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The autonomic sympathetic nervous system innervates peripheral resistance arteries, thereby controlling arterial diameter and modulating blood supply to organs and arterial tone. Despite its fundamental role in blood flow regulation and adaptive response of the cardiovascular system to challenging situations, how sympathetic arterial innervation develops remains poorly understood.We here show that sympathetic arterial innervation is regulated by the axonal guidance molecule EphrinA4 in arterial Smooth Muscle Cells (SMCs), which repels sympathetic axons via the EphA4 receptor. Specific inactivation of EphA4 in sympathetic axons induced a loss of repulsion and increased sympathetic innervation of peripheral arteries throughout life. Functional consequences were a significant increase in arterial tone (resistivity and vasoconstriction), leading to an elevated systemic arterial blood pressure that reached to hypertension under stressful circumstances. These findings identify a novel pathway that negatively regulates sympathetic arterial innervation, and could participate to the appearance of idiopathic resistant hypertension.

The binding of 17β-oestradiol to oestrogen receptor alpha (ERα) plays a crucial role in the control of reproduction, acting through both nuclear and membrane-initiated signalling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss of function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do become pregnant, allowing the investigation of the role of ERα during pregnancy for the first time. All neonatal deaths of the mutant offspring mice resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibited partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality resulted from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells into the utero-placental unit, which is associated with an imbalance in expression of angiogenic factors. Together, these processes control the trophoblast-mediated spiral arterial remodelling. Hence, loss of membrane ERα within maternal tissues clearly alters the activity of invasive trophoblast cells during placentogenesis. This previously unreported function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.

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.

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.

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

International audience; INTRODUCTION:Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biological functions, the contribution of endothelial TXNIP has not been well-defined in regards to endothelial and vascular function or in post-ischemic revascularisation. We postulated that inhibition of endothelial TXNIP with siRNA or in a Cre-LoxP system could be involved in protection from high fat, high protein, low carbohydrate (HFHPLC) diet-induced oxidative stress and endothelial dysfunction, leading to vascular damage and impaired revascularisation in vivo.METHODS AND RESULTS:To investigate the role of endothelial TXNIP, the TXNIP gene was deleted in endothelial cells using anti-TXNIP siRNA treatment or the Cre-LoxP system. Murine models were fed a HFHPLC diet, known to induce metabolic disorders. Endothelial TXNIP targeting resulted in protection against metabolic disorder-related endothelial oxidative stress and endothelial dysfunction. This protective effect mitigates media cell loss induced by metabolic disorders and hampered metabolic disorder-related vascular dysfunction assessed by aortic reactivity and distensibility. In aortic ring cultures, metabolic disorders impaired vessel sprouting and this alteration was alleviated by deletion of endothelial TXNIP. When subjected to ischemia, mice fed a HFHPLC diet exhibited defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. However, reducing endothelial TXNIP rescued metabolic disorder-related impairment of ischemia-induced revascularisation.CONCLUSION:Collectively, these results show that targeting endothelial TXNIP in metabolic disorders is essential to maintaining endothelial function, vascular function and improving ischemia-induced revascularisation, making TXNIP a potential therapeutic target for therapy of vascular complications related to metabolic disorders.

Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear. Objective: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms. Methods and Results: Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17β-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17β-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells. Conclusions: Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.

Exosomes are key regulators of cell-to-cell communication, becoming valuable tool as disease biomarker and in the development of new therapeutic strategies. We aimed to determine the role of endothelial progenitor cell (EPC)-derived exosomes (EXO) in vascular remodeling induced by flow. C57BL/6 mice underwent chronic changes in flow by mesenteric resistance arteries ligation. Arteries were thus submitted to high flow (HF), low flow (LF), or normal flow (NF). The day before surgery mice received I.P. injection with saline or exosomes (3x10 9 particle/kg), following the treatment every 3 days until sacrifice. After 14 days, arteries were studied in vitro in a pressure arteriograph. Increase in diameter and compliance by pressure found in HF arteries were not seen in EXO treated mice (Fig A). Decrease in diameter observed in saline treated LF arteries, was also abolished by EXO treatment. Network analysis of miRNA content in exosomes and arterial mRNA expression revealed an increased expression of components involved in angiogenesis/arteriogenesis (Fig B), which could contribute to flow maintenance in the tissue. Mesenteric arteries submitted to LF, HF and NF were isolated and placed in Matrigel matrix for angiogenesis analysis. We observed that change in flow is a trigger for vasculogenesis, which is enhanced by EXO treatment (Fig C). This study establishes the potential role of EPC-derived exosomes to regulate vascular remodeling during changes in flow. The mechanisms for these effects involve exosome-derived miRNA regulation of a network of mRNAs implicated in collateral angiogenesis/arteriogenesis, which in turn, may contribute to maintenance of tissue homeostasis.

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

Exosomes are key regulators of cell-to-cell communication, becoming valuable tool as disease biomarker and in the development of new therapeutic strategies. We aimed to determine the role of endothelial progenitor cell (EPC)-derived exosomes (EXO) in vascular remodelling induced by flow. C57BL/6 mice underwent chronic changes in flow by mesenteric resistance arteries ligation. Arteries were thus submitted to high flow (HF), low flow (LF), or normal flow (NF). The day before surgery mice received I.P. injection with saline or exosomes (3 × 109 particle/kg), following the treatment every 3 days until sacrifice. After 14 days, arteries were studied in vitro in a pressure arteriograph. Increase in diameter and compliance by pressure found in HF arteries were not seen in EXO treated mice (Figure 1A). Decrease in diameter observed in saline treated LF arteries, was also abolished by EXO treatment. Network analysis of miRNA content in exosomes and arterial mRNA expression revealed an increased expression of components involved in angiogenesis/arteriogenesis (Figure 1B), which could contribute to flow maintenance in the tissue. Mesenteric arteries submitted to LF, HF and NF were isolated and placed in Matrigel matrix for angiogenesis analysis. We observed that change in flow is a trigger for vasculogenesis, which is enhanced by EXO treatment (Figure 1C). This study establishes the potential role of EPC-derived exosomes to regulate vascular remodelling during changes in flow. The mechanisms for these effects involve exosome-derived miRNA regulation of a network of mRNAs implicated in collateral angiogenesis/arteriogenesis, which in turn, may contribute to maintenance of tissue homeostasis. Figure 1

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.

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.

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.

International audience; OBJECTIVES:Chronic increases in blood flow in resistance arteries induce outward remodeling associated with increased wall thickness and endothelium-mediated dilatation. This remodeling is essential for collateral arteries growth following occlusion of a large artery. As estrogens have a major role in this remodeling, we hypothesized that resveratrol, described as possessing phytoestrogen properties, could improve remodeling in ovariectomized rats.METHODS:Blood flow was increased in vivo in mesenteric arteries after ligation of adjacent arteries in 3-month old ovariectomized rats treated with resveratrol (5 or 37.5 mg/kg per day: RESV5 or RESV37.5) or vehicle. After 2 weeks arterial structure and function were measured in vitro in high flow (HF) and normal flow (NF) arteries isolated from each rat.RESULTS:Arterial diameter was greater in HF than in NF arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in vehicle-treated rats. In mice lacking estrogen receptor alpha diameter was equivalent in HF and NF arteries whereas in mice treated with RESV5 diameter was greater in HF than in NF vessels. A compensatory increase in wall thickness and a greater phenylephrine-mediated contraction were observed in HF arteries. This was more pronounced in HF arteries from RESV37.5-treated rats. ERK1/2 phosphorylation, involved in hypertrophy and contraction, were higher in RESV37.5-treated rats than in RESV5- and vehicle-treated rats. Endothelium-dependent relaxation was greater in HF than in NF arteries in RESV5-treated rats only. In HF arteries from RESV37.5-treated rats relaxation was increased by superoxide reduction and markers of oxidative stress (p67phox, GP91phox) were higher than in the 2 other groups.CONCLUSION:Resveratrol improved flow-mediated outward remodeling in ovariectomized rats thus providing a potential therapeutic tool in menopause-associated ischemic disorders. This effect seems independent of the estrogen receptor alpha. Nevertheless, caution should be taken with high doses inducing excessive contractility and hypertrophy in association with oxidative stress in HF arteries.

International audience; Epidemiologic studies have demonstrated that cardiovascular risk is not only determined by conventional risk factors in adulthood, but also by early life events which may reprogram vascular function. To evaluate the effect of maternal diabetes on fetal programming of vas-cular tone in offspring and its evolution during adulthood, we investigated vascular reactivity of third order mesenteric arteries from diabetic mother offspring (DMO) and control mother offspring (CMO) aged 3 and 18 months. In arteries isolated from DMO the relaxation induced by prostacyclin analogues was reduced in both 3-and 18-month old animals although endothelium (acetylcholine)-mediated relaxation was reduced in 18-month old DMO only. Endothelium-independent (sodium nitroprusside) relaxation was not affected. Pressure-induced myogenic tone, which controls local blood flow, was reduced in 18-month old CMO compared to 3-month old CMO. Interestingly, myogenic tone was maintained at a high level in 18-month old DMO even though agonist-induced vasoconstriction was not altered. These perturbations, in 18-months old DMO rats, were associated with an increased pMLC/MLC, pPKA/PKA ratio and an activated RhoA protein. Thus, we highlighted perturbations in the reactivity of resistance mesenteric arteries in DMO, at as early as 3 months of age, followed by the maintenance of high myogenic tone in older rats. These modifications are in favour of excessive vasoconstrictor tone. These results evi-denced a fetal programming of vascular functions of resistance arteries in adult rats exposed in utero to maternal diabetes, which could explain a re-setting of vascular functions and, at least in part, the occurrence of hypertension later in life.

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.

In resistance arteries, a chronic increase in blood flow induces hypertrophic outward remodeling. This flow-mediated remodeling (FMR) is absent in male rats aged 10 mo and more. As FMR depends on estrogens in 3-mo-old female rats, we hypothesized that it might be preserved in 12-mo-old female rats. Blood flow was increased in vivo in mesenteric resistance arteries after ligation of the side arteries in 3- and 12-mo-old male and female rats. After 2 wk, high-flow (HF) and normal-flow (NF) arteries were isolated for in vitro analysis. Arterial diameter and cross-sectional area increased in HF arteries compared with NF arteries in 3-mo-old male and female rats. In 12-mo-old rats, diameter increased only in female rats. Endothelial nitric oxide synthase expression and endothelium-mediated relaxation were higher in HF arteries than in NF arteries in all groups. ERK1/2 phosphorylation, NADPH oxidase subunit expression levels, and arterial contractility to KCl and to phenylephrine were greater in HF vessels than in NF vessels in 12-mo-old male rats only. Ovariectomy in 12-mo-old female rats induced a similar pattern with an increased contractility without diameter increase in HF arteries. Treatment of 12-mo-old male rats and ovariectomized female rats with hydralazine, the antioxidant tempol, or the angiotensin II type 1 receptor blocker candesartan restored HF remodeling and normalized arterial contractility in HF vessels. Thus, we found that FMR of resistance arteries remains efficient in 12-mo-old female rats compared with age-matched male rats. A balance between estrogens and vascular contractility might preserve FMR in mature female rats.

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

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.

International audience; Endothelial dysfunction in resistance arteries alters end organ perfusion in type 2 diabetes. Superoxides and cyclooxygenase-2 (COX-2) derivatives have been shown separately to alter endothelium-mediated relaxation in aging and diabetes but their role in the alteration of vascular tone in old diabetic subjects is not clear, especially in resistance arteries. Consequently, we investigated the role of superoxide and COX-2-derivatives on endothelium-dependent relaxation in 3 and 12 month-old Zucker diabetic fatty (ZDF) and lean (LZ) rats. Mesenteric resistance arteries were isolated and vascular tone was investigated using wire-myography. Endothelium (acetylcholine)-dependent relaxation was lower in ZDF than in LZ rats (60 versus 84% maximal relaxation in young rats and 41 versus 69% in old rats). Blocking NO production with L-NAME was less efficient in old than in young rats. L-NAME had no effect in old ZDF rats although eNOS expression level in old ZDF rats was similar to that in old LZ rats. Superoxide level and NADPH-oxidase subunits (p67phox and gp91phox) expression level were greater in ZDF than in LZ rats and were further increased by aging in ZDF rats. In young ZDF rats reducing superoxide level with tempol restored acetylcholine-dependent relaxation to the level of LZ rats. In old ZDF rats tempol improved acetylcholine-dependent relaxation without increasing it to the level of LZ rats. COX-2 (immunolabelling and Western-blot) was present in arteries of ZDF rats and absent in LZ rats. In old ZDF rats arterial COX-2 level was higher than in young ZDF rats. COX-2 blockade with NS398 restored in part acetylcholine-dependent relaxation in arteries of old ZDF rats and the combination of tempol and NS398 fully restored relaxation in control (LZ rats) level. Accordingly, superoxide production and COX-2 derivatives together reduced endothelium-dependent relaxation in old ZDF rats whereas superoxides alone attenuated relaxation in young ZDF or old LZ rats.

International audience; Shear stress due to blood flow is the most important force stimulating vascular endothelium. Acute stimulation of the endothelium by shear stress induces a vasodilatation mainly due to the release of nitric oxide (NO) among other relaxing agents. After a chronic increase in blood flow (shear stress), the endothelium triggers diameter enlargement, medial hypertrophy and improvement of arterial contractility and endothelium-mediated dilation. Shear stress-mediated outward remodeling requires an initial inflammatory response followed by the production of reactive oxygen species (ROS) and peroxinitrite anions, which activate MMPs and extracellular matrix digestion allowing diameter expansion. This outward remodeling occurs in collateral growth following occlusion of a large artery. In diabetes, an excessive ROS production is associated with the formation of advanced glycation end-products (AGEs) and the glycation of enzymes involved in vascular tone. The balance between inflammation, AGEs and ROS level determines the ability of resistance arteries to develop outward remodeling whereas AGEs and ROS contribute to decrease endothelium-mediated dilation in remodeled vessels. This review explores the interaction between ROS, AGEs and the endothelium in shear stress-mediated outward remodeling of resistance arteries in diabetes. Restoring or maintaining this remodeling is essential for an efficient blood flow in distal organs.

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

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; AIMS: Resistance arteries have a key role in the control of local blood flow and pressure, and chronic increases in blood flow induce endothelium-dependent outward hypertrophic remodelling. The incidence of metabolic syndrome increases with age, and the combination of these two risk factors impairs endothelium integrity, in part through an inflammatory process. We hypothesized that cyclooxygenase-2 (COX2) would affect remodelling in 12-month-old obese rats compared with young rats.METHODS AND RESULTS: Mesenteric arteries of obese and lean Zucker rats were alternatively ligated to generate high flow (HF) in the median artery. After 21 days, arteries were isolated for in vitro analysis. After 21 days, outward hypertrophic remodelling occurred in HF arteries in obese (498 +/- 20 vs. 443 +/- 18 mum intraluminal diameter in normal flow (NF) arteries, P < 0.01), but not in lean rats (454 +/- 17 vs. 432 +/- 14, NS; n = 12 per group). Endothelium-dependent (acetylcholine)-mediated relaxation (AMR) was lower in obese than in lean rats. AMR was reduced by NO-synthase blockade in all groups, and eNOS expression was higher in HF than in NF arteries without difference between lean and obese rats. Indomethacin further reduced AMR in HF arteries from obese rats only. Obesity increased COX2 immunostaining in mesenteric arteries. Acute COX2 inhibition (NS398) significantly reduced AMR in HF arteries from obese rats only, suggesting production of vasodilator prostanoid(s). In obese rats chronically treated with the COX2 inhibitor celecoxib, outward remodelling did not occur in HF arteries and AMR was improved without reaching the level found in lean rats.CONCLUSION: COX2 preserved in part flow-mediated arterial remodelling in old obese rats. Nevertheless, this effect was not sufficient to keep endothelium-dependent relaxation to the level obtained in lean rats.

International audience; Endothelial dysfunction in resistance arteries leads to end organ damages in type 2 diabetes. The capacity of the microcirculation to adapt or remodel in response to changes in blood flow or shear stress is essential for an optimal organ perfusion. Chronic increases in blood flow enhance arterial diameter and endothelium-dependent dilation.Since type 2 diabetes impairs endothelial sensitivity to flow (shear stress) and increases oxidative stress, we hypothesized that Zucker diabetic fatty rats (ZDF) would present an impaired flow-induced remodeling in resistance artery. Aim: The goal of the study was to compare the structural and functional adaptations of mesenteric arteries from lean (LZ) and ZDF rats, to chronic changes in blood flow. Methods: Two mesenteric resistance arteries were ligated in order to increase blood flow in a third artery. This artery was thus submitted, in vivo, to high flow (HF) and compared to normal flow (NF) arteries located at distance. After 3 weeks arteries were studied in vitro (n= 10 rats per group).Results: Arterial diameter (468 vs 394±8µm) and endothelial (acetylcholine)-dependent dilation (91±8 vs 75±6% dilation) were higher in HF than in NF arteries in LZ rats. In ZDF rats, arterial diameter (396±9 vs 440±17µm) and acetylcholine-mediated dilation (42±8 vs 75±7% dilation) were lower in HF than in NF arteries. Nevertheless, endothelial NO-synthase and NADP(H)-oxidase subunits (gp91, p67) expression level and superoxide production were higher in HF than in NF arteries in both LZ and ZDF rats HF suggesting an efficient flow-sensing process in both ZDF and LZ rats. But, in ZDF rats basal oxidative stress was higher compared to LZ rats: a) dihydroethydium staining was higher in both NF and HF arteries in ZDF than in LZ rats and b) acetylcholine-mediated dilation was improved by an acute antioxidant (tempol) in NF and HF arteries from ZDF rats (no effect in LZ rats NF arteries). As a consequence, oxidative stress, slightly increased in LZ rats HF arteries was strongly increased in ZDF rats HF vessels. This led to an excessive superoxide production impairing NO-dependent dilation and HF-remodeling. Finally, a chronic treatment with tempol restored HF arteries diameter (426±13 in NF vs 471±16µm in HF arteries) and endothelium-dependent dilation in ZDF rats. Conclusion: In type 2 diabetic rats increasing blood flow chronically failed to induce outward remodeling and to improve endothelium-dependent dilation; mainly because of superoxide overproduction.

The role of angiotensin II type 2 receptors (AT2R) is not fully understood, especially in vascular diseases. Although its vasodilatory and antitrophic properties are well accepted, in hypertensive rats AT2R stimulation induces vasoconstriction. Abnormalities in the renin-angiotensin system have a key role in cardiovascular disorders associated with the evolution of type II diabetes. Thus, we hypothesized that AT2R function might be altered in diabetic rats resistance arteries. Mesenteric resistance arteries (250 μm diameter) were isolated from type II diabetic Zucker (ZDF) and control rats (LZ) rats receiving the antioxidant tempol (16 mg/kg/day) or water for 3 weeks. Dilation to angiotensin II (due AT2R stimulation) in the presence of candesartan (angiotensin II type 1 receptor blocker) was studied in ring segment of arteries using a wire-myograph mesenteric wall force. AT2R induced endothelium and NO-dependent dilation in LZ (inhibited by L-NAME). It was not affected by cyclooxygenase (indomethacin) or reactive oxygen species (ROS) inhibition (TEMPOL). In ZDF rats AT2R-induced dilation was lower than in LZ rats and independent of NO (not inhibited by L-NAME). It was restored to control level after ROS (tempol) and cycloxygenase inhibition (indomethacin). Cycloxygenase-2 (NS398) and Thromboxane A2 receptor (SQ29548) inhibition also restored AT2R-dilation in ZDF rats to control (LZ) level. Cycloxygenase-2 and ROS were overexpressed (immuno-histological detection using confocal microscopy) in ZDF rats arteries. In ZDF rats chronically treated with the antioxydant Tempol (SOD mimetic) AT2R-induced dilation was equivalent to that in LZ (control) rats and it involved NO and cycloxygenase-2-derivatives in equivalent proportion (dilation inhibited in part by L-NAME and by NS398). In tempol-treated rats SQ29548 had no effect. We conclude that in type II diabetic rats AT2R induced dilation is reduced due to ROS and thromboxane A2-dependent vasoconstriction due to COX-2 activation. Chronic treatment of diabetic rats with tempol restored AT2R-dependent dilation through the suppression of ROS and TxA2 production. These findings might be important to consider in the choice of vasoactive drugs in diabetic patients.

Chronic increases in blood flow increase arterial diameter and NO-dependent dilation in resistance arteries. Because endothelial dysfunction accompanies metabolic syndrome, we hypothesized that flow-mediated remodeling might be impaired in obese rat resistance arteries. Obese and lean Zucker rat mesenteric resistance arteries were exposed to chronic flow increases through arterial ligation in vivo: arteries exposed to high flow were compared with normal flow arteries. Diameter was measured in vitro in cannulated arteries using pressure arteriography. After 7 days, outward remodeling (diameter increased from 346±9 to 412±11 μm at 100 mm Hg) occurred in lean high-flow arteries. Endothelium-dependent tone was reduced in high-flow arteries from obese rats by contrast with lean animals. On the other hand, diameter enlargement occurred similarly in the 2 strains. The involvement of NO in endothelium-dependent dilation (evidenced by NO blockade) and endothelial NO synthase phosphorylation was smaller in obese than in lean rats. Superoxide anion and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression (p67phox and gp91phox) increased in obese rats and were higher in high-flow than in control arteries. Acute Tempol (a catalase mimetic), catalase plus superoxide dismutase, and l -arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat normal and high-flow arteries to the level found in lean control arteries. Thus, flow-induced remodeling in obese resistance arteries was associated with a reduced endothelium-mediated dilation because of a decreased NO bioavailability and an excessive superoxide production. This dysfunction might have negative consequences in ischemic diseases in patients with obesity or metabolic syndrome.

Beside calcification, the impact of ABCC6 deficiency on the vasculature remains unclear. We investigated arterial structure and function in Abcc6-/- mice, a model of the human Pseudoxanthoma Elasticum (PXE). Arterial calcium accumulation determined by atomic absorption spectrometry was 1.5 – to 2-fold higher in Abcc6-/- than in wild-type mice. Calcium also accumulated locally leading to a specific punctuated pattern. Abcc6-/- mesenteric arteries mounted on a wire myograph displayed slight increase in arterial vasoconstrictor tone in response to phenylephrine and thromboxane A2. Interestingly, myogenic tone (Bayliss effect) determined using a pressure myograph was significantly elevated in Abcc6-/- compared to wild type arteries. Arterial blood pressure was not significantly modified in Abcc6-/- animals, despite higher variability. These changes were accompanied with deregulated gene expression (RTqPCR) in both liver and resistance arteries. Old Abcc6-/- mouse mesenteric arteries expressed markers of both osteogenic (Runx2, opn) and chondrogenic lineage (Sox9, col2a1). Surprisingly Enpp1 and Alpl genes encoding ectonucleotide pyrophosphatase/phosphodiesterase 1 and alkaline phosphatase were deregulated within Abcc6-/- liver and this was corroborated with reduced alkaline phosphatase circulating levels in PXE patients. As a conclusion, scattered calcium depositions result from osteochondrogenic transdifferentiation of vascular cells. The lower elasticity and increased myogenic tone evidenced in aged Abcc6-/- mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis. Our findings argue in favor of a deregulated arterial function and may help to decipher consequences of ABCC6 deficiency since PXE is a significant risk factor for small vessel disease and particularly ischemic stroke.

Introduction Le systeme Renine-Angiontensine est un regulateur majeur du systeme cardiovasculaire. Les effets de l’Angiotensine II se font via ses deux principaux recepteurs : AT1R et AT2R. AT1R est bien connu pour ses proprietes hypertensives et hypertrophiques alors qu’AT2R est decrit comme ayant des proprietes vasodilatatrices et hypotrophiques. Il a ete montre que la stimulation d’AT2R chez des rats ZDF entrainait un accroissement des especes reactives oxygenees (ROS) ainsi qu’une augmentation de synthese de thromboxane A2 (TXA2). Nous souhaitons maintenant determiner le role d’AT2R sur la fonction vasculaire dans le diabete. Materiels et methodes Des souris wild-type (WT) ou AT2R-/- sont rendues diabetiques par une injection de streptozotocine (150 mg/kg, ip). Apres 45 jours, les animaux sont sacrifies. Les arteres mesenteriques et l’aorte thoracique sont prelevees et montees sur myographe pour des etudes de reactivite vasculaire. Au niveau aortique, les niveaux d’expression des genes impliques ont ete determines par qPCR. Les donnees histomorphometriques ont ete obtenues a partir d’arteres mesenteriques fixees. Resultats La relaxation endothelium-dependante est significativement diminuee chez les animaux diabetiques WT. Le blocage pharmacologique d’AT2R, des ROS (tempol + catalase) ou l’inhibition de COX-2 t ameliorent cette relaxation. Les animaux AT2R-/- ont une relaxation equivalente chez les animaux diabetiques ou non, Le diabete a egalement induit une remodelage arteriel au niveau des arteres des animaux WT mais pas chez les animaux AT2R-/-. Conclusion Le recepteur AT2R s’oppose a la relaxation endotheliale dans notre modele de diabete, via les ROS et les derives prostanoides. Il est aussi implique dans le remodelage induit par le diabete.

Introduction L’epidemie actuelle d’obesite et de diabete de type II augmente la probabilite de diabete chez la femme enceinte. Si les complications maternelles, obstetricales et fœtales d’une grossesse diabetique sont bien connues, peu d’informations sont disponibles sur le devenir a long terme des enfants nes de meres diabetiques, notamment en terme de pathologie cardiovasculaire. Materiels et methodes Nous avons donc developpe un modele d’exposition in utero au diabete maternel. Le suivi postnatal des rats issus de ces meres diabetiques (DMO) a permis de mettre en evidence une programmation de l’hypertension arterielle (HTA) des l’âge de 6 mois ainsi qu’un profil d’expression genique aortique specifique des rats DMO au stade pre-hypertensif (3 mois) favorisant un phenotype vasoconstricteur. En effet, nous avons montre que differents acteurs du metabolisme de l’acide arachidonique - systeme clef dans la physiologie vasculaire - etaient differentiellement exprimes par rapport aux animaux temoins, avec une baisse de 50% de l’expression du gene du recepteur de la prostacycline et une augmentation de l’expression de plusieurs sous-unites du cytochrome P450. Notre objectif etait ensuite de determiner si la re-programmation vasculaire observee pouvait etre impliquee dans le developpement de l’HTA en comparant la reactivite vasculaire des vaisseaux de resistance des DMO et des rats controles mâles et femelles aux stades pre - (3 mois) et post-hypertensifs (18 mois) et en analysant leur structure histomorphometrique. Resultats Les premiers resultats ne montrent pas, a 18 mois comme a 3 mois, de modification de reactivite vasculaire aux agents pharmacologiques classiques (phenylephrine, achetylcholine…) mais une diminution de la reponse vasodilatatrice a un analogue de la prostacycline (beraprost) aussi bien chez les mâles que chez les femelles En revanche une augmentation du tonus myogenique n’est observe que chez les mâles. Conclusion Ces resultats montrent que la re-programmation vasculaire chez les rats issus de meres diabetiques serait impliquee dans la physiopathologie de l’HTA dans ce modele.

Resistance arteries (RA) have a key role in the control of local blood flow. They undergo outward expansive remodeling in response to a chronic increase in blood flow as seen in post-ischemic collateral arteries growth. This remodeling is associated with improved endothelium-dependent dilation. We hypothesized that estrogens play a role in flow-mediated improvement of endothelium-dependent dilation. Local increase in blood flow in one mesenteric artery was obtained local surgery in three-month old ovariectomized female rats treated or not with 17-b-estradiol (E2). Changes in arterial function and structure were measured after 2 weeks. After 2 weeks, arterial diameter increased in high flow (HF) compared to normal flow (NF) arteries in ovariectomized rats treated with E2, not in untreated rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries in control and OVX rats treated with E2. In untreated rats, the relaxation was lower in HF than in NF vessels. eNOS expression level was higher in HF than in NF vessels in E2-treated rats only. Acetylcholine-mediated relaxation was fully dependent on the production of NO in E2-treated rats (total inhibition by the NO-synthesis blocker L-NAME). In untreated OVX rats L-NAME blocked only partly the relaxation. Endothelium-independent relaxation (sodium nitroprusside) was not affect by OVX or by E2. Serotoninand phenylephrine-mediated contraction was higher in HF than in NF arteries in both treated and untreated OVX rats. Thus, we demonstrated the essential role of endogenous E2 in flowmediated improvement of endothelium (NO)-mediated dilation in mesenteric resistance arteries.