subjects although only experimental data is available. Indeed, the main issue is the reversibility of the vascular damage induced over time, and to date promoting prevention and limiting exposure to the risk factors remain the best options in this regard.

Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner.

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action, whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.

Deficiency or blockade of angiotensin II type 2 receptor delays tumorigenesis by inhibiting malignant cell proliferation and angiogenesis.

Despite significant expression level in cancer cells, the role of the angiotensin II Type 2 receptor (AT2R) in cancer progression remains poorly understood. We aimed to investigate the involvement of AT2R in tumorigenesis, hypothesizing a role in tumor cell proliferation and/or tumor angiogenesis. Two animal tumor models were used: fibrosarcoma induced by 3-methylcholanthrene (3-MCA) in FVB/N mice invalidated for AT2R (AT2R-KO) and carcinoma LL/2 cells injected in C57BL/6N mice treated with AT2R antagonist PD123,319. Tumor growth was monitored, microvascular density (MVD) evaluated by CD31 staining. Proliferation index of LL/2 and 3-MCA tumor cells was evaluated by expression of Ki-67. Angiogenesis was assessed by aorta ring assay and angiogenic mediators' expression by real-time RT-PCR. Tumor induction by 3-MCA was significantly delayed in AT2R-KO compared to wild-type mice (56 days vs. 28 days). Tumorigenesis following LL/2 cell injection in mice was also significantly reduced by early administration of the antagonist PD123,319. In vitro, inactivation or invalidation of AT2R inhibited proliferation of LL/2 and 3-MCA tumor cells, respectively. Tumor MVD was reduced in mice treated early with PD123,319. Ex vivo experiments revealed a significant decrease in angiogenesis after PD123,319 treatment or in AT2R-KO mice. Finally, we identified vascular endothelial growth factor (VEGF) as a soluble proangiogenic factor produced by LL/2 cells and we showed that in LL/2 and 3-MCA tumor cells, inhibition or deficiency of AT2R was associated with impaired production of proangiogenic factors included VEGF. This study uncovered novel mechanisms by which AT2R would promote tumor development, favoring both malignant cell proliferation and tumor angiogenesis.

Nuclear Activation Function 2 Estrogen Receptor α Attenuates Arterial and Renal Alterations Due to Aging and Hypertension in Female Mice.

Background The cardiovascular protective effects of estrogens in premenopausal women depend mainly on estrogen receptor α (ERα). ERα activates nuclear gene transcription regulation and membrane-initiated signaling. The latter plays a key role in estrogen-dependent activation of endothelial NO synthase. The goal of the present work was to determine the respective roles of the 2 ERα activities in endothelial function and cardiac and kidney damage in young and old female mice with hypertension, which is a major risk factor in postmenopausal women. Methods and Results Five- and 18-month-old female mice lacking either ERα (ERα-/-), the nuclear activating function AF2 of ERα (AF2°), or membrane-located ERα (C451A) were treated with angiotensin II (0.5 mg/kg per day) for 1 month. Systolic blood pressure, left ventricle weight, vascular reactivity, and kidney function were then assessed. Angiotensin II increased systolic blood pressure, ventricle weight, and vascular contractility in ERα-/- and AF2° mice more than in wild-type and C451A mice, independent of age. In both the aorta and mesenteric resistance arteries, angiotensin II and aging reduced endothelium-dependent relaxation in all groups, but this effect was more pronounced in ERα-/- and AF2° than in the wild-type and C451A mice. Kidney inflammation and oxidative stress, as well as blood urea and creatinine levels, were also more pronounced in old hypertensive ERα-/- and AF2° than in old hypertensive wild-type and C451A mice. Conclusions The nuclear ERα-AF2 dependent function attenuates angiotensin II-dependent hypertension and protects target organs in aging mice, whereas membrane ERα signaling does not seem to play a role.