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The COVID‐19 pandemic extended all around the world causing millions of deaths. In addition to acute respiratory distress syndrome, many patients with severe COVID‐19 develop thromboembolic complications associated to multiorgan failure and death. Here, we review evidence for the contribution of neutrophils, platelets, and extracellular vesicles (EVs) to the thromboinflammatory process in COVID‐19. We discuss how the immune system, influenced by pro‐inflammatory molecules, EVs, and neutrophil extracellular traps (NETs), can be caught out in patients with severe outcomes. We highlight how the deficient regulation of the innate immune system favors platelet activation and induces a vicious cycle amplifying an immunothrombogenic environment associated with platelet/NET interactions. In light of these considerations, we discuss potential therapeutic strategies underlining the modulation of purinergic signaling as an interesting target.

This study addressed the hypothesis that epoxyeicosatrienoic acids (EETs) synthesized by CYP450 and catabolized by soluble epoxide hydrolase (sEH) are involved in the maintenance of renal allograft function, either directly or through modulation of cardiovascular function. The impact of single nucleotide polymorphisms (SNPs) in the sEH gene EPHX2 and CYP450 on renal and vascular function, plasma levels of EETs and peripheral blood monuclear cell sEH activity was assessed in 79 kidney transplant recipients explored at least one year after transplantation. Additional experiments in a mouse model mimicking the ischemia–reperfusion (I/R) injury suffered by the transplanted kidney evaluated the cardiovascular and renal effects of the sEH inhibitor t-AUCB administered in drinking water (10 mg/l) during 28 days after surgery. There was a long-term protective effect of the sEH SNP rs6558004, which increased EET plasma levels, on renal allograft function and a deleterious effect of K55R, which increased sEH activity. Surprisingly, the loss-of-function CYP2C9*3 was associated with a better renal function without affecting EET levels. R287Q SNP, which decreased sEH activity, was protective against vascular dysfunction while CYP2C8*3 and 2C9*2 loss-of-function SNP, altered endothelial function by reducing flow-induced EET release. In I/R mice, sEH inhibition reduced kidney lesions, prevented cardiac fibrosis and dysfunction as well as preserved endothelial function. The preservation of EET bioavailability may prevent allograft dysfunction and improve cardiovascular disease in kidney transplant recipients. Inhibition of sEH appears thus as a novel therapeutic option but its impact on other epoxyfatty acids should be carefully evaluated.

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.

Rationale: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P 1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P 1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P 1 modulation in stroke. Objective: To address roles and mechanisms of engagement of endothelial cell S1P 1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. Methods and Results: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P 1 in the mouse brain. With an S1P 1 signaling reporter, we reveal that abluminal polarization shields S1P 1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P 1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P 1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P 1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P 1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P 1 -selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. Conclusions: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P 1 agonists.

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 17β-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.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Mechanotransduction plays a crucial role in vascular biology. One example of this is local regulation of vascular resistance via flow-mediated vasodilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction, and a precursor to a wide array of vascular diseases such as hypertension and atherosclerosis. And yet, the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium, and is a critical signaling component in cardiovascular pathophysiology.

Background Although estrogen receptor α ( ER α) acts primarily as a transcription factor, it can also elicit membrane‐initiated steroid signaling. Pharmacological tools and transgenic mouse models previously highlighted the key role of ER α membrane‐initiated steroid signaling in 2 actions of estrogens in the endothelium: increase in NO production and acceleration of reendothelialization. Methods and Results Using mice with ER α mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ER α plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20 = ERaAF2°), we sought to fully characterize the respective roles of nuclear versus membrane‐initiated steroid signaling in the arterial protection conferred by ER α. ERaC451A mice were fully responsive to estrogens to prevent atheroma and angiotensin II –induced hypertension as well as to allow flow‐mediated arteriolar remodeling. By contrast, ER α AF 20 mice were unresponsive to estrogens for these beneficial vascular effects. Accordingly, selective activation of nuclear ER α with estetrol was able to prevent hypertension and to restore flow‐mediated arteriolar remodeling. Conclusions Altogether, these results reveal an unexpected prominent role of nuclear ER α in the vasculoprotective action of estrogens with major implications in medicine, particularly for selective nuclear ER α agonist, such as estetrol, which is currently under development as a new oral contraceptive and for hormone replacement therapy in menopausal women.

We have previously shown that protein tyrosine phosphatase 1B (PTP1B) inactivation in mice [PTP1B-deficient (PTP1B−/−) mice] improves left ventricular (LV) angiogenesis, perfusion, remodeling, and function and limits endothelial dysfunction after myocardial infarction. However, whether PTP1B inactivation slows aging-associated cardiovascular dysfunction remains unknown. Wild-type (WT) and PTP1B−/− mice were allowed to age until 18 mo. Compared with old WT mice, in which aging increased the LV mRNA expression of PTP1B, old PTP1B−/− mice had 1) reduced cardiac hypertrophy with decreased LV mRNA levels of hypertrophic markers and atrial and brain natriuretic peptides, 2) lower LV fibrosis (collagen: 16 ± 3% in WT mice and 5 ± 3% in PTP1B−/− mice, P < 0.001) with decreased mRNA levels of transforming growth-factor-β1 and matrix metalloproteinase-2, and 3) higher LV capillary density and lower LV mRNA level of hypoxic inducible factor-1α, which was associated over time with a higher rate of proangiogenic M2 type macrophages and a stable LV mRNA level of VEGF receptor-2. Echocardiography revealed an age-dependent LV increase in end-diastolic volume in WT mice together with alterations of fractional shortening and diastole (transmitral Doppler E-to-A wave ratio). Invasive hemodynamics showed better LV systolic contractility and better diastolic compliance in old PTP1B−/− mice (LV end-systolic pressure-volume relation: 13.9 ± 0.9 in WT mice and 18.4 ± 1.6 in PTP1B−/− mice; LV end-diastolic pressure-volume relation: 5.1 ± 0.8 mmHg/relative volume unit in WT mice and 1.2 ± 0.3 mmHg/relative volume unit in PTP1B−/− mice, P < 0.05). In addition, old PTP1B−/− mice displayed a reduced amount of LV reactive oxygen species. Finally, in isolated resistance mesenteric arteries, PTP1B inactivation reduced aging-associated endothelial dysfunction (flow-mediated dilatation: −0.4 ± 2.1% in WT mice and 8.2 ± 2.8% in PTP1B−/− mice, P < 0.05). We conclude that PTP1B inactivation slows aging-associated LV remodeling and dysfunction and reduces endothelial dysfunction in mesenteric arteries. NEW & NOTEWORTHY The present study shows that protein tyrosine phosphatase 1B inactivation in aged mice improves left ventricular systolic and diastolic function associated with reduced adverse cardiac remodeling (hypertrophy, fibrosis, and capillary rarefaction) and limits vascular endothelial dysfunction. This suggests that protein tyrosine phosphatase 1B inhibition could be an interesting treatment approach in age-related cardiovascular dysfunction.

International audience; Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation ofvascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.

International audience; 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)-1β 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.

Objective Flow-mediated dilation (FMD) of resistance arteries (RAs) plays a key role in tissue perfusion. A reduced FMD is the hallmark of the endothelium dysfunction occurring early in cardiovascular and metabolic disorders. Estrogen therapy improves FMD in diseased conditions without major effect in healthy subjects. Nevertheless, estrogen receptor alpha (ERα) mutation in a man was associated with severe and selective FMD reduction. Methods Thus, we evaluated the role of ERα in FMD in healthy young male mice. FMD was determined in vitro in mesenteric RAs isolated from mice lacking ERα (ERα−/−), the ERα nuclear activation function AF2 (AF2-ERα−/−) or the ERα palmitoylation site (C451A-ERα−/−, inactivation of the membrane effects or ERα), compared to littermate wild-type (WT or+/+) mice. Results We found that FMD was significantly reduced in ERα−/− compared to ERα+/+ male and female (ovariectomized or not) mice. Exogenous ERα stimulation or blockade did not affect FMD. FMD was not affected in AF2-ERα−/− mice whereas it was significantly decreased in C451A-ERα−/− mice. NO-synthesis blockade with L-NNA strongly reduced FMD in WT and AF2-ERα−/− mice without major effect in ERα-/- and C451A-ERα−/− mice. Acetylcholine-mediated relaxation, not affected by the absence of ERα was similarly reduced by L-NNA in all groups. After ROS reduction in vivo, NO-dependent FMD was equivalent in C451A-ERα−/− and C451A-ERα+/+ mice. Conclusion Thus, membrane ERα is important for FMD through activation of eNOS activity and reduction of ROS production, possibly in a ligand-independent manner.

Introduction In total, 17β-Estradiol (E2) exerts numerous beneficial effects in vascular disease. It regulates gene transcription through nuclear estrogen receptor α (ERα) via 2 activation functions, AF1 and AF2, and can also activate membrane ERα. Objective and method Using mice with ERα mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ERα plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20), we sought to fully characterize the respective roles of nuclear versus membrane-initiated steroid signaling in the arterial protection conferred by ERα. Results Whereas the increase in NO production and the acceleration of endothelial healing in response to E2 were abrogated in ERaC451A mice, the protection against atheroma, neointimal hyperplasia, angiotensin II-induced hypertension, as well as the flow-mediated arteriolar remodelling, were preserved. In contrast, these last four beneficial vascular effects were lost in ERαAF20 mice in response to E2. In line with these results, selective activation of nuclear ERα with estetrol (E4) was capable of inducing arterial protection, except for the increase in NO production and the acceleration of endothelial healing. Conclusion Altogether, these results reveal a prominent role for nuclear ERα in the vasculoprotective action of estrogens. This will have major implications in medicine, since the selective nuclear ERα agonist, estetrol, is currently developed as a new oral contraceptive, and as hormone replacement therapy in menopausal women.

Diabetes induces a decrease in the number and function of different pro-angiogenic cell types generically designated as putative endothelial progenitor cells (EPC), which encompasses cells from myeloid origin that act in a paracrine fashion to promote angiogenesis and putative "true" EPC that contribute to endothelial replacement. This not only compromises neovasculogenesis in ischemic tissues but also impairs, at an early stage, the reendotheliziation process at sites of injury, contributing to the development of endothelial dysfunction and cardiovascular complications. Hyperglycemia, insulin resistance and dyslipidemia promote putative EPC dysregulation by affecting the SDF-1/CXCR-4 and NO pathways and the p53/SIRT1/p66Shc axis that contribute to their mobilization, migration, homing and vasculogenic properties. To optimize the clinical management of patients with hypoglycemic agents, statins and renin-angiotensin system inhibitors, which display pleiotropic effects on putative EPC, is a first step to improve their number and angiogenic potential but specific strategies are needed. Among them, mobilizing therapies based on G-CSF, erythropoietin or CXCR-4 antagonism have been developed to increase putative EPC number to treat ischemic diseases with or without prior cell isolation and transplantation. Growth factors, genetic and pharmacological strategies are also evaluated to improve ex vivo cultured EPC function before transplantation. Moreover, pharmacological agents increasing in vivo the bioavailability of NO and other endothelial factors demonstrated beneficial effects on neovascularization in diabetic ischemic models but their effects on endothelial dysfunction remain poorly evaluated. More experiments are warranted to develop orally available drugs and specific agents targeting p66Shc to reverse putative EPC dysfunction in the expected goal of preventing endothelial dysfunction and diabetic cardiovascular complications.

Background Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the Von Willebrand factor (VWF) cleaving protease ADAMTS13. Endothelial injury is believed to be a key initiating event in the pathogenesis leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and seems to differ among the microvascular territory. Especially, a cardiac injury has recently been identified and hypothesized as a major cause of death in TTP. Methods We used an animal model in which some TTP-like symptoms have been triggered in ADAMTS13 knockout mice (B6.129-ADAMTS13tm1Dgi ) by challenge with 2000 units/kg body weight of recombinant human VWF containing ultra large VWF (rhVWF, Baxter, Vienna Austria). rhVWF was also injected in 6 wild type mice (C57/bl6-B129 WT+VWF) while seven other wild type mice received placebo (WT). Cardiac involvement was assessed by transthoracic echocardiography using validated parameters i.e left ventricular ejection fraction (LVEF), fractional shortening (FS) and cardiac index (CI). Cardiac perfusion was determined by arterial spin labeling MRI (Biospec 47/40 advanced II, Brucker, Ettlingen, Germany). Ex vivo endothelial function analysis in isolated coronary, mesenteric and renal segments were performed using a myograph. Arteries were precontracted by phenylephrine or serotonin and the relaxations induced by increasing concentrations of acetylcholine (receptor-mediated, endothelium-dependent relaxation) or the nitric oxide (NO) donor sodium nitroprusside (SNP; endothelium-independent- relaxation) were assessed. Arteries were also incubated with the NO synthase-inhibitor N-Nitro L-Arginine (LNNA) to assess the contribution of NO to the relaxing responses to acetylcholine (endothelium-dependent NO-independent-relaxation). Results On day 1, VWF-challenged ADAMTS13 KO mice rapidly developed symptoms and severe thrombocytopenia (41.8G/L ± 6.9G/L vs 480.7G/L ± 86.5G/L (WT+VWF) and 1118.6G/L ± 160.8G/L (WT); p Conclusions This experimental study reproduced an animal model in which TTP like symptoms are triggered in ADAMTS13 KO mice by administration of VWF and demonstrated that the heart is the most sensitive target organ with myocardial necrosis and systolic dysfunction. We showed for the first time a strong mesenteric and coronary endothelial dysfunction in an induced-TTP model. It may represent an important trigger of the systemic organs failure occurring in this disease, strengthening the need to define the mechanisms involved in this early vascular injury. Disclosures: Schiviz: Baxter: Employment.

Resistance arteries play a key role in the control of local blood flow. They undergo outward remodeling in response to a chronic increase in blood flow as seen in collateral artery growth in ischemic disorders. We have previously shown that mesenteric artery outward remodeling depends on the endothelial estrogen receptor alpha. As outward arterial remodeling is associated with improved endothelium-dependent dilation, we hypothesized that estrogens might also play a role in flow-mediated improvement of endothelium-dependent dilation. Local increase in blood flow in first order mesenteric arteries was obtained after ligation of adjacent arteries in three-month old ovariectomized female rats treated with 17-beta-estradiol (OVX+E2) or vehicle (OVX). After 2 weeks, diameter was equivalent in high flow (HF) than in normal flow (NF) arteries with a greater wall to lumen ratio in HF vessels in OVX rats. Acetylcholine-mediated relaxation was lower in HF than in NF vessels. eNOS and caveolin-1 expression level was equivalent in HF and NF arteries. By contrast, arterial diameter was 30% greater in HF than in NF arteries and the wall to lumen ratio was not changed in OVX+E2 rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries. The expression level of eNOS was higher and that of caveolin-1 was lower in HF than in NF arteries. Acetylcholine (NO-dependent)-mediated relaxation was partly inhibited by the NO-synthesis blocker L-NAME in OVX rats whereas L-NAME blocked totally the relaxation in OVX+E2 rats. Endothelium-independent relaxation (sodium nitroprusside) was equivalent in OXV and OVX+E2 rats. Similarly, serotonin- and phenylephrine-mediated contractions were higher in HF than in NF arteries in both OVX and OVX+E2 rats in association with high ratio of phosphorylated ERK1/2 to ERK1/2. Thus, we demonstrated the essential role of endogenous E2 in flow-mediated improvement of endothelium (NO)-mediated dilatation in rat mesenteric arteries.

Background Cardiovascular diseases (CVD) incidence increases with aging and numerous risk factors like arterial hypertension. Estrogens reduce vascular damaging triggered by CVD like hypertension, mainly through estrogen receptor alpha (ERα). However, recent studies have shown that a fraction of ERα is located at the cell membrane where it initiates a Membrane Initiated Steroid Signaling (MISS). Both membrane and nuclear pathways of ERα seem to participate to the protective effect of estrogens but their respective role in hypertension is unknown. Objective This study aims to better understand the protective role of ERα in hypertension and its vascular consequences. Method Five groups of female mice were used in this study, each one lacking either estrogens (WT-OVX), ERα (ERα-KO), membrane-ERα (C451A-ERα) or ligand-dependent-transcriptional function of ERα (AF2°). Half of the mice received a chronic infusion of angiotensin II (0.5 mg/kg/d during 1 month) to induce moderate hypertension. Systolic blood pressure (SBP) and vascular structure were measured in KO mice and compared to wild-type mice (WT). Studies were performed in 5 months mice and 16 months mice in order to evaluate the impact of aging in the estrogens protection. Results SBP increased by 14 mmHg (ns) in WT mice. A similar increased was observed in C451A mice (20 mmHg, ns). By contrast, SBP increased significantly more in WT-OVX mice (31 mmHg, p=0,0007), ERα-KO (45 mmHg, p Conclusion Taken together, these data suggest that the protective effect of ERα against angiotensin II-induced hypertension depend on ERα gene transcription through AF2 and not membrane ERα signaling by contrast with most described vasculoprotective effects of estrogens. The author hereby declares no conflict of interest

Ischemia-reperfusion (I/R) injury is an immediate complication of renal transplantation and a determinant of persistent allograft injury and related cardiovascular outcomes. Epoxyeicosatrienoic acids (EETs) are endothelium- derived hyperpolarizing factors with anti-inflammatory and vasodilator properties. The objective of our study was to evaluate the effects of t-AUCB, a pharmacological inhibitor of EETs degradation, on the shortand potential long-term consequences of renal I/R. C57Bl/6J mice were subjected to 30-min renal bilateral I/R, and subsequently received t-AUCB (15 mg/l PO) or vehicle. Kidney function and pathology were evaluated after 48 hours (h48), 28 days (d28) and 8 months (m8). Vascular function on isolated renal artery, and cardiac function were evaluated at d28 (echocardiography, invasive hemodynamics), and m8(echocardiography). Renal I/R produced severe renal failure at h48, and mild renal failure at d28 and m8, non significantly reduced by t-AUCB (plasma creatinine sham 5.0±1.7 μmol/l; d28 I/R 14.9±2.7; d28 I/R+t-AUCB 10.8±1.1; m8 I/R 6.6±1.2; m8 I/R+t-AUCB 7.0±1.3). Kidney histology revealed severe tubulointerstitial lesions at d28, reduced by t-AUCB (sham 0.3±0.1; I/R 1.5±0.2, p The author hereby declares no conflict of interest

Resistance arteries are sensitive to mechanical forces exerted on the vessel wall. While endothelial shear stress triggers flow-mediated dilation (FMD), chronic increase in shear forces drives expansive arterial remodeling. We showed previously that endothelial estrogen receptor alpha (ER‹) controlled this adaptive remodeling. We aimed to evaluate ER‹contribution in acute response to flow, by using various genetic models of ER‹deficiency in male mice, reducing estrogen hormonal influence. FMD was evaluated following step increase in flow applied to pressurized mesenteric arteries (arteriography). Arteries were isolated from wild-type (WT), (i) ER‹KO, or mice invalidated for (ii) the ligand-dependent transactivation function AF2 of ERa (AF2°) and (iii) the plasma membrane-located ER‹(mutated for the palmitoylation site C451A). In mice deficient in ER‹, FMD was attenuated (dilation 50μl/min WT: 59+/–4% vs. ER‹KO: 41+/–4% p The author hereby declares no conflict of interest

Eur J Clin Invest 2012 Abstract Background The role of bone marrow–derived cells in stimulating angiogenesis, vascular repair or remodelling has been well established, but the nature of the circulating angiogenic cells is still controversial. Design The existing literature on different cell types that contribute to angiogenesis in multiple pathologies, most notably ischaemic and tumour angiogenesis, is reviewed, with a focus on subtypes of angiogenic mononuclear cells and their local recruitment and activation. Results A large number of different cells of myeloid origin support angiogenesis without incorporating permanently into the newly formed vessel, which distinguishes these circulating angiogenic cells (CAC) from endothelial progenitor cells (EPC). Although CAC frequently express individual endothelial markers, they all share multiple characteristics of monocytes and only express a limited set of discriminative surface markers in the circulation. When cultured ex vivo, or surrounding the angiogenic vessel in vivo, however, many of them acquire similar additional markers, making their discrimination in situ difficult. Conclusion Different subsets of monocytes show angiogenic properties, but the distinct microenvironment, in vitro or in vivo, is needed for the development of their pro-angiogenic function.

International audience; C hronic heart failure (HF) after myocardial infarction (MI) is steadily increasing worldwide and remains a major cause of death. Mineralocorticoid receptor (MR) antagonists (MRAs) improve survival in patients with HF as illustrated by spironolactone in the RALES (Randomized Aldactone Evaluation Study) 1 trial and by eplerenone in the EPHESUS (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) trial, 2 which only included patients with post-MI. Recently, the EMPHASIS (Eplerenone in Mild Patients Hospitalization and Survival Study in Heart Failure) study even showed that eplerenone is effective in slowing down the progression of mild-to-moderate HF. 3 However, MRAs are associated with side effects, such as hyperkalemia or gynecomastia; the latter is caused by the antiandrogenic properties of their steroidal structure. These adverse effects are responsible for the underuse of MRAs. 4 Understanding of the cell-specific contribution of MR to HF and of the effects of MRA on various cell subtypes will be useful for the future development of tissue-selective MR targeting approaches that would improve the benefit/risk ratio. 5 A crucial role of cardiomyocyte MR has been demonstrated in mice with cardiomyocyte-specific MR deletion, which allows improving left ventricular (LV) function after either MI 6 or pressure overload induced by transverse aortic constriction. 7 The deletion of MR specifically in fibroblasts does not affect cardiac failure after aortic constriction. 7 The role of vascular MR has been underlined recently: the MR expressed in vas-cular smooth muscle cells (VSMCs) is involved in age-related Abstract-Mineralocorticoid receptor (MR) antagonists slow down the progression of heart failure after myocardial infarction (MI), but the cell-specific role of MR in these benefits is unclear. In this study, the role of MR expressed in vascular smooth muscle cells (VSMCs) was investigated. Two months after coronary artery ligation causing MI, mice with VSMC-specific MR deletion (MI-MR SMKO) and mice treated with the MR antagonist finerenone (MI-fine) had improved left ventricular compliance and elastance when compared with infarcted control mice (MI-CTL), as well as reduced interstitial fibrosis. Importantly, the coronary reserve assessed by magnetic resonance imaging was preserved (difference in myocardial perfusion before and after induction of vasodilatation, mL mg −1 min −1 : MI-CTL: 1.1±0.5, nonsignificant; MI-MR SMKO : 4.6±1.6 [P

n-3 polyunsaturated fatty acids (omega-3) supplementation is associated with reduced cardiovascular mortality and post-infarction death. However, the impact of omega-3 supplementation in congestive heart failure (CHF) is still unknown. This study assesses the effects of omega-3 supplementation on left ventricular (LV) function and remodelling. We assessed, in rats with CHF induced by left coronary ligation, the effects of a 1-week and a 12-week supplementation with omega-3 (450 mg/kg per day) on LV hemodynamics, function and structure. Chronic omega-3 reduces total peripheral resistance due to an increase in cardiac output without modification of arterial pressure. Only chronic omega-3 reduces LV end-diastolic pressure and LV relaxation constant. Moreover, chronic omega-3 decreases LV systolic and diastolic diameters, LV weight and collagen density. Acute and chronic omega-3 increase LV γ-glutamyl-cysteine synthetase and oppose glutathione deficiency resulting in a reduction of myocardial oxidized glutathione. In experimental CHF, long-term omega-3 supplementation improves LV hemodynamics and function and prevents LV remodelling and glutathione deficiency. The latter might be one of the mechanisms involved, but whether other mechanism, independent of myocardial redox 'status', such as reduced inflammation, are implicated remains to be confirmed.

Cytochrome-derived epoxyeicosatrienoic acids may be important endothelium-derived hyperpolarizing factors, opening calcium-activated potassium channels, but their involvement in the regulation of arterial stiffness during changes in blood flow in humans is unknown. In healthy volunteers, we measured arterial pressure, radial artery diameter, wall thickness, and flow (NIUS02) during hand skin heating in the presence of saline or inhibitors of NO synthase ( N G -monomethyl- l -arginine), calcium-activated potassium channels (tetraethylammonium), and cytochrome epoxygenases (fluconazole). Arterial compliance and elastic modulus were calculated and fitted as functions of midwall stress to suppress the confounding influence of geometric changes. Under saline infusion, heating induced an upward shift of the compliance-midwall stress curve and a downward shift of the modulus-midwall stress curve demonstrating a decrease in arterial tone and stiffness when blood flow increases. These shifts were reduced by N G -monomethyl- l -arginine and abolished by the combinations of N G -monomethyl- l -arginine+tetraethylammonium and N G -monomethyl arginine+fluconazole. In parallel, in isolated mice coronary arteries, fluconazole and tetraethylammonium reduced the relaxations to acetylcholine. However, fluconazole did not affect the relaxations to the openers of calcium-activated potassium channels of small- and intermediate-conductance NS309 and of large-conductance NS1619 excluding a direct effect on these channels. Moreover, tetraethylammonium reduced the relaxations to NS1619 but not to NS309, suggesting that the endothelium-derived hyperpolarizing factor involved mainly acts on large-conductance calcium-activated potassium channels. These results show in humans that, during flow variations, arterial stiffness is regulated by the endothelium through the release of both NO and cytochrome-related endothelium-derived hyperpolarizing factor.

Systemic sclerosis (SSc) is a systemic inflammatory disorder, resulting in severe vascular dysfunction. The endothelin (ET) system has vasoconstrictor and profibrotic properties and has been shown to be activated in SSc. ET antagonists are currently used in SSc-related pulmonary arterial hypertension, but the endothelial impact of ET antagonists remains less known in SSc. We thus assessed the effects of the dual ET(A)-ET(B) antagonist, bosentan, on endothelial dysfunction in a murine model of SSc, the heterozygous tight-skin mice 1 (TSK1(+)). Six-week-old TSK1(+) were either untreated or treated for 6 weeks with bosentan (100 mg/kg/day), and compared with controls. Endothelial function was evaluated in isolated mesenteric resistance arteries, using a small vessel myograph. TSK1(+) displayed endothelial dysfunction, as shown by a decreased response of mesenteric arteries to acetylcholine, especially in the presence of L-nitro-arginine methyl ester (L-NAME), corresponding to NO-independent, prostaglandin-mediated relaxation. The NO-independent relaxation was partially restored in bosentan-treated TSK1(+), and this was abolished by a cyclo-oxygenase inhibitor. Therefore, the murine model of SSc, TSK1(+) exhibits severe endothelial dysfunction of peripheral resistance arteries. The ET antagonist bosentan prevents endothelial alterations, suggesting a major role of ET in the adverse vascular effects of SSc.

A premature atherosclerosis has been presumed in patients with antiphospholipid syndrome. The potential role of antiphospholipid antibodies in the development of atheroma is rather controversial. In this study, we tested the hypothesis that antiphospholipid antibodies could induce atherosclerosis via vascular functional changes. CD1 mice received one single injection of antiphospholipid monoclonal antibodies derived from male (BXSB × NZW) F1 mice with a lupus-like disease associated with an antiphospholipid syndrome and coronary artery disease. One week later, first-order mesenteric arteries (diameter 220–260 μm) were isolated and mounted on a small-vessel myograph for the measurement of the relaxation responses to acetylcholine or the NO donor nitroprusside after precontraction by phenylephrine. Five out of eight antiphospholipid monoclonal antibodies reduced the response to acetylcholine compared with control mice, and this effect was especially marked with one of them. No change in the response to nitroprusside was observed. The impairment was maintained after 3 weeks of treatment and appeared related to a moderate decrease in NO-mediated responses and a marked decrease in prostanoid-mediated relaxations. These vascular functional changes could be prevented by chronic treatment with statins or aspirin. These data could constitute additional elements supporting a direct pathogenic role of antiphospholipid antibodies. We suggest that a sub-population of these autoantibodies could be responsible for the endothelial dysfunction observed in antiphospholipid syndrome.

Background— Cardiomyocyte-specific overexpression of aldosterone synthase in male (MAS) mice induces a nitric oxide–independent coronary dysfunction. Because calcium-activated potassium (BKCa) channels are essential for vascular smooth muscle cell (VSMC) relaxation, we hypothesized that aldosterone alters their expression and/or function in VSMCs. Methods and Results— Left coronary artery segments were isolated from MAS or male wild-type mice and mounted in a wire myograph. Responses to acetylcholine were assessed (in the presence of a nitric oxide synthase inhibitor) without or with the cyclooxygenase inhibitor diclofenac, the KCa inhibitors charybdotoxin plus apamin, or the BKCa inhibitor iberiotoxin. Expression of BKCa was quantified in hearts by real-time quantitative polymerase chain reaction and Western blot and in isolated coronary arteries by polymerase chain reaction. The effect of aldosterone on BKCa expression also was studied in cultured rat aortic VSMCs. Acetylcholine-mediated coronary relaxation was markedly decreased in MAS mice and was prevented by spironolactone. Diclofenac did not affect the MAS-induced impairment in the responses to acetylcholine, whereas charybdotoxin plus apamin virtually abolished the relaxation in both male wild-type and MAS mice. After iberiotoxin, relaxation to acetylcholine was decreased to a larger extent in male wild-type than in MAS, leading to similar levels of relaxation. BKCa-α and -β1 subunit expressions were significantly decreased in MAS heart and coronary arteries. In cultured VSMCs, aldosterone induced a concentration-dependent decrease in BKCa expression, which was prevented by spironolactone. Conclusions— Aldosterone overexpression altered VSMC BKCa expression and coronary BKCa-dependent relaxation. The resulting alteration of relaxing responses may contribute to the deleterious effects of aldosterone in cardiovascular diseases. BKCa channels may therefore be useful therapeutic targets in cardiovascular diseases.

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation. Among the candidate proteins harbored by MPs, we recently showed that sonic hedgehog (Shh) is present in MPs generated from activated/apoptotic human T lymphocytes [Martínez et al., Blood (2006) vol. 108, 3012-3020]. We show here that Shh carried by MPs induces nitric oxide (NO) release from endothelial cells, triggers changes in the expression and phosphorylation of enzymes related to the NO pathway, and decreases production of reactive oxygen species. When PI3-kinase and ERK signaling were specifically inhibited, the effects of MPs were reversed. In vivo injection of MPs in mice was also able to improve endothelial function by increasing NO release, and it reversed endothelial dysfunction after ischemia/reperfusion. Silencing the effects of Shh with cyclopamine, a specific inhibitor of Shh, or siRNA, an inhibitor of the Shh receptor Patched, strongly reduced production of NO elicited by MPs. Taken together, we propose that the biological message carried by MPs harboring Shh may represent a new therapeutic approach against endothelial dysfunction during acute severe endothelial injury.

A defect in neo-vascularization process involving circulating angiogenic mononuclear cells (CACs) dysfunction is associated with diabetes. We showed that oxidative stress was elevated in CACs cultured from blood of individuals with metabolic syndrome (MetS) and diabetes. We then assessed the action of palmitic acid (PA), a deregulated and increased NEFA in metabolic disorders, focusing on its oxidant potential. We observed that the phyto-polyphenol resveratrol normalized oxidative stress both in CACs isolated from MetS patients or treated with PA. Resveratrol further decreased the deleterious action of PA on gene expression of vascularization factors (TNFα, VEGF-A, SDF1α, PECAM-1, VEGFR2, Tie2 and CXCR4) and improved CAC motility. Particularly, resveratrol abolished the PA-induced over-expression of the pro-oxidant protein p66Shc. Neither KLF2 nor SIRT1, previously shown in resveratrol and p66Shc action, was directly involved. Silencing p66Shc normalized PA action on VEGF-A and TNFα specifically, without abolishing the PA-induced oxidative stress, which suggests a deleterious role of p66Shc independently of any major modulation of the cellular oxidative status in a high NEFA levels context. Besides showing that resveratrol reverses PA-induced harmful effects on human CAC function, certainly through profound cellular modifications, we establish p66Shc as a major therapeutic target in metabolic disorders, independent from glycemic control.

Circulating angiogenic cells (CACs) are monocyte-derived cells with endothelial characteristics, which contribute to both angiogenesis and arteriogenesis in a paracrine way. Interferon-β (IFN-β) is known to inhibit these divergent processes in animals and patients. We hypothesized that IFN-β might act by affecting the differentiation and function of CACs. CACs were cultured from peripheral blood mononuclear cells and phenotypically characterized by surface expression of monocytic and endothelial markers. IFN-β significantly reduced the number of CACs by 18–64%. Apoptosis was not induced by IFN-β, neither in mononuclear cells during differentiation, nor after maturation to CACs. Rather, IFN-β impaired adhesion to, and spreading on, fibronectin, which was dependent on α5β1(VLA-5)-integrin. IFN-β affected the function of VLA-5 in mature CACs, leading to rounding and detachment of cells, by induction of calpain 1 activity. Cell rounding and detachment was completely reversed by inhibition of calpain 1 activity in mature CACs. During in vitro capillary formation, CAC addition and calpain 1 inhibition enhanced sprouting of endothelial cells to a comparable extent, but were not sufficient to rescue tube formation in the presence of IFN-β. We show that the IFN-β-induced reduction of the numbers of in vitro differentiated CACs is based on activation of calpain 1, resulting in an attenuated adhesion to extracellular matrix proteins via VLA-5. In vivo, this could lead to inhibition of vessel formation due to reduction of the locally recruited CAC numbers and their paracrine angiogenic factors.

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 × 2-min ischemia/5-min reperfusion and 2 × 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 ± 3; I/R 44 ± 6; PC 74 ± 5%). This protective effect was abolished by l-NAME (41 ± 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.

Background Estrogens have protective effects in the cardiovascular system, as evidenced by the decreased incidence of cardiovascular diseases (CVD) in premenopausal compared with postmenopausal women. Estrogens reduce vascular damaging triggered by CVD like hypertension, through their nuclear receptors, mainly estrogen receptor alpha (ERα). However, recent studies have shown that a fraction of ERα is located at the cell membrane where it initiates a Membrane Initiated Steroid Signaling (MISS). Both membrane and nuclear pathways of ERα seem to participate to the protective effect of estrogens but their respective role in hypertension is unknown. This study aims to better understand the protective role of ERα in hypertension and its vascular consequences. Methods Five groups of female mice were used in this study, each one lacking either estrogens (WT-OVX), ERβ (ERβ-KO), ERα (ERα-KO), mem-brane-ERα (C451A-ERα) or ligand-dependent-transcriptional function of ERα (AF2°). Half of the mice received a chronic infusion of angiotensin II (0,5 mg/kg/d during 1 month) to induce moderate hypertension. Systolic blood pressure (SBP), heart rate, vascular structure and reactivity were measured in KO mice and compared to wild-type mice (WT). Results SBP increased from 117±3,05 to 131±2,90 mmHg (ns) in WT mice. A similar increased was observed in C451A mice (124±7,18 vs 103±5,59 mmHg, ns). By contrast, SBP increased significantly more in WT-OVX mice (144±4,97 vs 113±3,17 mmHg, p=0,0007), ERα-KO (155±4,02 mmHg vs 110±2,36 mmHg, p Conclusions Consistently with the literature, we found that estrogens through ERa activation and not ERβ are protective against angiotensin II-induced hypertension. Interestingly, mice deficient in ligand dependent-tran-scriptional-function also developed hypertension whereas mice lacking membrane ERα did not. Taken together, these date suggest that the protective effect of ERα against angiotensin II-induced hypertension depend on ERα gene transcription through AF2 and not membrane ERα signaling by contrast with most described vasculoprotective effects of estrogens.

Women, before menopause, are less affected than men by hypertension and atherosclerosis, the main causes of cardiovascular diseases. Estrogens have been shown to protect the vascular endothelium through their nuclear receptors, mainly via the estrogen receptor alpha (ERα). However, recent studies have shown that a fraction of ERα is located at the cell membrane where it initiates a Membrane Initiated Steroid Signaling (MISS). Both membrane and nuclear pathways of ERα play a protective role in endothelial cells but their respective role in hypertension is unknown. To investigate the protective effects of ERα in hypertension, we used (i) mice lacking ERα (ERα-/-), (ii) mice with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function (iii) mice lacking the AF2 ligand-dependant-transcriptional function domain of ERα (AF2°) and (iv) wild type (WT) mice. Hypertension was induced using Angiotensin II (Alzet minipumps) and blood pressure measured using tail cuff. After 28 days vascular structure and function were measured in vitro. Angiotensin II-induced hypertension was greater in ERα-/- and in AF2° mice than in WT and C451AERα mice. These data suggest that the protective effect of ERα against elevated blood pressure involved nuclear AF2-mediated transcriptional functions rather than MISS-mediated function. Changes in pressure were associated with proportional changes in endothelium-dependent dilation and wall thickness. Thanks to this work, we hope to better understand how ERα is able to mediate the protective effects of estrogen against hypertension.

Mineralocorticoid receptor (MR) antagonists slow down the progression of heart failure after myocardial infarction (MI), but the cell-specific role of MR in these benefits is unclear. In the present study, the role of MR expressed in vascular smooth muscle cells (VSMC) was investigated. Two months after coronary artery ligation causing MI, mice with VSMC-specific MR deletion (MI-MRSMKO) and mice treated with the MR antagonist finerenone (MIfine) had improved left ventricular (LV) compliance and elastance when compared to infarcted control mice (MI-CTL), as well as reduced interstitial fibrosis. Importantly, the coronary reserve assessed by magnetic resonance imaging was preserved (difference in myocardial perfusion before and after induction of vasodilatation, ml/mg/min: MI-CTL: 1.1±0.5, NS; MIMRSMKO: 4.6±1.6, P In conclusion, deletion of MR in VSMC improved LV dysfunction after MI, likely through maintenance of the coronary reserve and improvement of coronary endothelial function. MR blockage by finerenone had similar effects. The author hereby declares no conflict of interest

The role of bone marrow-derived cells in stimulating angiogenesis, vascular repair or remodelling has been well established, but the nature of the circulating angiogenic cells is still controversial.The existing literature on different cell types that contribute to angiogenesis in multiple pathologies, most notably ischaemic and tumour angiogenesis, is reviewed, with a focus on subtypes of angiogenic mononuclear cells and their local recruitment and activation.A large number of different cells of myeloid origin support angiogenesis without incorporating permanently into the newly formed vessel, which distinguishes these circulating angiogenic cells (CAC) from endothelial progenitor cells (EPC). Although CAC frequently express individual endothelial markers, they all share multiple characteristics of monocytes and only express a limited set of discriminative surface markers in the circulation. When cultured ex vivo, or surrounding the angiogenic vessel in vivo, however, many of them acquire similar additional markers, making their discrimination in situ difficult.Different subsets of monocytes show angiogenic properties, but the distinct microenvironment, in vitro or in vivo, is needed for the development of their pro-angiogenic function.

Background Small resistance arteries regulate peripheral tissue perfusion following variations in arterial pressure and blood flow. An altered flow-mediated dilation (FMD) in response to intraluminal shear stress is the hallmark of early vascular dysfunction. We recently showed that the adaptive flow-dependent arterial remodeling was controlled by the endothelial estrogen receptor alpha (ERa). Our goal was to evaluate the role of ERa in endothelial mechanosensitive mechanisms related to the acute response to flow, by using multiple models of ERa deficiency in male mice, avoiding the hormonal influence of estrogens encountered in females. Methods Evaluation of the FMD was performed on pressurized mesenteric resistance arteries mounted on an arteriograph following step increase in intraluminal flow (6-100 μl/min). Arteries were isolated from wild-type (WT) and ERa genetically modified male mice deficient in either (i) total ERa (ERaKO), (ii) its ligand-dependent transactivation function AF2 (AF2°) and (iii) the plasmic membrane-located ERa following a point mutation of the palmitoylation (C451A) site of the receptor. Results We first observed a selective attenuation of FMD, without any major modification in response to vasodilator agonists (acetylcholine), in mice deficient in ERa (% dilation 50 μl/min: ERaKO: 41 ± 5 vs. WT: 59 ± 4 p Conclusions We thus show, for the first time, that membrane ERa contributes to arterial shear-sensing irrespective of the presence of an agonist or an antagonist. ERa at the membrane could contribute to vascular homeostasis and the regulation of its expression should now be studied.

The deleterious effects of aldosterone excess demonstrated in cardiovascular diseases might be linked in part to coronary vascular dysfunction. However, whether such vascular dysfunction is a cause or a consequence of the changes occurring in the cardiomyocytes is unclear. Moreover, the possible link between mineralocorticoid receptor (MR)-mediated effects on the cardiomyocyte and the coronary arteries is unknown. Thus we used a mouse model with conditional, cardiomyocyte-specific overexpression of human MR (hMR) and observed the effects on endothelial function in isolated coronary segments. hMR overexpression decreased the nitric oxide (NO)-mediated relaxing responses to acetylcholine in coronary arteries (but not in peripheral arteries), and this was prevented by a 1-mo treatment either with an MR antagonist, vitamin E/vitamin C, or a NADPH oxidase inhibitor. hMR overexpression did not affect coronary endothelial NO synthase content nor its level of phosphorylation on serine 1177, but increased cardiac levels of reactive oxygen species, cardiac NADPH oxidase (NOX) activity, and expression of the NOX subunit gp91phox, which was limited to endothelial cells. Thus an increase in hMR activation, restricted to cardiomyocytes, is sufficient to induce a severe coronary endothelial dysfunction. We suggest a new paracrine mechanism by which cardiomyocytes trigger a NOX-dependent, reactive oxygen species-mediated coronary endothelial dysfunction.

Objective— To assess the coronary endothelial protective effects of 17β-estradiol (E2) and the role of estrogen receptor (ER) α in ischemia/reperfusion (I/R). Methods and Results— E2 exerts protective effects in cardiac I/R. However, the implication in vivo of the endothelium and the cellular targets of the anti-ischemic effects of E2 are unknown. Mice were subjected to I/R (30 minutes of I and 1 hour of R) in vivo, after which acetylcholine-induced relaxation of isolated coronary segments was assessed ex vivo. I/R induced a coronary endothelial dysfunction in untreated ovariectomized mice that was prevented by long-term treatment with E2 in wild-type, but not in ERα −/− , mice. Chimeric mice inactivated for ERα in the hematopoietic compartment remained protected by E2. Further inactivation of endothelial ERα abolished the protective action of E2 on coronary endothelial function in Tie2-Cre(+) ERα f/f mice. More importantly, E2 significantly limited infarct size in wild-type mice but not in mice deficient in endothelial ERα, even in the presence of hematopoietic ERα. Conclusion— Endothelial ERα plays a crucial role in the E2-induced prevention of endothelial dysfunction after I/R. To our knowledge, we demonstrate for the first time, by using unique genetically modified mice, that targeting endothelial protection per se can confer cardiomyocyte protection in I/R.

International audience; Estrogens, and in particular 17beta-estradiol (E2), play a pivotal role in sexual development and reproduction and are also implicated in a large number of physiological processes, including the cardiovascular system. Both acetylcholine-induced and flow-dependent vasodilation are preserved or potentiated by estrogen treatment in both animal models and humans. Indeed, E2 increases the endothelial production of nitric oxide and prostacyclin and prevents early atheroma through endothelial-mediated mechanisms. Furthermore, whereas it prevents endothelial activation, E2 potentiates the ability of several subpopulations of the circulating or resident immune cells to produce proinflammatory cytokines. The balance between these 2 actions could determine the final effect in a given pathophysiological process. E2 also promotes endothelial healing, as well as angiogenesis. Estrogen actions are essentially mediated by 2 molecular targets: estrogen receptor-alpha (ERalpha) and ERbeta. The analysis of mouse models targeted for ERalpha or ERbeta demonstrated a prominent role of ERalpha in vascular biology. ERalpha directly modulates transcription of target genes through 2 activation functions (AFs), AF-1 and AF-2. Interestingly, an AF-1-deficient ERalpha isoform can be physiologically expressed in the endothelium and appears sufficient to mediate most of the vasculoprotective actions of E2. In contrast, AF-1 is necessary for the E2 actions in reproductive targets. Thus, it appears conceivable to uncouple the vasculoprotective and sexual actions with appropriate selective ER modulators.

n-3 polyunsaturated fatty acids (omega-3) supplementation is associated with reduced cardiovascular mortality and post-infarction death. However, the impact of omega-3 supplementation in congestive heart failure (CHF) is still unknown. This study assesses the effects of omega-3 supplementation on left ventricular (LV) function and remodelling. We assessed, in rats with CHF induced by left coronary ligation, the effects of a 1-week and a 12-week supplementation with omega-3 (450 mg/kg per day) on LV hemodynamics, function and structure. Chronic omega-3 reduces total peripheral resistance due to an increase in cardiac output without modification of arterial pressure. Only chronic omega-3 reduces LV end-diastolic pressure and LV relaxation constant. Moreover, chronic omega-3 decreases LV systolic and diastolic diameters, LV weight and collagen density. Acute and chronic omega-3 increase LV γ-glutamyl-cysteine synthetase and oppose glutathione deficiency resulting in a reduction of myocardial oxidized glutathione. In experimental CHF, long-term omega-3 supplementation improves LV hemodynamics and function and prevents LV remodelling and glutathione deficiency. The latter might be one of the mechanisms involved, but whether other mechanism, independent of myocardial redox 'status', such as reduced inflammation, are implicated remains to be confirmed.