Absence of dystrophin in mice reduces NO-dependent vascular function and vascular density: total recovery after a treatment with the aminoglycoside gentamicin.

International audienceOBJECTIVE: Mutations in the dystrophin gene causing Duchenne's muscular dystrophy (DMD) lead to premature stop codons. In mice lacking dystrophin (mdx mice), a model for DMD, these mutations can be suppressed by aminoglycosides such as gentamicin. Dystrophin plays a role in flow (shear stress)-mediated endothelium-dependent dilation (FMD) in arteries. We investigated the effect of gentamicin on vascular contractile and dilatory functions, vascular structure, and density in mdx mice. METHODS AND RESULTS: Isolated mice carotid and mesenteric resistance arteries were mounted in arteriographs allowing continuous diameter measurements. Mdx mice showed lower nitric oxide (NO)-dependent FMD and endothelial NO synthase (eNOS) expression as well as decreased vascular density in gracilis and cardiac muscles compared with control mice. Treatment with gentamycin restored these parameters. In contrast, smooth muscle-dependent contractions as well as endothelium-dependent or -independent dilation were not affected by dystrophin deficiency or by gentamicin treatment. CONCLUSIONS: Dystrophin deficiency induces a selective defect in flow-dependent mechanotransduction, thus attenuating FMD and eNOS expression, and may contribute to low arteriolar density. These findings open important perspectives regarding the mechanism involved in the pathophysiology of genetic diseases related to premature stop codons such as DMD

Cyclooxygenase-2 Inhibition Restored Endothelium-Mediated Relaxation in Old Obese Zucker Rat Mesenteric Arteries

Metabolic syndrome is associated with reduced endothelial vasodilator function. It is also associated with the induction of cyclooxygenase-2 (COX2), which produces vasoactive prostanoids. The frequency of metabolic syndrome increases with age and aging per se is a risk factor associated with reduced endothelium-mediated relaxation. Nevertheless, the combined effect of aging and metabolic syndrome on the endothelium is less known. We hypothesized that COX2 derived prostanoids may affect endothelium function in metabolic syndrome associated with aging. We used obese Zucker rats, a model of metabolic syndrome. First order mesenteric arteries were isolated from 4- and 12-month-old rats and acetylcholine (endothelium)-dependent relaxation determined using wire-myography. Endothelium-mediated relaxation, impaired in young Zucker rats (89 versus 77% maximal relaxation; lean versus Zucker), was further reduced in old Zucker rats (72 versus 51%, lean versus Zucker). The effect of the nitric oxide-synthesis inhibitor L-NAME on the relaxation was reduced in both young and old Zucker rats without change in eNOS expression level. COX inhibition (indomethacin) improved acetylcholine-mediated relaxation in old obese rats only, suggesting involvement of vasoconstrictor prostanoids. In addition, COX2 inhibition (NS398) and TxA2/PGH2 receptor blockade (SQ29548) both improved relaxation in old Zucker rat arteries. Old Zucker rats had the highest TxB2 (TxA2 metabolite) blood level associated with increased COX2 immunostaining. Chronic COX2 blockade (Celecoxib, 3 weeks) restored endothelium-dependent relaxation in old Zucker rats to the level observed in old lean rats. Thus the combination of aging and metabolic syndrome further impairs endothelium-dependent relaxation by inducing an excessive production of COX2-derived vasoconstrictor(s); possibly TxA2

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

Effects of hydrogen sulfide on hemodynamics, inflammatory response and oxidative stress during resuscitated hemorrhagic shock in rats

Introduction Hydrogen sulfide (H2S) has been shown to improve survival in rodent models of lethal hemorrhage. Conversely, other authors have reported that inhibition of endogenous H2S production improves hemodynamics and reduces organ injury after hemorrhagic shock. Since all of these data originate from unresuscitated models and/or the use of a pre-treatment design, we therefore tested the hypothesis that the H2S donor, sodium hydrosulfide (NaHS), may improve hemodynamics in resuscitated hemorrhagic shock and attenuate oxidative and nitrosative stresses. Methods Thirty-two rats were mechanically ventilated and instrumented to measure mean arterial pressure (MAP) and carotid blood flow (CBF). Animals were bled during 60 minutes in order to maintain MAP at 40 ± 2 mm Hg. Ten minutes prior to retransfusion of shed blood, rats randomly received either an intravenous bolus of NaHS (0.2 mg/kg) or vehicle (0.9% NaCl). At the end of the experiment (T = 300 minutes), blood, aorta and heart were harvested for Western blot (inductible Nitric Oxyde Synthase (iNOS), Nuclear factor-κB (NF-κB), phosphorylated Inhibitor κB (P-IκB), Inter-Cellular Adhesion Molecule (I-CAM), Heme oxygenase 1(HO-1), Heme oxygenase 2(HO-2), as well as nuclear respiratory factor 2 (Nrf2)). Nitric oxide (NO) and superoxide anion (O2 -) were also measured by electron paramagnetic resonance. Results At the end of the experiment, control rats exhibited a decrease in MAP which was attenuated by NaHS (65 ± 32 versus 101 ± 17 mmHg, P < 0.05). CBF was better maintained in NaHS-treated rats (1.9 ± 1.6 versus 4.4 ± 1.9 ml/minute P < 0.05). NaHS significantly limited shock-induced metabolic acidosis. NaHS also prevented iNOS expression and NO production in the heart and aorta while significantly reducing NF-kB, P-IκB and I-CAM in the aorta. Compared to the control group, NaHS significantly increased Nrf2, HO-1 and HO-2 and limited O2 - release in both aorta and heart (P < 0.05). Conclusions NaHS is protective against the effects of ischemia reperfusion induced by controlled hemorrhage in rats. NaHS also improves hemodynamics in the early resuscitation phase after hemorrhagic shock, most likely as a result of attenuated oxidative stress. The use of NaHS hence appears promising in limiting the consequences of ischemia reperfusion (IR)

Role of the cytoskeleton in flow (shear stress)-induced dilation and remodeling in resistance arteries.

International audienceCytoskeletal proteins determine cell shape and integrity and membrane-bound structures connected to extracellular components allow tissue integrity. These structural elements have an active role in the interaction of blood vessels with their environment. Shear stress due to blood flow is the most important force stimulating the endothelium. The role of cytoskeletal proteins in endothelial responses to flow has been studied in resistance arteries using pharmacological tools and transgenic models. Shear stress activates extracellular "flow sensing" elements associated with a thick glycocalyx communicating the signal to membrane-bound complexes (integrins and/or dystrophin-dystroglycans) and to eNOS through a pathway involving the intermediate filament vimentin, the microtubule network and actin. When blood flow increases chronically the endothelium triggers diameter enlargement and medial hypertrophy. This is facilitated by the genetic absence of the intermediate filaments, vimentin and desmin suggesting that these elements oppose the process

Etude physiopathologique du tonus myogénique vasculaire (implication de Rhoa, de Notch3 et du facteur de réponse au sérum)

Le tonus myogénique (TM) est la capacité des artères de résistance à se contracter suite à une élévation de la pression intraluminale. Il permet la protection des capillaires contre une augmentation excessive de pression, et régule les débits sanguins locaux. L'implication du TM dans le développement de certaines pathologies fait de lui une cible thérapeutique intéressante. Cela nécessite une identification précise des protagonistes moléculaires. La voie RhoA/Rho-kinase joue un rôle important dans le TM en régulant la sensibilisation au calcium de l'appareil contractile. Nous nous sommes donc particulièrement intéressés à des éléments pouvant moduler ou être modulés par cette voie. Ainsi, nous avons mis en évidence que l'intervention de la voie RhoA/Rho-kinase dans le TM implique tout d'abord une localisation membranaire de RhoA, afin d'interagir avec la cavéoline-1 et d'activer ses effecteurs tels que Rho-kinase. De plus l'activation de RhoA par la pression est régulée par le récepteur Notch3 intervenant ainsi comme un mécanosenseur. Nos travaux montrent aussi le rôle du facteur de réponse au sérum (SRF) dans le TM via la régulation du cytosquelette et de l'activité des canaux mécanosensibles. Pour finir, l'inhibition de l'activité de RhoA, lors d'un traitement avec du Sildénafil, entraine une surexpression de RhoA qui est responsable d'une élévation du TM et d'une hypertension à l'arrêt du traitement. Cette thèse apporte de nouvelles connaissances sur l'implication de RhoA, Notch3 et SRF dans le TM permettant une meilleure compréhension de ce mécanisme. Cela offre de nouvelles cibles thérapeutiques pour combattre les pathologies impliquant des dysfonctions vasculaires.The myogenic tone (MT) is the ability of the resistance arteries to contract in response to an increase in intraluminal pressure. MT protects capillaries against excessive rises in pressure, and regulates local blood flow. Due to its role in the development of pathologies, MT is an interesting therapeutic target. However the molecular mechanisms involved in MT still remain to be elucidated. The RhoA/Rho-kinase pathway plays an important role in the MT in particular via its role in the calcium sensitization of the contractile apparatus. Thus, we were particularly interested in determining factors that can modulate or be modulated by this pathway. First, we demonstrated that MT requires RhoA to be localized at the cellular membrane, where it interacts with caveolin-1 and activates its effectors such as Rho-kinase. Furthermore the activation of RhoA by the pressure is regulated by the Notch3 receptor, so occurring as a mechanosensor. Our works also showed the role of the serum response factor (SRF) in the MT via the regulation of the cytoskeleton and the activity of mecanosensitive channels. Finally, we showed in sildenafil treated rats, that the inhibition of RhoA activity induced a overexpression of it, that is responsible to an increase of the MT and an hypertension after this treatment. These works bring new and additional knowledges on the implication of proteins such as RhoA, Notch3 and SRF in the MT providing a better understanding of this mechanism and its regulation. These new elements offer new therapeutic targets for the treatment of pathologies involving vascular dysfonctions.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Key role of the NO-pathway and matrix metalloprotease-9 in high blood flow-induced remodeling of rat resistance arteries.: flow-remodeling in resistance arteries

International audienceOBJECTIVE: 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