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Background/objectives Maternal obesity impacts vascular functions linked to metabolic disorders in offspring, leading to cardiovascular diseases during adulthood. Even if the relation between prenatal conditioning of cardiovascular diseases by maternal obesity and vascular function begins to be documented, little is known about resistance arteries. They are of particular interest because of their specific role in the regulation of local blood flow. Then our study aims to determine if maternal obesity can directly program fetal vascular dysfunction of resistance arteries, independently of metabolic disorders. Methods With a model of rats exposed in utero to mild maternal diet-induced obesity (OMO), we investigated third-order mesenteric arteries of 4-month old rats in absence of metabolic disorders. The methylation profile of these vessels was determined by reduced representation bisulfite sequencing (RRBS). Vascular structure and reactivity were investigated using histomorphometry analysis and wire-myography. The metabolic function was evaluated by insulin and glucose tolerance tests, plasma lipid profile, and adipose tissue analysis. Results At 4 months of age, small mesenteric arteries of OMO presented specific epigenetic modulations of matrix metalloproteinases (MMPs), collagens, and potassium channels genes in association with an outward remodeling and perturbations in the endothelium-dependent vasodilation pathways (greater contribution of EDHFs pathway in OMO males compared to control rats, and greater implication of PGI2 in OMO females compared to control rats). These vascular modifications were detected in absence of metabolic disorders. Conclusions Our study reports a specific methylation profile of resistance arteries associated with vascular remodeling and vasodilation balance perturbations in offspring exposed in utero to maternal obesity, in absence of metabolic dysfunctions.

Aneurysm is the second-most common disease affecting the aorta worldwide after atherosclerosis. While several clinical metabolomic studies have been reported, no study has reported deep metabolomic phenotyping in experimental animal models of aortic aneurysm. We performed a targeted metabolomics study on the blood and aortas of an experimental mice model of aortic aneurysm generated by high-cholesterol diet and angiotensin II in Ldlr

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.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

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.

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.

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.

BackgroundCardiopulmonary bypass (CPB) induces a systemic inflammatory response (SIRS) and affects the organ vascular bed. Experimentally, the lack of pulsatility alters myogenic tone of resistance arteries and increases the parietal inflammatory response. The purpose of this study was to compare the vascular reactivity of the internal thoracic arteries (ITAs) due to the inflammatory response between patients undergoing coronary artery bypass grafting (CABG) under CPB with a roller pump or with a centrifugal pump.MethodsEighty elective male patients undergoing CABG were selected using one or two internal thoracic arteries under CPB with a roller pump (RP group) or centrifugal pump (CFP group). ITA samples were collected before starting CPB (Time 1) and before the last coronary anastomosis during aortic cross clamping (Time 2). The primary endpoint was the endothelium-dependent relaxation of ITAs investigated using wire-myography. The secondary endpoint was the parietal inflammatory response of arteries defined by the measurements of superoxide levels, leukocytes and lymphocytes rate and gene expression of inflammatory proteins using. Terminal complement complex activation (SC5b-9) and neutrophil activation (elastase) analysis were performed on arterial blood at the same times.ResultsExposure time of ITAs to the pump flow was respectively 43.3 minutes in the RP group and 45.7 minutes in the CFP group. Acetylcholine-dependent relaxation was conserved in the two groups whatever the time. Gene expression of C3 and C4a in the artery wall decreased from Time 1 to Time 2. No oxidative stress was observed in the graft. There was no difference between the groups concerning the leukocytes and lymphocytes rate. SC5b-9 and elastase increased between Time 1 and Time 2.ConclusionEndothelium-dependent relaxation of the internal thoracic arteries was preserved during CPB whatever the type of pump used. The inflammatory response observed in the blood was not found in the graft wall within this time frame.Trial registrationName of trial study protocol: IPITA Registration number (ClinicalTrials.gov): NCT04168853.

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.

Introduction Mitochondria are dynamic organelles altering their inner and outer membranes through fission and fusion mechanisms. In a response to shear stress, the mitochondria in endothelial cells (ECs) continuously undergo fusion and fission mechanisms. Objective We hypothesized that alteration in mitochondria dynamic could alter EC functions leading to vascular damage. Methods We investigated the role of Opa1 deficiency in ECs and vascular homeostasis using mice lacking the fusion protein Opa1 (heterozygous Opa1 ± mice). Results Endothelium-mediated dilatation was lower in Opa1 ± than in Opa1 +/+ mice, without change in vascular contractility in isolated arteries. Flow-mediated ECs alignment was reduced in Opa1± compared to Opa1 +/+ mice. We then investigated mitochondrial dynamics-related proteins expression level in ECs submitted to a disturbed shear stress (evidenced by eNOS down-regulation and endothelin-1 up-regulation) and observed a reduction in fusion protein Opa1 mRNA expression and an increase in fission protein Drp1 mRNA expression level. As shear stress determines atherogenesis in large arteries, we investigated mitochondrial dynamics-related proteins in various areas in the mouse aorta. In the area submitted to low shear stress, Opa1 mRNA expression was reduced and Drp1 mRNA expression was increased compared to other parts of the aorta submitted to higher shear stress. These observations led to investigate atherosclerosis in Opa1± mice. These mice, when crossed with LDLr-/- mice and fed high-fat diet, develop more atherosclerosis than Opa1 +/+ mice. Conclusion Thus, reduced mitochondrial fusion altered endothelium-dependent functions and exacerbated plaque formation in atherosclerotic mice. And, in order to investigate the mechanism by which the ECs respond to shear stress, we are studying the effect of mitochondrial dynamic proteins on endothelial cells alignment and functions.

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-β-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.

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.

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

On vascular endothelium, shear stress produces vasorelaxation and outward arterial remodeling. A chronic increase in blood flow induces arterial diameter expansion (normalizing shear stress), and is associated with a compensatory hypertrophy to normalize wall strain. This remodeling is due to Inflammation and nitric oxide (NO) production in resistance arteries. A large number of studies have demonstrated that Thrombospondin-1 (TSP1), a homotrimeric glycoprotein, can have an inhibitory action on the NO pathway and a positive effect on inflammation in vitro. Nevertheless, the role of TSP1 on remodeling is poorly understood. We hypothesized that TSP1 is able to participate in this outward arterial remodeling and hypertrophy by immune cells recruitment. We investigated this remodeling in mesenteric resistance arteries of WT or TSP1-KO mice aged to 12-16 weeks were used. An IgG2a isotype control mAb 2A3 or Ly6G-specific mAb 1A8 are used for neutrophil depletion by in vivo administration. Ligation of mesenteric resistance arteries of WT/TSP1- KO and IgG2a/neutrophil-depleted mice allowed modifying blood flow in vivo, thus exposing arteries to low (LF), normal (NF), or high flow (HF). After 7 days, arteries were isolated for in vitro study. Phenylephrine-mediated contraction and Acetylcholine-mediated relaxation were stronger in HF than in NF vessels on WT and IgG2a control mice. Any difference between HF or NF in TSP1-KO and neutrophil-depleted mice were found. Furthermore, passive diameter analysis revealed a higher diameter in HF than NF in WT and IgG2a mice. These differences are lost in TSP1-KO and neutrophil-depleted mice. These results suggest that TSP1 are essential for remodeling in mesenteric arteries by promoting neutrophils recruitment. 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

Patients with sickle cell disease suffer from painful crises associated with disseminated vaso-occlusions, increased circulating erythrocyte microparticles (MPs), and thrombospondin-1 (TSP1). MPs are submicron membrane vesicles shed by compromised or activated cells. We hypothesized that TSP1 mediates MP shedding and participates in vaso-occlusions. We injected TSP1 to transgenic SAD mice with sickle cell disease and characterized circulating phosphatidylserine+ MPs by FACS. TSP1 stimulated MPs in plasma and initiated vaso-occlusions within minutes. In vitro, TSP1 triggered rapid erythrocyte conversion into spicule-covered echinocytes, followed by MP shedding. MP shedding was recapitulated by peptides derived from the TSP1 carboxyterminus. We purified MPs shed by erythrocytes in vitro and administered them back to SAD mice. MPs triggered immediate renal vaso-occlusions. In vitro, MPs triggered the production of radical oxygen species by endothelial monolayers, favored erythrocyte adhesion, and induced endothelial apoptosis. MPs also compromised vasodilation in perfused microvessels. These effects were inhibited by saturating MP phosphatidylserine with annexin-V, or with inhibitors of endothelial ROS production. We conclude that TSP1 triggers erythrocyte MP shedding. These MPs induce endothelial injury and facilitate acute vaso-occlusive events in transgenic SAD mice. This work supports a novel concept that toxic erythrocyte MPs may connect sickle cell anemia to vascular disease.

Diabetes mellitus is associated with severe cardiovascular disorders involving the renin-angiotensin system, mainly through angiotensin II type 1 receptor (AT1R) activation. Although the angiotensin type 2 receptor (AT2R) opposes the effects of AT1R, with vasodilator and anti-trophic properties, its role in diabetes remains debatable. However, AT2R expression level is activated by estrogens and is involved in adipose tissue maturation and in vasodilatation in type 1 diabetes. We investigated the role of AT2R in perivascular adipose tissue (PVAT)-dependent tone in the aorta in a model of type 2 diabetes in female mice with AT2R(AT2R+/+) or without(AT2R−/−). We used 3 month-old female AT2R+/+ and AT2R−/− mice fed a diabetogenic diet in order to induce type 2 diabetes after 4 months. Mice were intact or ovariectomized. Arteries were isolated for in vitro study of vascular reactivity and left with or without PVAT. In AT2R+/+ mice, in the presence of PVAT, phenylephrine-mediated contraction was increased, but PVAT did not affect endothelium dependent (acetylcholine) and independent (sodium nitroprusside)-relaxation. This effect of PVAT was abolished after ovariectomy. PVAT-dependent contraction was also absent in AT2R−/− mice. On the other hand, in type 2 diabetic mice, PVAT was associated with a reduced vasoconstriction and increased vasodilation. In these diabetic mice, PVAT-dependent relaxation was abolished in AT2R−/− mice whereas it was not affected by ovariectomy. Thus, PVAT, in diabetic female mice, opposes vasoconstriction through a mechanism involving AT2R. Therefore AT2R might represent a new therapeutic target in type 2 diabetes in females.

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins Gαq/11 also mediate flow-induced responses. Here, we investigated whether PECAM-1 and Gαq/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of Gαq/11 and PECAM-1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gαq/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gαq/11–PECAM-1 complex within 30 s and a partial relocalization of the Gαq/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced Gαq/11–PECAM-1 dissociation. These results allow us to conclude that Gαq/11–PECAM-1 forms a mechanosensitive complex and its localization suggests the Gαq/11–PECAM-1 complex is a critical mediator of vascular diseases.

Resistance arteries are the site of the earliest manifestations of many cardiovascular and metabolic diseases. Flow (shear stress) is the main physiological stimulus for the endothelium through the activation of vasodilatory pathways generating flow-mediated dilation (FMD). The role of FMD in local blood flow control and angiogenesis is well established, and alterations in FMD are early markers of cardiovascular disorders. α1-Integrin, which has a role in angiogenesis, could be involved in FMD. FMD was studied in mesenteric resistance arteries (MRA) isolated in arteriographs. The role of α1-integrins in FMD was tested with selective antibodies and mice lacking the gene encoding for α1-integrins. Both anti-α1blocking antibodies and genetic deficiency in α1-integrin in mice (α1−/−) inhibited FMD without affecting receptor-mediated (acetylcholine) endothelium-dependent dilation or endothelium-independent dilation (sodium nitroprusside). Similarly, vasoconstrictor tone (myogenic tone and phenylephrine-induced contraction) was not affected. In MRA phosphorylated Akt and phosphatidylinositol 3-kinase (PI3-kinase) were significantly lower in α1−/−mice than in α1+/+mice, although total Akt and endothelial nitric oxide synthase (eNOS) were not affected. Pharmacological blockade of PI3-kinase-Akt pathway with LY-294002 inhibited FMD. This inhibitory effect of LY-294002 was significantly lower in α1−/−mice than in α1+/+mice. Thus α1-integrin has a key role in flow (shear stress)-dependent vasodilation in resistance arteries by transmitting the signal to eNOS through activation of PI3-kinase and Akt. Because of the central role of flow (shear stress) activation of the endothelium in vascular disorders, this finding opens new perspectives in the pathophysiology of the microcirculation and provides new therapeutic targets.

Resume Le benefice de la perfusion pulsee en circulation extra-corporelle (CEC) reste encore debattu. Au-dela de la difficulte a quantifier la pulsatilite en CEC, l’absence de preuve d’un flux pulse dans la microcirculation a longtemps contribue a cette controverse. Les techniques de microscopie intra vitale ont permis dans les annees 90 de demontrer la realite de la pulsatilite dans la microcirculation. La perfusion pulsee permet la transmission d’une puissance energetique laterale aux tissus assurant la permeabilite capillaire et les echanges entre les milieux vasculaire et extra-vasculaire. Le metabolisme cellulaire s’en trouve ainsi ameliore comme en temoignent de nombreux marqueurs physiologiques et biochimiques.

Objective— Blood flow is altered in metabolic and ischemic diseases with dramatic consequences. Resistance arteries structure and function remodel in response to chronic blood flow changes through a mechanism remaining mainly unknown. We hypothesized that the NO pathway and matrix metalloproteases (MMPs) activation might play a role in flow (shear stress)-induced microvascular remodeling. Methods and Results— Mesenteric resistance arteries were ligated to alter blood flow in vivo for 4 or 14 days: arteries were submitted to high (HF), low (LF), or normal flow (NF). Rats were treated with L-NAME, the angiotensin converting enzyme inhibitor perindopril or the MMPs inhibitor doxycycline. After 14 days, outward hypertrophic remodeling occurred in HF arteries in association with eNOS overexpression. MMP9 activity increased in the early phase (day 4). HF-remodeling was prevented by L-NAME, eNOS gene knockout, and doxycycline. L-NAME prevented eNOS overexpression and MMPs activation whereas doxycycline only prevented MMPs activation. In LF arteries diameter reduction was associated with a decreased eNOS expression without change in MMPs expression and activation. LF-remodeling was reduced by perindopril. Conclusions— In resistance arteries, high flow induced diameter enlargement and wall hypertrophy associated with the sequential activation of eNOS and MMP9.

Modifications of the intra-uterine environment are now recognized as an important cause of fetal stress. Leading to several responses such as loss of structure/function and pre-emptive adaptations to an adverse post-natal environment, and finally to adult diseases. It has been demonstrated that children of diabetic mothers have an increased risk of developing cardiovascular diseases (i.e. hypertension) during adulthood. Blood vessels are able to reorganize their structure in response to physiological alteration of blood flow or pathological stimuli. Constrictive vascular remodeling is currently associated with the occurrence of cardiovascular diseases. Thus, our objective was to study vascular remodeling in case of in utero exposure to maternal diabetes. This is in order to investigate the effect of intra-uterine environment perturbations on vascular fetal programming. We have developed an animal model of rats exposed in utero to moderated maternal hyperglycemia (DMO), which developed an hypertension around 6 months of age. We analyzed structure of elastic and resistance arteries (internal diameter, MCSA, intima-media thickness and remodeling parameters) in absence or presence of established hypertension in male DMO compared to controls (CMO) (at 3 and 18 months of age). Moreover, by in vivo sequential ligation of mesenteric arteries, we have studied vascular remodeling of resistance arteries in response to low (LF), normal (NF) or high flow (HF). In old DMO, we did not observe any vascular remodeling induced by hypertension neither in aorta nor in mesenteric arteries. This could be related to the increase of smooth muscle cell attachments that we observed. Moreover, after 1 or 3 weeks of mesenteric arteries ligation, DMO did not exhibit any constrictive remodeling in LF although expansive remodeling in HF is maintained. Interestingly, we measured an increase of eNOS activity and GP91 in LF arteries of theses animals although transglutaminase-2 protein expression was not modified. Our results demonstrate that in utero exposure to maternal diabetes induce modification of vascular remodeling mechanisms in adulthood. Absence of vascular response could be a pre-emptive adaptation to fetal programmed-hypertension in these animals.

Background: Defects in mitochondrial dynamics have been associated with various disorders, including cardiovascular diseases. OPA1 is essential for mitochondrial inner membrane fusion and mutation in OPA1 is associated with autosomal dominant optic atrophy. Since OPA1 has been reported to be associated with cell apoptosis, cell proliferation, mitochondrial ATP synthesis, calcium homeostasis and reactive oxygen species (ROS) production, we investigated its role in vascular function and/or structure in physiological and pathological condition like hypertension. Methods and Results: We used the heterozygous Opa1 mouse model carrying the recurrent Opa1 delTTAG mutation and OPA1 silencing in artery smooth muscle and endothelial cells. Electron microscopy revealed altered mitochondrial cristae structure in vascular smooth muscle cells and endothelial cells of Opa1+/- mice, 6 month-old Opa1+/- mice had a normal baseline blood pressure and vascular function (contraction and dilation). A chronic treatment with L-NAME induced hypertension in mice. Systolic blood pressure was significantly greater in Opa1+/- than in wild type (WT) mice. This was associated with a stronger reduction in endothelium-dependent relaxation to acetylcholine of resistance arteries in Opa1+/- than in WT animal. On the other hand, hypertension-induced wall hypertrophy in the aorta was absent in Opa1+/- in association with reduced proliferation and increased apoptosis of vascular cells. Conclusions: Thus mitochondria alteration due to OPA1 down-regulation did not affect baseline blood pressure and vascular tone but induced an excessive elevation in blood pressure in hypertension. These results suggest for the first time that OPA1 may play an important role in protection of the vasculature in pathological conditions such as hypertension.

Objective: We examined the arterial phenotype of semicarbazide-sensitive amine-oxidase null mouse (SSAO −/−) using various techniques including high resolution echotracking. Methods and results: SSAO −/− mice showed no change in arterial pressure under anesthesia. The in vivo arterial diameter, only measured in the carotid artery (CA), was higher in SSAO −/− than in SSAO +/+ animals. Elastic modulus–wall stress curves and CA rupture pressure were similar between SSAO −/− and +/+ mice, indicating no change in arterial wall stiffness or mechanical strength. There was no significant difference in insoluble elastin, total collagen content and elastic lamellar morphology between the two genotypes. No alteration in vascular reactivity was observed in aortic rings and mesenteric arteries from SSAO −/− mice. Aortic lysyl oxidase (LO) activity remained unaltered, indicating that SSAO invalidation is not accompanied by a compensatory increase in LO activity. Conclusion: This is the first functional study of arteries lacking SSAO. Our results indicate that SSAO −/− mice present an increased arterial diameter associated with normal arterial mechanical properties, suggesting that SSAO deficiency might contribute to arterial wall remodeling. However, these results argue against the hypothesis that SSAO intervenes in elastic fibre organization, elastin cross-linking processes and vasoreactivity.

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The influence of cyclooxygenase pathway activation following thromboxane-endoperoxide (TP) receptor stimulation was studied in rat mesenteric resistance arteries (n=6 to 10 per group). We studied isolated, perfused, and pressurized mesenteric resistance arteries (mean internal diameter 214 μm) using an arteriograph, enabling us to study arteries in physiological conditions of flow and pressure. Changes in diameter were continuously recorded, and contractions measured as internal diameter reduction. Release of cyclooxygenase pathway metabolites was also assessed by enzyme immunoassay (EIA) analysis of mesenteric bed perfusions. The thromboxane A2 (TxA 2 ) analog U-46619 (1 μmol/L) induced a significant contraction (108 μm maximal diameter reduction). Inhibition by 3 chemically different cyclooxygenase inhibitors (ie, flurbiprofen, indomethacin, and aspirin) potently reduced the contraction to 27%, 25%, and 6% of control, respectively. The selective cyclooxygenase-1 inhibitor SC-58560 inhibited U-46619 contraction, whereas selective cyclooxygenase-2 inhibition (SC-58236) had no effect. Thromboxane synthase inhibition (furegrelate) did not affect U-46619-induced contraction, but it was reduced by cytosolic phospholipase A2 inhibition. Measurement of cyclooxygenase derivatives produced by the isolated mesenteric bed showed that PGE 2 was produced after TxA 2 -receptor stimulation with U-46619. Exogenous prostaglandin E 2 (in the presence of the TxA 2 receptor antagonist SQ 29 548) and U-46619 contracted mesenteric arteries with a similar potency (EC 50 : 0.30 and 0.48 μmol/L, respectively). This study provides the first evidence that TxA 2 -receptor-dependent contraction in a resistant artery involved cyclooxygenase stimulation and, at least in part, a PGE 2 formation. This mechanism of TxA 2 -dependent contraction in resistant arteries might be of importance in the understanding of diseases affecting resistant arteries and involving TxA 2 , such as hypertension.

Dystrophin has a key role in striated muscle mechanotransduction. In mice lacking the gene encoding for dystrophin (mdx mice), the absence of dystrophin and several other proteins of the dystrophin-glycoprotein complex induces a defect in flow (shear stress)–mediated NO-dependent dilation (FMD). Because the endothelium is essential for the adaptation of arteries to chronic changes in blood flow, the long-term consequences of this vascular deficiency might affect flow-induced vascular remodeling. Thus, we submitted mouse mesenteric resistance arteries to chronic changes in flow by alternatively ligating arteries. Arteries were thus submitted to high flow (HF), low flow (LF), or normal flow. After 2 weeks, arteries were studied in vitro in an arteriograph. Increases in diameter (from 174±10 to 210±15 μm, pressure 75 mm Hg) found in HF arteries were not significant in mdx mice. Arterial diameters in LF arteries decreased similarly in control and mdx mice. FMD increased in HF arteries and decreased in LF arteries. FMD was not increased in HF arteries in mdx mice. NO-dependent FMD and NO synthase expression increased in the HF arteries of control mice but not in those of mdx mice. Dilatory and contractile tone, depending on the smooth muscle, was unaffected in HF arteries but decreased in LF arteries of both strains. We conclude that resistance arteries of mdx mice do not adapt properly to chronic changes in flow, inasmuch as the increases in diameter, endothelial NO synthase expression, and FMD did not occur in mdx mice submitted to HF for 2 weeks. This study suggests that blood flow regulation might be disturbed in dystrophin-related myopathies, possibly increasing organ damage.

Objective— Desmin, an intermediate filament, has a key role in the integrity of myocytes, and its absence induces cardiomyopathies. Mice lacking desmin (Des−/− group) exhibit microvascular dysfunction leading to smooth muscle hyporeactivity. We investigated the effect of the absence of desmin in mice (Des−/− mice versus Des+/+ mice) on the adaptation of mesenteric arteries to changes in blood flow. Methods and Results— With the use of selective ligations of second-order mesenteric arteries, blood flow was either diminished (low flow [LF]) or elevated (high flow [HF]); respective LF to HF values were 136±18 to 206±29 μL/min for Des+/+ mice and 119±14 to 189±24 μL/min for Des−/−mice in daughter arteries. Two weeks after ligation, arteries were mounted in an arteriograph, allowing the measurement of diameter under controlled conditions of pressure and flow. In HF arteries, diameter changes in response to increases in pressure were higher in Des−/− mice than in Des+/+ mice. Conversely, in LF arteries, diameter was lower in Des−/− mice. Flow-dependent dilation was higher in HF arteries and lower in LF arteries than in control arteries. This adaptation was lower in Des−/− mice than in Des+/+ mice (11.6±3.1% versus 25.5±4.8% dilation, respectively). Endothelial NO synthase expression increased in HF arteries in both strains. Conclusions— These findings provide a demonstration of the role of the intermediate filament desmin in microvascular remodeling. This dysfunction might take place in desmin-related myopathies.

Thromboxane A2 (TxA2) is a potent proaggregating, vasoconstrictor agent produced in many physiological and pathological situations. Although mitogen-activated protein (MAP) kinases [MAPK (ERK1/2 and p38)] have been shown to be activated after endoperoxide/thromboxane receptor (TP) stimulation, no study has investigated their potential role in resistance arteries, especially in physiological conditions of pressure and flow in which the arteries can contract. Thus, responses to TP stimulation by the stable agonist U46619 were studied in isolated rat mesenteric resistance arteries (inner diameter 262 ± 5 µm) mounted in an arteriograph. Changes in diameter were recorded under physiological levels of flow (90 µl/min) and pressure (50 mm Hg). TP stimulation induced a concentration-dependent contraction (EC50 value of 1.94 ± 0.22 × 10–7M), without desensitization. U46619-induced contraction was inhibited by calcium entry blockade (nifedipine) and protein kinase C inhibition (GF109203X), but it was not affected by tyrosine kinase inhibition (tyrphostin A25). MAPKK (MEK) inhibition (PD98059) did not alter U46619-dependent contraction, although ERK1/2 MAPK were activated. By contrast, p38 MAPK inhibition (SB203580) dose-dependently inhibited the contraction, and Western blot analysis showed activation of p38 MAPK in arteries contracted with U46619. Activation of p38 MAPK by U46619 was inhibited by nifedipine and in the absence of extracellular calcium. This study brings new insights in the transduction pathway involved in the contractile response of resistance arteries to TxA2/endoperoxide receptor stimulation. This contraction requires p38 MAPK activation, but did not involve ERK1/2 MAPK activation although both were activated.

Kinin B1-receptors are induced by various inflammatory stimuli. Since myocardial ischaemia-reperfusion results in inflammation, we questioned whether it could induce B1-receptor-dependent responses to des-Arg9-bradykinin (DBK). Thirty-six rabbits were submitted either to a 30 min coronary occlusion followed by a 3 h reperfusion or to a sham operation. The response to DBK was then tested in vivo on mean arterial pressure (MAP) and in vitro on isolated hearts and arterial rings. DBK induced a dose-dependent decrease in MAP in the ischaemia-reperfusion group (DBK, 10 μg kg−1, intra-arterial: -12 ± 2 vs. -5 ± 2 mmHg in the sham group, P < 0.02), which was significantly antagonised by [Leu8]-des-Arg9-bradykinin (LBK), a B1-receptor antagonist. Following ischaemia-reperfusion, isolated hearts responded to DBK by a decrease in coronary perfusion pressure greater than that of the sham group. DBK dose-dependently decreased the isometric force of isolated carotid rings (DBK, 10−5m: -9 ± 2 vs. -1 ± 2 % in the sham group, P < 0.02) and mesenteric arteries (DBK, 10−6m: -38 ± 7 %vs. -3 ± 2 % in the sham group, P < 0.001). The vascular effects of DBK seen after ischaemia-reperfusion were significantly antagonised by LBK. The presence of B1-receptors in ischaemia-reperfusion animals was confirmed by immunolocalisation and Western blot analysis. This study demonstrates that myocardial ischaemia-reperfusion induces a global induction of functional kinin B1-receptors in the endothelium. B1-receptors are inducible receptors. They were first described by Regoli et al. (1977) who showed that large rabbit arteries, which normally do not respond to des-Arg9-bradykinin (DBK), gradually increased their sensitivity to this B1-receptor agonist when incubated in vitro in normal conditions. These authors also demonstrated that intravenous administration of bacterial lipopolysaccharide (LPS) 5 or 20 h earlier is able to induce hypotension or vasorelaxation in response to DBK by promoting the de novo formation of B1-receptors (Regoli et al. 1981). Various inflammatory stimuli are able to induce B1-receptors (Marceau et al. 1997). In particular, cytokines such as interleukin-1 (IL-1) have been shown to enhance the development of the response to DBK (de Bloiset al. 1988) and could be one of the mediators of the effect of LPS. The induction of B1-receptors is also enhanced by angiotensin converting enzyme (ACE) inhibition or bradykinin (BK) administration, possibly because of the inflammatory effect of the increase in kinin concentrations (Nwator et al. 1989). Ischaemia-reperfusion is known to induce an inflammatory response that contributes to tissue injury; it is associated with the activation of monocytes and neutrophils, and the production of cytokines (Entman et al. 1991). In particular, IL-1 is released by endothelial cells upon hypoxia-reoxygenation (Ala et al. 1992), and its mRNA expression is increased in ischaemic myocardium (Herskowitz et al. 1995). Also, kinins are released during ischaemia in different species (Hashimoto et al. 1978; Matsuki et al. 1987; Baumgarten et al. 1993) and could contribute to the inflammatory response. Thus, the aim of this study was to investigate whether myocardial ischaemia-reperfusion could induce B1-receptor-dependent responses. The effect of DBK following myocardial ischaemia-reperfusion was evaluated in vivo in anaesthetised rabbits and in vitro on isolated rabbit hearts and arterial rings. The presence of cardiovascular B1-receptors was also confirmed by Western blot analysis and immunohistochemical localisation.

Abstract —The aim of this study was to evaluate the involvement of the mitogen-activated protein kinase (ERK1/2) pathway in response to stretch in a blood vessel developing myogenic tone on stretch. Indeed, in resistance arteries and veins, the main effect of pressure is to induce a maintained vasoconstrictor (myogenic) tone. Isolated segments of rabbit facial vein were mounted in organ baths and submitted to isometric stretch. In this experimental model, myogenic tone was absent when the bath temperature was 33°C. ERK1/2 activity was determined in each isolated segment by an in-gel kinase assay. Wall tension and ERK1/2 activity were measured in the same samples in the presence (39°C) or in the absence of myogenic tone (33°C). At 39°C, a 5-mN wall tension induced myogenic tone (5.7±1.8 mN) and an increase in ERK1/2 activity (282±52% versus unstretched vessels, P −7 mol/L), but not the calcium-dependent PKC blocker Go-6976 (10 −6 mol/L), inhibited myogenic tone. However, ERK1/2 activity was not affected by either PKC blocker. Genistein (10 −7 mol/L), a general tyrosine kinase inhibitor, but not herbimycin A (5×10 −7 mol/L), a cSrc-family tyrosine kinase inhibitor, suppressed stretch-induced ERK1/2 activation ( P −6 mol/L), a voltage-dependent calcium entry inhibitor, and ryanodine (10 −6 mol/L), which depletes calcium stores, both inhibited ERK1/2 activity (113±12% and 121±7%, respectively; P −6 mol/L) also inhibited ERK1/2 activation without affecting myogenic tone. The present results suggest that stretching the rabbit facial vein induced 2 distinct pathways, one leading to myogenic tone (via a non–calcium-dependent PKC activation) and one leading to ERK1/2 activation through a calcium-dependent pathway involving tyrosine kinase.

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.

Resistance arteries control local blood flow. Chronic increases in blood flow in RA occur in growth, pregnancy, exercising as well as to compensate ischemia and induce outward hypertrophic remodeling associated with improved endothelium dilation. As estrogens have a key role in flow-mediated remodeling, we hypothesized that resveratrol, suggested to act as phytoestrogen, may improve flow-mediated remodeling in ovariectomized rats. Blood flow was increased in vivo in one mesenteric resistance artery without other changes in pressure or circulating factors in three-month old ovariectomized female rats treated with resveratrol (5 or 37.5mg/kg per day in osmotic minipumps: RESV5 or RESV37.5) or solvent (DMSO). After 2 weeks arterial structure and function were measured in vitro. Arterial diameter increased in high flow (HF) compared to normal flow (NF) arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in control rats. Hypertrophy in HF arteries occurred in the 3 groups but was greater in RESV37.5-treated rats so that wall / lumen ratio was elevated in this group. ERK1/2 activation, involved in flow-mediated hypertrophy was greater in RESV37.5-treated rats than in RESV5 – and DMSO-treated rats. RESV5, not RESV37.5, improved L-NAME sensitive arterial relaxation. eNOS expression level was equivalent in HF and NF vessels in all groups animals. Markers of oxidative stress (p67phox, GP91phox) were greater in arteries of RESV37.5-treated rats than in RESV5 – and solvent-treated rats. They were also higher in HF arteries in RESV37.5-treated rats. Thus, although resveratrol improved flow-mediated outward hypertrophic remodeling in OVX rats, the high dose induced oxidative stress and hypertrophy.

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.

Objective: Flow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25 μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days. Results: After 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=9 per group, P Conclusion: we demonstrate for the first time the involvement of lymphocytes T cells polarization into TH17 by angiotensin II type 2 receptor in flow-mediated outward remodeling of resistance arteries.

Rationale: Vascular smooth muscle (SM) cell phenotypic modulation plays an important role in arterial stiffening associated with aging. Serum response factor (SRF) is a major transcription factor regulating SM genes involved in maintenance of the contractile state of vascular SM cells. Objective: We investigated whether SRF and its target genes regulate intrinsic SM tone and thereby arterial stiffness. Methods and Results: The SRF gene was inactivated SM-specific knockout of SRF (SRF SMKO ) specifically in vascular SM cells by injection of tamoxifen into adult transgenic mice. Fifteen days later, arterial pressure and carotid thickness were lower in SRF SMKO than in control mice. The carotid distensibility/pressure and elastic modulus/wall stress curves showed a greater arterial elasticity in SRF SMKO without modification in collagen/elastin ratio. In SRF SMKO , vasodilation was decreased in aorta and carotid arteries, whereas a decrease in contractile response was found in mesenteric arteries. By contrast, in mice with inducible SRF overexpression, the in vitro contractile response was significantly increased in all arteries. Without endothelium, the contraction was reduced in SRF SMKO compared with control aortic rings owing to impairment of the NO pathway. Contractile components (SM-actin and myosin light chain), regulators of the contractile response (myosin light chain kinase, myosin phosphatase target subunit 1, and protein kinase C–potentiated myosin phosphatase inhibitor) and integrins were reduced in SRF SMKO . Conclusions: SRF controls vasoconstriction in mesenteric arteries via vascular SM cell phenotypic modulation linked to changes in contractile protein gene expression. SRF-related decreases in vasomotor tone and cell-matrix attachment increase arterial elasticity in large arteries.

International audience; Background: Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain.Objectives: To investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries.Methods and Results: We examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p

Background Mitochondrial dynamics have been involved in cardiovascular diseases, particularly in ischemia-reperfusion process. While excessive mitochondrial fission has been described as pejorative, the role of fusion proteins (OPA1, MFN2) in this context remains uncertain. Aims To determine the effects of OPA1 (protein implicated in mitochondrial inner membrane fusion) deficiency on cardiac ischemia-reperfusion (I/R) injury. Methods and results We investigated cardiac structure and function (assessed by TTE) of OPA1+/– mutant mice (50% of OPA1 expression decreasing) and found that they displayed a significant alteration of left ventricular systolic function at 6 months, but were similar to Wild-type (WT) at 3 months. 3-month-old OPA1+/– mutant mice and theirs controls were then submitted to I/R in vivo (coronary artery ligature during 45 min/ 2h reperfusion) and ex vivo (30 min of global ischemia / 2h reperfusion). In vivo, infarct size was significantly higher in OPA1+/– mutant mice compared to WT group (43.2±4.1% vs. 28.4±3.5% respectively; p Conclusion Deficiency in the fusion protein OPA1 was associated with higher susceptibility to myocardial I/R injury. Physiopathological mechanisms seem to involve calcium transients modulation, but need further explorations. The author hereby declares no conflict of interest

Background— Patients with sickle cell disease suffer from painful vaso-occlusive crises. These crises are associated with disseminated vaso-occlusions, raised levels of circulating erythrocyte microparticles as well as thrombospondin-1 (TSP1). Microparticules (MP) are submicron membrane vesicles shed by compromised or activated cells. We hypothesized that TSP1 mediates the shedding of erythrocyte MP and participates in vaso-occlusions. Methods and Results— We injected TSP1 to transgenic SAD mice with sickle cell disease, sensitive to renal vaso-occlusions. We characterized circulating MP by FACS after phosphatidylserine labeling. TSP1 injection strongly stimulated MP levels (+260%) and initiated renal vaso-occlusions within 5 minutes, without impacting systemic parameters (heart rate, cardiac output). This was consistent with increased local vascular resistance. In vitro, TSP1 triggered a rapid conversion of purified erythrocytes into echinocytes carrying long and apparently fragile spicules. This was followed by MP shedding. These modifications of erythrocyte phenotype and MP shedding were recapitulated by peptides derived from TSP1 carboxyterminus. We purified the MP shed by erythrocytes in vitro, and administered them back into SAD mice. Erythrocyte MP triggered immediate renal vaso-occlusions, similar to those induced by TSP1. In vitro, purified erythrocyte MP triggered the production of radical oxygen species by endothelial monolayers, favored erythrocyte adhesion to them and induced endothelial apoptosis. Purified erythrocyte MP also compromised acetylcholine-dependent vasodilation in perfused microvessels. These effects of MP were inhibited by saturating surface phosphatidylserine with annexin-V, or with inhibitors of endothelial ROS production. Conclusions— We conclude that TSP1 can trigger the production of microparticles by erythrocytes in sickle cell disease. Second, our data reveal that erythrocyte MP can induce endothelial injury, and this mechanism facilitates the occurrence of vaso-occlusive crises in mice. Therefore, our work supports a novel concept that toxic erythrocyte microparticles constitute a direct connection between sickle cell anemia and vascular disease.

ObjectiveFlow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days.ResultsAfter 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=xx per group, P

International audience; ObjectiveResistance arteries remain subject to pulsatility, a potent regulator of large elastic artery tone and structure, but the effect is incompletely understood. Extracorporeal circulation during cardiac surgery is often associated with absence of pulsatility, which may affect vascular tone. To define the role of the vascular wall in the inflammatory process that may occur with or without pulsatility, we studied resistance arteries functions exvivo. We measured vascular reactivity, oxidative stress, and inflammation in the arterial wall. Methods Isolated rat mesenteric resistance arteries were mounted in an arteriograph and subjected to pulsatility or not invitro. Arteries were perfused with a physiologic salt solution without circulating cells. Results After 180 minutes, flow-mediated dilation was higher and pressure-induced myogenic tone lower in arteries subjected to pulsatility. Without pulsatility, reactive oxygen species and markers of inflammation (monocyte chemotactic protein 1 and tumor necrosis factor α) were higher than baseline. In perfused mesenteric beds under similar conditions, tumor necrosis factor α was higher in perfusate after 180 minutes of nonpulsatility (5.7 ± 1.6 pg/mL vs 1.1 ± 0.4 pg/mL; P&nbsp

International audience; BACKGROUND: Aging is associated with reduced structural and functional adaptation to chronic changes in blood flow (shear stress) in small arteries. As heme oxygenase-1 (HO-1) is induced by hemodynamic forces in vascular smooth muscle and endothelial cells, we hypothesized that it might improve flow-dependent remodeling in aging.METHOD: First-order mesenteric arteries from 3 and 16-month-old rats were exposed to high, low, or normal flow by alternate ligation in vivo. Rats were treated with the HO-1 inducer, cobalt protoporphyrin (CoPP, 5 mg/kg) or vehicle. 14 days later, local blood flow was measured in vivo, and arteries were studied in vitro.RESULTS: Despite an equivalent change in blood flow, diameter enlargement in the high-flow arteries was blunted in old compared to young rats and was associated with decreased endothelium-dependent relaxation to acetylcholine. In old rats, HO-1 induction with CoPP restored outward remodeling, via a paradoxical reactive oxygen species-dependent mechanism, and was associated with a Mn-superoxide dismutase (SOD) overexpression, as well as a significant reduction of mitochondrial aconitase activity, used as a biomarker for oxidative stress. The heme oxygenase activity inhibitor, Sn-protoporphyrin, and the SOD-mimetic, TEMPOL, prevented the effect of CoPP on remodeling and oxidative status in old rats. Furthermore, HO-1 induction improved endothelial function, in association with increased endothelial nitric oxide synthase protein expression and phosphorylation (Ser-1177). In low-flow arteries, inward remodeling was unaffected by aging or by CoPP. Thus, in old rats, CoPP-induced up-regulation of HO-1 restored high-flow-dependent remodeling (diameter enlargement) and improved endothelial function in mesenteric arteries.CONCLUSION: This opens new perspectives in the treatment of ischemic diseases in aging.

International audience; Recent studies reported cardioprotective effects of erythropoietin (EPO) against ischemia–reperfusion (I/R) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been reported to be impaired in diabetes and insulin resistance syndrome, we examined whether EPO-induced cardioprotection was maintained in rat models of type 1 diabetes and insulin resistance syndrome. Isolated hearts were obtained from three rat cohorts: healthy controls, streptozotocin (STZ)-induced diabetes, and high-fat diet (HFD)-induced insulin resistance syndrome. All hearts underwent 25 min ischemia and 30 min or 120 min reperfusion. They were assigned to receive either no intervention or a single dose of EPO at the onset of reperfusion. In hearts from healthy controls, EPO decreased infarct size (14.36 ± 0.60 and 36.22 ± 4.20% of left ventricle in EPO-treated and untreated hearts, respectively, p < 0.05) and increased phosphorylated forms of Akt, ERK1/2, and their downstream target GSK-3β. In hearts from STZ-induced diabetic rats, EPO did not decrease infarct size (32.05 ± 2.38 and 31.88 ± 1.87% in EPO-treated and untreated diabetic rat hearts, respectively, NS) nor did it increase phosphorylation of Akt, ERK1/2, and GSK-3β. In contrast, in hearts from HFD-induced insulin resistance rats, EPO decreased infarct size (18.66 ± 1.99 and 34.62 ± 3.41% in EPO-treated and untreated HFD rat hearts, respectively, p < 0.05) and increased phosphorylation of Akt, ERK1/2, and GSK-3β. Administration of GSK-3β inhibitor SB216763 was cardioprotective in healthy and diabetic hearts. STZ-induced diabetes abolished EPO-induced cardioprotection against I/R injury through a disruption of upstream signaling of GSK-3β. In conclusion, direct inhibition of GSK-3β may provide an alternative strategy to protect diabetic hearts against I/R injury.