Laurent Loufrani

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.

The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure

Hypertension is one of the most frequent pathologies in the industrialized world. Although recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. Increased activity of the monomeric G protein RhoA in arteries is a common feature of hypertension. However, how RhoA is activated and whether it has a causative role in hypertension remains unclear. Here we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for angiotensin II–induced activation of RhoA signaling in arterial smooth muscle cells. We found that angiotensin II activates Arhgef1 through a previously undescribed mechanism in which Jak2 phosphorylates Tyr738 of Arhgef1. Arhgef1 inactivation in smooth muscle induced resistance to angiotensin II–dependent hypertension in mice, but did not affect normal blood pressure regulation. Our results show that control of RhoA signaling through Arhgef1 is central to the development of angiotensin II–dependent hypertension and identify Arhgef1 as a potential target for the treatment of hypertension.

Activation of AT(2) receptors by endogenous angiotensin II is involved in flow-induced dilation in rat resistance arteries.

Pressure-induced tone (myogenic, MT) and flow (shear stress)-induced dilation (FD) are potent modulators of resistance artery tone. We tested the hypothesis that locally produced angiotensin II interacts with MT and FD. Rat mesenteric resistance arteries were perfused in situ. Arterial diameter was measured by intravital microscopy after a bifurcation on 2 distal arterial branches equivalent in size (150 microm, n=7 per group). One was ligated distally and thus submitted to pressure only (MT, no FD). The second branch was submitted to flow and pressure (MT and FD). The difference in diameter between the 2 vessels was considered to be FD. Flow-diameter-pressure relationship was established in the absence and then in the presence of 1 of the following agents. In the nonligated segment (MT+FD), angiotensin II type 1 (AT(1)) receptor blockade (losartan) had no significant effect, whereas angiotensin II type 2 (AT(2)) receptor blockade (PD123319) or saralasin (AT(1)+AT(2) blocker) decreased the diameter significantly, by 9+/-1 and 10+/-0.8 microm, respectively. Angiotensin II in the presence of losartan increased the diameter by 18+/-0.6 microm (inhibited by PD123319). PD123319 or saralasin had no effect after NO synthesis blockade or after endothelial disruption. In the arterial segment ligated distally (MT only), AT(1) or AT(2) receptor blockade had no significant effect. AT(2)-dependent dilation represented 20% to 39% of FD (shear stress from 22 to 37 dyn/cm(2)), and AT(2)-receptor mRNA was found in mesenteric resistance arteries. Thus, resistance arterial tone was modulated in situ by locally produced angiotensin II, which might participate in flow-induced dilation through endothelial AT(2) receptor activation of NO release.

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.

The endothelial mineralocorticoid receptor regulates vasoconstrictor tone and blood pressure

Pathophysiological aldosterone (aldo)/ mineralocorticoid receptor (MR) signaling has significant effects on the cardiovascular system, resulting in hypertension and cardiovascular remodeling; however, the specific contribution of the vascular MR to blood pressure regulation remains to be established. To address this question, we generated a mouse model with conditional overexpression of the MR in endothelial cells (MR‐EC). In basal conditions, MR‐EC mice developed moderate hypertension that could be reversed by canrenoate, a pharmacological MR antagonist. MR‐EC mice presented increased contractile response of resistance arteries to vasoconstrictors (phenylephrine, thromboxane A2 analog, angiotensin II, and endothelin 1) in the absence of vascular morphological alterations. The acute blood pressure response to angiotensin II or endothelin 1 infusion was increased in MR‐EC mice compared with that in littermate controls. These observations demonstrate that enhanced MR activation in the endothelium generates an increase in blood pressure, independent of stimulation of renal tubular Na+ transport by aldo/MR or direct activation of smooth muscle MR and establish one mechanism by which endothelial MR activation per se may contribute to impaired vascular reactivity.—Nguyen Dinh Cat, A., Griol‐Charhbili, V., Loufrani, L., Labat, C, Benjamin, L., Farman, N., Lacolley, P., Henrion, D., and Jaisser, F. The endothelial mineralocorticoid receptor regulates vasoconstrictor tone and blood pressure. FASEB J. 24, 2454–2463 (2010). www.fasebj.org

High blood pressure reduction reverses angiotensin II type 2 receptor-mediated vasoconstriction into vasodilation in spontaneously hypertensive rats.

Background—We have previously shown that angiotensin II type 2 receptor (AT2R) stimulation causes endothelium-dependent vasodilation that does not desensitize after chronic angiotensin II type 1 receptor (AT1R) blockade, suggesting a role for AT2R in antihypertensive treatment. Methods and Results—We recorded mean arterial pressure (MAP) and investigated AT2R by Western blot analysis, immunohistochemistry, and function in isolated mesenteric resistance arteries (205 &mgr;m in diameter) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) receiving the following for 4 weeks in drinking water: placebo, AT1R blockade (candesartan; 2 mg/kg per day), ACE inhibitor (perindopril; 3 mg/kg per day), nonselective vasodilator (hydralazine; 16 or 24 mg/kg per day), or candesartan plus hydralazine (16 mg/kg per day). In precontracted isolated arteries, AT2R stimulation (angiotensin II in the presence of candesartan) caused vasodilation in WKY rats (MAP=118 mm Hg) and vasoconstriction in SHR (MAP=183 mm Hg). In SHR treated with candesartan (MAP=146 mm Hg) or hydralazine (16 mg/kg per day; MAP=145 mm Hg), AT2R-induced contraction was reduced by 50%. In SHR treated with perindopril (MAP=125 mm Hg), AT2R stimulation induced vasodilation. In SHR treated with hydralazine (24 mg/kg per day; MAP=105 mm Hg) and in SHR treated with hydralazine (16 mg/kg per day) plus candesartan (MAP=102 mm Hg), an AT2R-mediated vasodilation was restored. Immunochemistry and Western blot analysis showed that AT2R expression, lower in SHR than in WKY rats, was restored to normal levels by treatments reducing arterial pressure in SHR. Conclusions—Our results suggest that in resistance arteries of SHR, (1) AT2R is downregulated by hypertension, and (2) specific and nonspecific antihypertensive treatments restore AT2R expression and vasodilator functions.

Key Role of the NO-Pathway and Matrix Metalloprotease-9 in High Blood Flow-Induced Remodeling of Rat Resistance Arteries

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

Flow (Shear Stress)–Induced Endothelium-Dependent Dilation Is Altered in Mice Lacking the Gene Encoding for Dystrophin

Background —Dystrophin has a key role in striated muscle mechanotransduction of physical forces. Although cytoskeletal elements play a major role in the mechanotransduction of pressure and flow in vascular cells, the role of dystrophin in vascular function has not yet been investigated. Thus, we studied endothelial and muscular responses of arteries isolated from mice lacking dystrophin (mdx mice). Methods and Results —Carotid and mesenteric resistance arteries 120 &mgr;m in diameter were isolated and mounted in vitro in an arteriograph to control intraluminal pressure and flow. Blood pressure was not affected by the absence of dystrophin. Pressure-induced (myogenic), phenylephrine-induced, and KCl-induced forms of tone were unchanged. Flow (shear stress)–induced dilation in arteries isolated from mdx mice was decreased by 50% to 60%, whereas dilation to acetylcholine or sodium nitroprusside was unaffected. NG-nitro-L-arginine methyl ester–sensitive flow dilation was also decreased in arteries from mdx mice. Thus, the absence of dystrophin was associated with a defect in signal transduction of shear stress. Dystrophin was present in vascular endothelial and smooth muscle cells, as shown by immunolocalization, and localized at the level of the plasma membrane, as seen by confocal microscopy of perfused isolated arteries. Conclusions —This is the first functional study of arteries lacking the gene for dystrophin. Vascular reactivity was normal, with the exception of flow-induced dilation. Thus, dystrophin could play a specific role in shear-stress mechanotransduction in arterial endothelial cells. Organ damage in such diseases as Duchenne dystrophy might be aggravated by such a defective arterial response to flow.

Defect in Microvascular Adaptation to Chronic Changes in Blood Flow in Mice Lacking the Gene Encoding for Dystrophin

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

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.