Véronique Eder

Multipotential Mesenchymal Stem Cells Are Mobilized into Peripheral Blood by Hypoxia

MSCs constitute a population of multipotential cells giving rise to adipocytes, osteoblasts, chondrocytes, and vascular‐smooth muscle‐like hematopoietic supportive stromal cells. It remains unclear whether MSCs can be isolated from adult peripheral blood under stationary conditions and whether they can be mobilized in a way similar to hematopoietic stem cells. In this report, we show that MSCs are regularly observed in the circulating blood of rats and that the circulating MSC pool is consistently and dramatically increased (by almost 15‐fold) when animals are exposed to chronic hypoxia. The immunophenotype and the adipocytic, osteoblastic, and chondrocytic differentiation potential of circulating MSCs were similar to those of bone marrow MSCs. Hypoxia‐induced mobilization appears to be specific for MSCs since total circulating hematopoietic progenitor cells were not significantly increased. Our data provide an in vivo model amenable to analysis of MSC‐mobilizing factors.

Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions.

Findings from animal studies have suggested that bone remodeling is under beta‐adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.1 mg/Kg/day), medium (5 mg/Kg/day) or high (20 mg/Kg/day) doses of propranolol given 5 days per week for 10 weeks in ovariectomized (OVX) rats. Characteristics of bone microarchitecture, biomechanical properties and bone turnover were investigated, whilst heart functions were assessed by echocardiography and catheterization of the left ventricle. We first confirmed the expression of Adrβ2R and the absence of Adrβ1R on osteoblasts by PCR and confocal microscopy. We then showed that low dose propranolol prevented OVX induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (−52% of osteoclast surface on bone surface vs. placebo, P = 0.01). Consequently, rats receiving 0.1 mg/kg/day propranolol displayed higher stress (+27%), intrinsic energy (+28.7%) and Youngs Modulus in compression versus placebo (all, P < 0.05). No significant effects on heart hemodynamic parameters were found in rats receiving this dose. In contrast, medium and high doses of propranolol had a negative effect on heart functions but no significant protective effects on bone mass in ovariectomized rats. These results, consistent with the dominant nature of the high bone mass phenotype and normal heart function of Adrβ2R‐deficient mice, suggest that low doses of β‐blockers may have a therapeutic utility in the treatment of osteoporosis with high selectivity for bone tissues. J. Cell. Physiol. 217: 819–827, 2008.

Influence of hypoxia on the domiciliation of mesenchymal stem cells after infusion into rats: possibilities of targeting pulmonary artery remodeling via cells therapies?

BackgroundBone marrow (BM) cells are promising tools for vascular therapies. Here, we focused on the possibility of targeting the hypoxia-induced pulmonary artery hypertension remodeling with systemic delivery of BM-derived mesenchymal stem cells (MSCs) into non-irradiated rats.MethodsSix-week-old Wistar rats were exposed to 3-week chronic hypoxia leading to pulmonary artery wall remodeling. Domiciliation of adhesive BM-derived CD45- CD73+ CD90+ MSCs was first studied after a single intravenous infusion of Indium-111-labeled MSCs followed by whole body scintigraphies and autoradiographies of different harvested organs. In a second set of experiments, enhanced-GFP labeling allowed to observe distribution at later times using sequential infusions during the 3-week hypoxia exposure.ResultsA 30% pulmonary retention was observed by scintigraphies and no differences were observed in the global repartition between hypoxic and control groups. Intrapulmonary radioactivity repartition was homogenous in both groups, as shown by autoradiographies. BM-derived GFP-labeled MSCs were observed with a global repartition in liver, in spleen, in lung parenchyma and rarely in the adventitial layer of remodeled vessels. Furthermore this global repartition was not modified by hypoxia. Interestingly, these cells displayed in vivo bone marrow homing, proving a preservation of their viability and function. Bone marrow homing of GFP-labeled MSCs was increased in the hypoxic group.ConclusionAdhesive BM-derived CD45- CD73+ CD90+ MSCs are not integrated in the pulmonary arteries remodeled media after repeated intravenous infusions in contrast to previously described in systemic vascular remodeling or with endothelial progenitor cells infusions.

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.

Hemin decreases cardiac oxidative stress and fibrosis in a rat model of systemic hypertension via PI3K/Akt signalling

AIMS Angiotensin II induces cardiac myocyte apoptosis and hypertrophy, which contribute to heart failure, possibly through enhanced oxidative stress. The aim of this work was to assess the impact of hemin (heme oxygenase-1 inducer) on NADPH oxidase activation, cardiac oxidative stress, and development of fibrosis in a rat model of renovascular hypertensive cardiomyopathy in comparison to an anti-hypertensive reference treatment with losartan. METHODS AND RESULTS A 3 week hemin treatment was tested in an angiotensin II-dependent hypertensive rat model and a cellular model of neonatal rat cardiomyocytes stimulated by angiotensin II. Our findings demonstrate that hemin prevented development of intercellular fibrosis, expression of collagen I, and disorganization of intracellular fibres. Oxidative stress and apoptosis evaluated in hypertensive myocardial tissue were decreased by hemin. The reference treatment with the angiotensin II receptor (AT(1)) antagonist (losartan) was less effective than hemin in prevention of fibrosis and oxidative stress, although it was more effective in reducing hypertension. Rac-1 activation and, subsequently, NADPH oxidase activity were further decreased with hemin than with losartan. Hemin enhanced the expression of phosphoinositide 3-kinase (PI3K) p85 regulatory subunit, in contrast to losartan. The PI3K/Akt signalling pathway activation by hemin was related to heme oxygenase-1 activation and an increase in biliverdin reductase, and its inhibition by LY294002 reversed the effects of hemin on collagen I and caspase-3 expression. Finally, hemin increased Akt activation, and concomitantly decreased RhoA and p38 mitogen-activated protein kinase activation. CONCLUSION We confirmed a positive effect of hemin on oxidative cardiac damage, apoptosis, and fibrosis induced by hypertension by modulating the NADPH oxidase activation through enhanced expression of the PI3K p85 regulatory subunit.

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.

Initial color Doppler findings in retinal vein occlusion

We assessed early hemodynamic characteristics of various types of retinal vein occlusion using color Doppler imaging and spectral analysis.

Heme Oxygenase-1 Inducer Hemin Attenuates the Progression of Remnant Kidney Model

Backgrounds/Aims: Heme oxygenase-1 (HO-1) has been shown to protect against fibrotic proliferation and apoptosis in several models of renal damage. The purpose of this study was to evaluate the impact of a treatment with the HO-1 inducer hemin on the progression of chronic kidney disease in nephrectomized rats versus the AT1 receptor antagonist losartan. Methods: The rats underwent 5/6 renal ablation and after the procedure received either losartan (20 mg/kg/day; n = 9), hemin (50 mg/kg/twice a week; n = 8) or vehicle (n = 8) over a 12-week period. At week 12, blood pressure was measured, urine, blood and remnant kidney were collected for biochemical (proteinuria, urea, creatinine) and histopathological (degrees of glomerulosclerosis and tubular atrophy) analysis. The expressions of HO-1, bone morphogenic protein 7 (BMP-7) and TGF-β were assessed by immunochemistry, and the level of the apoptosis marker protein caspase-3 by Western blot, on the remnant kidney. Results: Hemin significantly reduced blood pressure, proteinuria, inhibited the expression of TGF-β and caspase-3 protein and increased BMP-7 expression protein. Hemin-treated rats had lower glomerulosclerosis and tubular atrophy indexes than controls. Conclusion: Hemin treatment attenuates the progression of chronic kidney disease and appears more efficient than losartan in our rat model hypothetically because of the impact of hemin on the renal expression of BMP-7.

New Insights into Sexual Dimorphism during Progression of Heart Failure and Rhythm Disorders

Neurohormonal imbalance is a key determinant of the progression of heart failure (HF), which results in an elevated risk of mortality. A better understanding of mechanisms involved may influence treatment strategies. The incidence and prevalence of HF are lower in women. We explored sexual dimorphism in the progression of HF using a mice model of neurohormonal-dependent HF. Male and female mice overexpressing the human beta2-adrenergic receptor (TG4 strain) develop HF. We compared TG4 animals with age-matched wild-type controls. Cardiac function was studied in vivo by echocardiography and electrocardiography. Histological studies were performed. Conduction parameters were assessed by intracardiac electrophysiological exploration, as was the occurrence of spontaneous and inducible arrhythmias. The patch-clamp technique was used to determine the cellular electrophysiological profile. The role of hormonal status in HF progression was investigated by surgical gonadectomy. High mortality rate was observed in TG4 mice with a dramatic difference between males and females. Male TG4 mice exhibited intraventricular conduction abnormalities, as measured by infrahisian interval and QRS durations potentially determining reentrant circuits and increasing susceptibility to arrhythmia. The severity of HF was correlated with the degree of fibrosis, which was modulated by the gonadal hormones. Action potentials recorded from male and female left ventricular cardiomyocytes were indistinguishable, although both sexes exhibited delayed repolarization when compared with their wild-type counterparts. In conclusion, female TG4 mice were better protected than males against cardiac remodeling and rhythm disorders. A link between fibrosis, conduction time, and mortality was established in relation with sex hormones.