Jean-Marc Hyvelin

Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation.

Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases, which increase morbidity and mortality. Hypoxic PH has previously been attributed to structural changes in the pulmonary vasculature including narrowing of the vascular lumen and loss of vessels, which produce a fixed increase in resistance. Using quantitative stereology, we now show that chronic hypoxia caused PH and remodeling of the blood vessel walls in rats but that this remodeling did not lead to structural narrowing of the vascular lumen. Sustained inhibition of the RhoA/Rho-kinase pathway throughout the period of hypoxic exposure attenuated PH and prevented remodeling in intra-acinar vessels without enlarging the structurally determined lumen diameter. In chronically hypoxic lungs, acute Rho kinase inhibition markedly decreased PVR but did not alter the alveolar to arterial oxygen gap. In addition to increased vascular resistance, chronic hypoxia induced Rho kinase–dependent capillary angiogenesis. Thus, hypoxic PH was not caused by fixed structural changes in the vasculature but by sustained vasoconstriction, which was largely Rho kinase dependent. Importantly, this vasoconstriction had no role in ventilation-perfusion matching and optimization of gas exchange. Rho kinase also mediated hypoxia-induced capillary angiogenesis, a previously unrecognized but potentially important adaptive response.

Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes

The effect of endothelin (ET)-1 on both cytosolic Ca2+ concentration ([Ca2+]i) and membrane current in freshly isolated myocytes, as well as on the contraction of arterial rings, was investigated in rat main pulmonary artery (RMPA) and intrapulmonary arteries (RIPA). ET-1 (5-100 nM, 30 s) induced a first [Ca2+]ipeak followed by 3-5 oscillations of decreasing amplitude. In RMPA, the ET-1-induced [Ca2+]iresponse was fully abolished by BQ-123 (0.1 μM). In RIPA, the response was inhibited by BQ-123 in only 21% of the cells, whereas it was abolished by BQ-788 (1 μM) in 70% of the cells. In both types of arteries, the response was not modified in the presence of 100 μM La3+ or in the absence of external Ca2+ but disappeared after pretreatment of the cells with thapsigargin (1 μM) or neomycin (0.1 μM). In RPMA myocytes clamped at -60 mV, ET-1 induced an oscillatory inward current, the reversal potential of which was close to the equilibrium potential for Cl-. This current was unaltered by the removal of external Ca2+ but was abolished by niflumic acid (50 μM). In arterial rings, the ET-1 (100 nM)-induced contraction was decreased by 35% in the presence of either niflumic acid (50 μM) or nifedipine (1 μM). These results demonstrate that ET-1 via the ETA receptor only in RMPA and both ETA and ETB receptors in RIPA induce [Ca2+]ioscillations due to iterative Ca2+release from an inositol trisphosphate-sensitive Ca2+ store. Ca2+ release secondarily activates an oscillatory membrane Cl-current that can depolarize the cell membrane, leading to an influx of Ca2+, this latter contributing to the ET-1-induced vasoconstrictor effect.The effect of endothelin (ET)-1 on both cytosolic Ca2+ concentration ([Ca2+]i) and membrane current in freshly isolated myocytes, as well as on the contraction of arterial rings, was investigated in rat main pulmonary artery (RMPA) and intrapulmonary arteries (RIPA). ET-1 (5-100 nM, 30 s) induced a first [Ca2+]i peak followed by 3-5 oscillations of decreasing amplitude. In RMPA, the ET-1-induced [Ca2+]i response was fully abolished by BQ-123 (0.1 microM). In RIPA, the response was inhibited by BQ-123 in only 21% of the cells, whereas it was abolished by BQ-788 (1 microM) in 70% of the cells. In both types of arteries, the response was not modified in the presence of 100 microM La3+ or in the absence of external Ca2+ but disappeared after pretreatment of the cells with thapsigargin (1 microM) or neomycin (0.1 microM). In RPMA myocytes clamped at -60 mV, ET-1 induced an oscillatory inward current, the reversal potential of which was close to the equilibrium potential for Cl-. This current was unaltered by the removal of external Ca2+ but was abolished by niflumic acid (50 microM). In arterial rings, the ET-1 (100 nM)-induced contraction was decreased by 35% in the presence of either niflumic acid (50 microM) or nifedipine (1 microM). These results demonstrate that ET-1 via the ETA receptor only in RMPA and both ETA and ETB receptors in RIPA induce [Ca2+]i oscillations due to iterative Ca2+ release from an inositol trisphosphate-sensitive Ca2+ store. Ca2+ release secondarily activates an oscillatory membrane Cl- current that can depolarize the cell membrane, leading to an influx of Ca2+, this latter contributing to the ET-1-induced vasoconstrictor effect.

Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall

BACKGROUND We evaluated the possibility of restoring a physiologic vascular wall using undifferentiated mesenchymal stem cells (MSCs) seeded on a polyurethane vascular prosthesis. METHODS Undifferentiated MSCs were seeded on a vascular prosthesis and implanted into Wistar male rats (weight, 350 g) to investigate differentiation into smooth muscle cells and to determine graft endothelialization in vivo. RESULTS Seeded or nonseeded grafts were surgically implanted. Undifferentiated MSCs were first labelled for green fluorescent protein. After 2 weeks in vivo, MSC that were initially self-expanded on the graft in a monolayer were organized in a multicellular layer mimicking media of aortic adjacent wall. They coexpressed green fluorescent protein and smooth muscle proteins that were not present before the in vivo engraftment, indicating that in vivo conditions induced smooth muscle protein maturation. Undifferentiated MSC showed an electrophysiologic profile quite different than mature smooth muscle cells. In both in vitro- and in vivo-differentiated MSCs, adenosine triphosphate, an IP(3)-dependent agonist, induced an increase in calcium similar to that which occurred in mature smooth muscle cells. However, MSCs failed to respond to caffeine, a ryanodine receptor activator, indicating the absence of mature calcium signaling, and finally, contraction was absent. Endothelialization attested by immunohistology and scanning electron microscopy was greater in MSC-seeded grafts that prevent thrombosis. CONCLUSION Only partial smooth muscle cell differentiation of MSCs resulted when seeded on vascular grafts, but MSCs spontaneously restore a media-like thick wall. Mesenchymal stem cells have a positive impact on in vivo endothelialization in rats that supports their potential for use in vascular surgery. CLINICAL RELEVANCE Thrombosis of vascular prostheses is a major complication of surgery. We showed on rat aorta that mesenchymal stem cells seeded on polyurethane patch restore endothelium. It also induced incomplete smooth muscle differentiation. In the future, stem cell could prevent thrombosis of vascular prostheses.

Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug toxicology and safety pharmacology.

Background: Echocardiography (EC) is a method used for the investigation of cardiac morphology and function. Two-dimensional EC gives a visualisation of the morphology of the heart. M-mode EC allows heart function to be monitored. Pulsed Doppler EC is the method of choice to measure blood flows. Objective: To describe the information EC can provide for cardiovascular investigation in laboratory animals, with a special focus on the potential helpfulness of EC in preclinical toxicology and safety pharmacology. Methods: This review includes publications describing the methodology of EC and its application to several animal species used in biological experimentation. Results/conclusion: EC has been established in dogs, monkeys, rodents, rabbits and pigs. As demonstrated by experiments in different species, EC can be particularly helpful in toxicology and safety pharmacology, based on the amount of information it can give on the causes and consequences of drug adverse effects on the cardiovascular system. Furthermore, EC does not require any surgery and is therefore a key refinement compared to invasive methods generally used for investigating the cardiovascular function in laboratory animals. Despite some limitations of the method (the need for trained people, time required for an accurate EC recording, lack of current validation), EC should be further developed in preclinical toxicology and safety pharmacology.

Heme oxygenase-1 inducer hemin prevents vascular thrombosis

Hemin is a heme oxygenase-1 (HO-1) inducer which provides endogenous carbon monoxide known for playing roles in cell proliferation, inflammation or aggregation process. The objective of the current study was to examine the effect of prophylactic treatment with hemin in a thrombosis vascular model. Three groups of Wistar rats, control (n = 6), hemin (n = 6) and hemin + HO-1 inhibitor (n = 6), were used for this study. Hemin-treated animals received hemin (50 mg/kg/d; I.P.) for seven days and HO-1 inhibitor group received hemin at the same dose and SnPP IX (60 mg/kg/d; I.P.). All animals were exposed to electric stimulation of the left carotid according to Kawasakis procedure to induce reproducible thrombus formation. The hemin treatment did not induce blood pressure disturbance. Effects of hemin on vascular thrombosis were quantified by histopathology and its influence on haemostasis was assessed by measuring prothrombin time (PT), activated partial thromboplastin time (APTT) and blood parameters at the end of treatment. The HO-1 mRNA and protein level variation were also checked out. Results showed that chronic treatment with hemin significantly (p < 0.01) reduced the vascular occlusion degree when compared to control and hemin SnPP groups with 7.2 +/- 4.6 vs. 71.1 +/- 14.7 and 74.0 +/- 8.8%, respectively. Moreover, we observed significant (p < 0.05) perturbations of blood parameters in hemin-treated and hemin-SnPP treated rats. Interestingly, hemin treatment did not significantly increase both PT and APTT. Finally, the HO-1 mRNA and protein levels were increased in hemin-treated carotid artery. In conclusion, hemin by inducing HO-1 expression may be a preventive agent against clinical disorders associated to an increased risk of thrombosis events and may limit haemorrhagic risks.

Short- and Long-Term Cardioprotective Effect of Darbepoetin-α: Role of Bcl-2 Family Proteins

The purpose of this study was to assess the short- and long-term cardioprotective effects of darbepoetin-α (DA) in a rat myocardial ischemia and reperfusion model and to investigate the signaling pathway through which DA limits cardiomyocytes apoptosis. Rats were subjected to 40 minutes of coronary artery ligation followed by 72 hours or 4 weeks reperfusion and received either DA (3 or 30 μg/kg, DA3 and D30 groups) or vehicle (control) prior to ischemia. In the DA groups reperfused for 72 hours, left ventricular shortening fraction and left ventricular ejection fraction were higher than that in the control rats (P < 0.05), in agreement with a smaller left ventricular (LV) infarct size. DA treatment activated the JAK2/Akt signaling pathway, lowered cleaved caspase-3, and increased both phosphorylated-Bad and phosphorylated-GSK-3β proteins. This was consistent with the decrease of reactive oxygen species production and the lowered binding of Bad to Bcl-xL and Bcl-2 in a DA30 group of rats. Similarly, in the DA-4-week group, LV function was greater compared to the control. Histology alterations implicated lower LV cardiac fibrosis and greater capillary density; furthermore, both Bcl-xL and Bcl-2 were upregulated. In conclusion, DA afforded short- and long-term cardioprotective effects. Antiapoptotic effects, through the activation of Akt that regulates the Bcl-2 family proteins and activates GSK-3β, are central in the DA cardioprotective mechanism.