Polycystin-1 and -2 dosage regulates pressure sensing.

Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

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.

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

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

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

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

Metabolomic Profiling of Angiotensin-II-Induced Abdominal Aortic Aneurysm in Ldlr-/- Mice Points to Alteration of Nitric Oxide, Lipid, and Energy Metabolisms.

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-/- mice. The mice model showed a significant increase in media/lumen ratio and wall area, which is associated with lipid deposition within the adventitia, describing a hypertrophic remodeling with an aneurysm profile of the abdominal aorta. Altered aortas showed increased collagen remodeling, disruption of lipid metabolism, decreased glucose, nitric oxide and lysine metabolisms, and increased polyamines and asymmetric dimethylarginine (ADMA) production. In blood, a major hyperlipidemia was observed with decreased concentrations of glutamine, glycine, taurine, and carnitine, and increased concentrations of the branched amino acids (BCAA). The BCAA/glycine and BCAA/glutamine ratios discriminated with very good sensitivity and specificity between aneurysmatic and non-aneurysmatic mice. To conclude, our results reveal that experimental induction of aortic aneurysms causes a profound alteration in the metabolic profile in aortas and blood, mainly centered on an alteration of NO, lipid, and energetic metabolisms.

Pathophysiological adaptations of resistance arteries in rat offspring exposed in utero to maternal obesity is associated with sex-specific epigenetic alterations.

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.

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

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

Dual Role of Thrombospondin-1 in Flow-Induced Remodeling.

(1) Background: Chronic increases in blood flow, as in cardiovascular diseases, induce outward arterial remodeling. Thrombospondin-1 (TSP-1) is known to interact with matrix proteins and immune cell-surface receptors, but its contribution to flow-mediated remodeling in the microcirculation remains unknown. (2) Methods: Mesenteric arteries were ligated in vivo to generate high- (HF) and normal-flow (NF) arteries in wild-type (WT) and TSP-1-deleted mice (TSP-1-/-). After 7 days, arteries were isolated and studied ex vivo. (3) Results: Chronic increases in blood flow induced outward remodeling in WT mice (increasing diameter from 221 ± 10 to 280 ± 10 µm with 75 mmHg intraluminal pressure) without significant effect in TSP-1-/- (296 ± 18 to 303 ± 14 µm), neutropenic or adoptive bone marrow transfer mice. Four days after ligature, pro inflammatory gene expression levels (CD68, Cox2, Gp91phox, p47phox and p22phox) increased in WT HF arteries but not in TSP-1-/- mice. Perivascular neutrophil accumulation at day 4 was significantly lower in TSP-1-/- than in WT mice. (4) Conclusions: TSP-1 origin is important; indeed, circulating TSP-1 participates in vasodilation, whereas both circulating and tissue TSP-1 are involved in arterial wall thickness and diameter expansion.

Painless local pressure application to test microvascular reactivity to ischemia.

BACKGROUND: Forearm cutaneous blood flux (CBF) measurement with post-occlusive reactive hyperemia (PORH) is uncomfortable and may not be devoid of risks. We aimed to investigate post-compression reactive hyperemia (PCRH) with a custom-made indenter that was designed to be easily used routinely by inexperienced observers. METHODS: Medical students evaluated PCRH with 1- to 4-min pressure applications of 16 to 34 kPa and PORH with 3-min forearm cuff occlusion using laser speckle contrast imaging in 15 healthy volunteers. Participants were asked to quantify their discomfort with a visual analogue scale (VAS) of 10 cm. Total ischemia (ISCH) was quantified by the product of CBF during ischemia and ischemia duration (min). We subtracted the CBF changes in the skin from a reference ipsilateral (PCRH) or contralateral (PORH) non-stimulated area. RESULTS: The average VAS was 1.0 for PCRH vs. 6.0 for PORH (p < 0.001). A strong linear relationship between ISCH and peak PCRH (r2 = 0.915, p < 0.001) was noted. Peak PORH values (63.9 laser perfusion units (LPU)) were significantly lower than all values of the 3-min PCRH (72.6 LPU), including the one obtained with 16 kPa. CONCLUSION: Inexperienced observers could test microvascular reactivity with PCRH without inducing the discomfort that is typically experienced with PORH. Further, PCRH elicits a higher peak response to ischemia compared with PORH. This extremely simple method could influence a broad spectrum of routine cutaneous microcirculation investigations, especially when a painful approach is particularly inadequate or if the patient is fragile. CLINICAL TRIAL REGISTRATION: NCT02861924.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone.

OBJECTIVE: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. APPROACH AND RESULTS: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries. CONCLUSIONS: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models.

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4(+/-) mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4(+/-) mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

Inactivation of serum response factor contributes to decrease vascular muscular tone and arterial stiffness in mice.

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.

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

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

Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease.

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.

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

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

Selective involvement of serum response factor in pressure-induced myogenic tone in resistance arteries.

OBJECTIVE: In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. METHODS AND RESULTS: We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9±2.3%) compared with control (16.3±3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. CONCLUSIONS: This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries.

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis.

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Arterial stiffness and stroke in sickle cell disease.

BACKGROUND AND PURPOSE: Large vessels are also affected in sickle cell disease. The aim of this study was to assess several parameters in adult patients with sickle cell disease compared with control subjects and in patients with sickle cell disease with stroke. METHODS: Carotid arterial stiffness, intima-media thickness, and transcranial Doppler ultrasonography were measured. RESULTS: Arterial stiffness and transcranial Doppler velocity were significantly increased in 49 patients with sickle cell disease compared with 47 control subjects (P<0.05) and especially in patients with stroke (P<0.05). CONCLUSIONS: These data suggest that transcranial Doppler and arterial stiffness might be associated to stroke in adult patients with sickle cell disease.

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

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