Anne-Laure Guihot

Reactive oxygen species are necessary for high flow (shear Stress)-induced diameter enlargement of rat resistance arteries

Objectives: Chronic increases in blood flow induce remodeling associated with increases in diameter and endothelium‐mediated dilation. Remodeling requires cell growth and migration, which may involve reactive oxygen species (ROS). Nevertheless, the role of ROS in flow‐mediated remodeling in resistance arteries is not known. Materials and Methods: Rat mesenteric resistance arteries (MRAs) were exposed to high flow (HF) by sequentially ligating second‐order MRAs in vivo. After three weeks, arteries were collected for structural, pharmacological, and biochemical analysis. Results: In HF arteries, luminal diameter (431±12 to 553±14 μm; n=10), endothelium (acetylcholine)‐mediated vasodilatation (61±6 to 77±6% relaxation) and NAD(P)H subunit (gp91phox and p67phox) expression levels, and ROS (dihydroethydine microphotography) and peroxynitrite (3‐nitro‐tyrosine) production were higher than in normal flow arteries. Acute ROS scavenging with tempol improved acetylcholine‐dependent relaxation (92±4% relaxation), confirming that ROS are produced in HF arteries. Chronic treatment with tempol prevented the increase in diameter, reduced ROS and peroxynitrite production, and improved endothelium‐mediated relaxation in HF arteries. Thus, ROS and NO were involved in HF‐induced diameter enlargement, possibly through the formation of peroxynitrite, while ROS reduced the increase in endothelium‐dependent relaxation. Conclusions: ROS production is necessary for flow‐mediated diameter enlargement of resistance arteries. However, ROS counteract, in part, the associated improvement in endothelium‐mediated relaxation.

The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production

AIMSnThe angiotensin II type 1 receptor (AT1R) through the activation of immune cells plays a key role in arterial inward remodelling and reduced blood flow in cardiovascular disorders. On the other side, flow (shear stress)-mediated outward remodelling (FMR), involved in collateral arteries growth in ischaemic diseases, allows revascularization. We hypothesized that the type 2 receptor (AT2R), described as opposing the effects of AT1R, could be involved in FMR.nnnMETHODS AND RESULTSnWe studied FMR using a model of ligation of feed arteries supplying collateral pathways in the mouse mesenteric arterial bed in vivo. Seven days after ligation, diameter increased by 30% in high flow (HF) arteries compared with normal flow vessels. FMR was absent in mice lacking AT2R. At Day 2, T lymphocytes expressing AT2R were present preferentially around HF arteries. FMR did not occur in athymic (nude) mice lacking T cells and in mice treated with anti-CD3ε antibodies. AT2R activation induced interleukin-17 production by memory T cells. Treatment of nude mice or AT2R-deficient mice with interleukin-17 restored diameter enlargement in HF arteries. Interleukin-17 increased NO-dependent relaxation and matrix metalloproteinases activity, both important in FMR. Remodelling of feeding arteries in the skin flap model of ischaemia was also absent in AT2R-deficient mice and in anti-interleukin-17-treated mice. Finally, remodelling, absent in 12-month-old mice, was restored by a treatment with the AT2R non-peptidic agonist C21.nnnCONCLUSIONnAT2R-dependent interleukin-17 production by T lymphocyte is necessary for collateral artery growth and could represent a new therapeutic target in ischaemic disorders.

Estrogens are needed for the improvement in endothelium-mediated dilation induced by a chronic increase in blood flow in rat mesenteric arteries.

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.

Predominant Role of Nuclear Versus Membrane Estrogen Receptor α in Arterial Protection: Implications for Estrogen Receptor α Modulation in Cardiovascular Prevention/Safety

Background Although estrogen receptor α (ERα) 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 ERα 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 ERα mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ERα plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20 = ERaAF2°), we sought to fully characterize the respective roles of nuclear versus membrane‐initiated steroid signaling in the arterial protection conferred by ERα. 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, ERαAF20 mice were unresponsive to estrogens for these beneficial vascular effects. Accordingly, selective activation of nuclear ERα 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 ERα in the vasculoprotective action of estrogens with major implications in medicine, particularly for selective nuclear ERα agonist, such as estetrol, which is currently under development as a new oral contraceptive and for hormone replacement therapy in menopausal women.

Cardioprotective Role of Colchicine Against Inflammatory Injury in a Rat Model of Acute Myocardial Infarction

Background: Inflammation plays a crucial role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. A clinical trial has recently reported a smaller infarct size in a cohort of patients with ST-segment elevation myocardial infarction (MI) treated with a short colchicine course. The mechanism underlying colchicine-induced cardioprotection in the early MI phase remains unclear. We hypothesized that a short pretreatment with colchicine could induce acute beneficial effects by protecting the heart against inflammation in myocardial I/R injury. Methods and Results: Rats were subjected to 40-minute left anterior descending coronary occlusion, followed by 120-minute reperfusion. Colchicine (0.3 mg/kg) or a vehicle was administered per os 24 hours and immediately before surgery. Infarct size was significantly reduced in the colchicine group (35.6% ± 3.0% vs 46.6% ± 3.3%, P < .05). The beneficial effects of colchicine were associated with an increased systemic interleukin-10 (IL-10) level and decreased cardiac transforming growth factor-β level. Interleukin-1β was found to increase in a “time of reperfusion”-dependent manner. Colchicine inhibited messenger RNA expression of caspase-1 and pro-IL-18. Interleukin-1β injected 10 minutes prior to myocardial ischemia induced greater infarct size (58.0% ± 2.0%, P < .05) as compared to the vehicle. Colchicine combined to IL-1β injection significantly decreased infarct size (47.1% ± 2.2%, P < .05) as compared to IL-1β alone, while colchicine alone exhibited a significantly more marked cardioprotective effect than the colchicine-IL-1β association. Conclusion: The cardioprotection induced by a short colchicine pretreatment was associated with an anti-inflammatory effect in the early reperfusion phase in our rat MI model.

0020 : Mechanisms of the protective effect of the estrogen receptor alpha in hypertension and aging

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 (0.5xa0mg/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 14xa0mmHg (ns) in WT mice. A similar increased was observed in C451A mice (20xa0mmHg, ns). By contrast, SBP increased significantly more in WT-OVX mice (31 mmHg, p=0,0007), ERα-KO (45xa0mmHg, 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

CO-37: Importance of the membrane estrogen receptor ALPHA (ERA) in the vascular response to shear stress in mice

BACKGROUNDnSmall resistance arteries regulate peripheral tissue perfusion following variations in arterial pressure and blood flow. An altered flow-mediated dilation (FMD) in response to intraluminal shear stress is the hallmark of early vascular dysfunction. We recently showed that the adaptive flow-dependent arterial remodeling was controlled by the endothelial estrogen receptor alpha (ERa). Our goal was to evaluate the role of ERa in endothelial mechanosensitive mechanisms related to the acute response to flow, by using multiple models of ERa deficiency in male mice, avoiding the hormonal influence of estrogens encountered in females.nnnMETHODSnEvaluation of the FMD was performed on pressurized mesenteric resistance arteries mounted on an arteriograph following step increase in intraluminal flow (6-100μl/min). Arteries were isolated from wild-type (WT) and ERa genetically modified male mice deficient in either (i) total ERa (ERaKO), (ii) its ligand-dependent transactivation function AF2 (AF2°) and (iii) the plasmic membrane-located ERa following a point mutation of the palmitoylation (C451A) site of the receptor.nnnRESULTSnWe first observed a selective attenuation of FMD, without any major modification in response to vasodilator agonists (acetylcholine), in mice deficient in ERa (% dilation 50μl/min: ERaKO: 41±5 vs. WT: 59±4 p<0.05 two-way ANOVA) or its AF2 function involved in its nuclear action (% dilation 100μl/min: AF2: 33±4 vs. WT: 56±5 p<0.01). Interestingly, the proportion of NO involved in FMD droped markedly with the total loss of ERa as NOS inhibition by LNNA only slightly affected FMD in ERaKO, in contrast to WT and AF2, suggesting a putative compensatory mechanism (% of inhibition at flow 100μl/min: ERaKO: 13%; AF2: 70%; WT: 71%). Ex vivo ligand-dependent modulation of ERa after incubation with its agonist 17beta estradiol (10-8M) or the antagonist ICI 182,780 (10-6M) had no major effect on FMD in WT arteries. However, a default in receptor membrane addressing in C451A male mice markedly altered FMD (% dilation 50μl/min: C451A: 26±4 vs. WT: 53±5; p<0.01) characterizing a major decrease in flow-mediated NO production.nnnCONCLUSIONSnWe thus show, for the first time, that membrane ERa contributes to arterial shear-sensing irrespective of the presence of an agonist or an antagonist. ERa at the membrane could contribute to vascular homeostasis and the regulation of its expression should now be studied.

CO-46: Protective role of the estrogen receptor alpha during hypertension

BACKGROUNDnEstrogens 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.nnnMETHODSnFive 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 (0,5mg/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).nnnRESULTSnSBP increased from 117±3,05 to 131±2,90mmHg (ns) in WT mice. A similar increased was observed in C451A mice (124±7,18 vs 103±5,59mmHg, ns). By contrast, SBP increased significantly more in WT-OVX mice (144±4,97 vs 113±3,17mmHg, p=0,0007), ERα-KO (155±4,02mmHg vs 110±2,36mmHg, p<0,0001) and AF2° mice (156±3,64 vs 117±2,03mmHg, p<0,0001). Changes in vascular structure were proportional to the evolution of SBP. Measurements of vascular reactivity showed no significant difference in contraction and relaxation in normotensive and hypertensive WT, ERα-KO, AF2° and C451A. However, in WT-OVX mice, angiotensin II dependant hypertension was associated to increased contraction and decreased dilation.nnnCONCLUSIONSnConsistently 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.

0362: Interleukin 17 production induced by angiotensin II type 2 receptor activation in T cells drives flow-mediated outward remodeling of mouse resistance arteries

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±25xa0μl/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=xx per group, P occurrence of remodeling in mice lacking AT2R or IL-17. In both AT2R-/- and IL-17-/- mice diameter expansion did not occur whereas T cells and myeloid cells accumulation around the HF artery was not affected. IL-17 infusion (5ng/h, Alzet osmotic minipump) to nude mice and to AT2R-/- mice restored HF remodeling to control level. In order to confirm the role of AT2R in flow-mediated remodeling in another vascular territory, neovascularization was examined using an ischemic skin flap model in AT2R-/- mice. As in mesenteric arteries, the absence of AT2R prevented diameter expansion in the arteries feeding the skin flap. 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.

K012 Pharmacological blockade or genetic deletion of AT2R abolishes flow-induced structural remodeling in rat and mice resistance arteries in vivo

The renin—angiotensin system (RAS) plays a major role in regulating the cardiovascular system. Angiotensin II (AngII), the main effector of the RAS, is involved in both endothelial and smooth muscle cells functions, acting by two receptors, type 1 (AT1R) and type 2 (AT2R). If the role of the AT1R has been widely investigated in vivo and in vitro, the role of the AT2R remains controversial. It is admitted that AT2R induces vasodilation and possesses anti-proliferative properties. Nevertheless, recent studies have shown that the AT2R is vasoconstrictor in hypertension and has proliferative effects in tumors. AT2R has a role in vascular reactivity to flow (shear stress) and in cell growth and migration. Moreover, the AT2R decreases MMP-2 and increases elastin. Thus we hypothesized that changes in vascular wall mechanical strain, extracellular matrix deposition, and MMPs activity could be modulated differentially by the AngII receptors. We investigated the role of the AT2R on the capacity of resistance arteries to remodel in response to chronic changes in local blood flow. We investigated flow-mediated remodeling in resistance arteries in PD123319 (AT2R blocker)-treated rats and in AT2R-knockout mice. Second order mesenteric arteries were ligated in vivo, generating low flow (LF) and high flow (HF) arteries, compared to normal flow (NF) vessels. In these vessels a biochemical (protein expression and mRNA level) and a structural study (lumen diameter, intima media thickness and media cross-sectional area) will be performed in order to determine the mechanism involved in the remodeling. After 2 weeks, outward hypertrophic remodeling occurred in HF arteries (increased diameter and medial cross-sectional area) in NaCl treated-rats or wild type mice. Nevertheless, high blood flow-induced remodeling in mesenteric resistance arteries failed to occur in PD123319-treated-rats and in AT2R-KO mice. Inward eutrophic remodeling, which occurred in LF arteries, was not affected to the absence of the AT2R or its blockade. Thus, the diameter enlargement due to a chronic rise in blood flow in resistance arteries involves the AT2R, suggesting a vasodilator and trophic effect of the receptor in arteriolar remodeling in vivo.