Daniel Antier

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.

Oxidative Stress Is Related to the Deleterious Effects of Heme Oxygenase-1 in an In Vivo Neuroinflammatory Rat Model

Heme oxygenase-1 (HO-1) induction is associated with beneficial or deleterious effects depending on the experimental conditions adopted and the neurodegenerative rodent models used. The present study aimed first to evaluate the effects of cerebral HO-1 induction in an in vivo rat model of neuroinflammation by intrastriatal injection of quinolinic acid (QA) and secondly to explore the role played by reactive oxygen species (ROS) and free iron (Fe2+) derived from heme catabolism promoted by HO-1. Chronic I.P. treatment with the HO-1 inductor and substrate hemin was responsible for a significant dose-related increase of cerebral HO-1 production. Brain tissue loss, microglial activation, and neuronal death were significantly higher in rats receiving QA plus hemin (H-QA) versus QA and controls. Significant increase of ROS production in H-QA rat brain was inhibited by the specific HO-1 inhibitor ZnPP which supports the idea that ROS level augmentation in hemin-treated animals is a direct consequence of HO-1 induction. The cerebral tissue loss and ROS level in hemin-treated rats receiving the iron chelator deferoxamine were significantly decreased, demonstrating the involvement of Fe2+in brain ROS production. Therefore, the deleterious effects of HO-1 expression in this in vivo neuroinflammatory model were linked to a hyperproduction of ROS, itself promoted by free iron liberation.

Stability of antibiotics in portable pumps used for bronchial superinfection: guidelines for prescribers.

OBJECTIVES. The aims of this study were (1) to collect data on the stability of antibiotics in portable pumps for the treatment of bronchial superinfection in patients with cystic fibrosis and (2) to provide guidelines for prescribers. METHODS. The stability over 72 hours, in portable pumps stored at 35°C, of piperacillin plus tazobactam, ticarcillin plus clavulanic acid, cefsulodin, cefepime, and aztreonam was checked at 3 different concentrations. Stability was assessed through visual examination, pH measurements, and direct measurements of drug concentrations by using high-performance liquid chromatography. All parameters were measured at time 0, time 0 plus 24 hours, and time 0 plus 72 hours. RESULTS. Degradation rates for penicillin plus β-lactamase inhibitor combinations remained <10% at time 0 plus 24 hours for all drugs, but the rate for piperacillin reached 12% for the highest concentration tested. The cephalosporins cefepime and cefsulodin had significant respective degradation rates of 18% and 28% at time 0 plus 24 hours and 60% and 68.5% at time 0 plus 72 hours, which were linked to the storage temperature. Aztreonam seemed to be stable over 72 hours. CONCLUSIONS. This work provides data on drug stability that were lacking, allowing recommendations for physicians to optimize the safety and efficacy of antibiotic treatment of patients with cystic fibrosis. Piperacillin plus tazobactam and ticarcillin plus clavulanic acid infusions must be limited to 24 hours, and patients receiving cefepime or cefsulodin must wear a cold pack close to the ambulatory drug-delivery device during the infusion.

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.

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.

Translocator Protein (18 kDa) Mapping with [125I]-CLINDE in the Quinolinic Acid Rat Model of Excitotoxicity: A Longitudinal Comparison with Microglial Activation, Astrogliosis, and Neuronal Death

Excitotoxicity leads to an inflammatory reaction involving an overexpression of: translocator protein 18 kDa (TSPO) in cerebral microglia and astrocytes. Therefore, we performed ex vivo explorations with [125]-CLINDE, a TSPO-specific radioligand, to follow the time course of TSPO expression, in parallel with lesion progression, over 90 days after induction of cerebral excitotoxicity in rats intrastriatally injected with quinolinic acid. Biodistribution data showed a significant increase in CLINDE uptake on the injured side from 1 days postlesion (dpl); the maximal striatal binding values evidenced a plateau between 7 and 30 dpl. [125I]-CLINDE binding was displaced from the lesion by PK11195, suggesting TSPO specificity. These results were confirmed by ex vivo autoradiography. Combined immunohistochemical studies showed a marked increase in microglial expression in the lesion, peaking at 14 dpl, and astrocytic reactivity enhanced at 7 and 14 dpl, whereas a prominent neuronal cell loss was observed. At 90 dpl, CLINDE binding and immunoreactivity targeting activated microglia, astrogliosis, and neuronal cell density returned to a basal level. These results show that both neuroinflammation and neuronal loss profiles occurred concomitantly and appeared to be transitory processes. These findings provide the possibility of a therapeutic temporal window to compare the differential effects of antiinflammatory treatments in slowing down neurodegeneration in this rodent model, with potential applications to humans.

Direct implication of carbon monoxide in the development of heart failure in rats with cardiac hypertrophy subjected to air pollution

Pollution is known to particularly affect patients with respiratory insufficiency and right ventricle abnormalities. We therefore hypothesized that carbon monoxide (CO) at low dose could be involved in cardiovascular disorders in patients with chronic hypoxic pulmonary hypertension secondary to chronic hypoxia. Ten-week-old male and female healthy Dark Agouti rats were randomly divided into two series—untrained (U) and trained (T)—of four groups of 18 animals each. Both U and T series were continuously exposed to ambient air (UAIR, and TAIR; n=16) or air plus 50 ppm CO (UAIR+CO and TAIR+CO; n=18). Similarly, rats initially subjected to right ventricle hypertrophy secondary to chronic hypoxia (H) were continuously exposed to ambient air (THAIR, and UHAIR; n=18) or air plus 50 ppm CO (UHAIR+CO, and THAIR+CO; n=18). Doppler-echocardiography and hemodynamic studies performed at rest both indicated that CO had no significant effect on cardiac morphology or functions in control rats (UAIR+CO vs UAIR). In contrast, cardiac dilation and large decreases in left ventricular ejection fraction, mitral early diastolic rapid inflow (E) deceleration, E/atrial contraction filling (A) ratio, +dP/dt, and −dP/dt were found in THAIR+CO compared with THAIR. After exposure, heart rate variability was unaffected in UAIR+CO, whereas total power spectra were markedly decreased and low frequency/high frequency power ratio was increased in THAIR+CO rats. CO pollution could be directly involved in cardiac disorders of patients with pre-existent hypertrophic cardiomyopathies.

Dexfenfluramine discontinuous treatment does not worsen hypoxia-induced pulmonary vascular remodeling but activates RhoA/ROCK pathway: Consequences on pulmonary hypertension

The anorectic drug, dexfenfluramine has been associated with an increase in the relative risk of developing pulmonary hypertension. 5-hydroxytryptamine (5-HT) is a mitogen for smooth muscle cell, an effect that relies on 5-HT transporter expression and which has been proposed to explain pulmonary side effect of dexfenfluramine, and more particularly its effect on vascular remodeling. However recent data supported a major role of pulmonary artery vasoconstriction through the RhoA/Rho-kinase pathway. We questioned whether or not anorectic treatment aggravates pulmonary hypertension through vascular remodeling and if RhoA/Rho-kinase (ROCK) was potentially involved. In rats exposed to hypoxia, concomitant dexfenfluramine treatment (5 mg/kg/day, i.v.) for 4 weeks had no effect on pulmonary hypertension development. When exposure to 2 weeks of chronic hypoxia followed discontinuation of dexfenfluramine treatment (dexfenfluramine-hypoxic rats), echocardiographic parameters of pulmonary artery flow and right ventricle were further altered (P<0.05) as well as right ventricle systolic pressure was further increased (P<0.001) when compared to hypoxic rats treated with vehicle (hypoxic rats). However, the total number of muscularized distal pulmonary arteries artery was similar in dexfenfluramine-hypoxic vs. hypoxic rats (P>0.05). Western blot, RT-PCR and immunofluorescence analysis revealed a greater expression of 5-HT transporter and ROCK, as well as a greater activation of RhoA in dexfenfluramine-hypoxic rats compared to hypoxic rats. These data show that increased 5-HT transporter expression that follows dexfenfluramine discontinuation is not associated to a greater vascular remodeling despite worsening the development of pulmonary hypertension. Furthermore dexfenfluramine discontinuation promotes a greater RhoA/ROCK pathway activation. This pathway, involved in many cardiovascular diseases, might explain the cardiac and pulmonary toxicity of serotoninergic agonists.

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.

Evaluation of the Sensitivity of an in vitro High Frequency Ultrasound Device to Monitor the Coagulation Process: Study of the Effects of Heparin Treatment in a Murine Model

This study evaluates the sensitivity of a new in vitro high frequency ultrasound test of the whole blood coagulation process. A rat model of anticoagulant treatment is reported. Many recent studies of the role of red blood cells in the whole blood coagulation process have revealed an increasing demand for global tests of the coagulation process performed on whole blood instead of plasma samples. In contrast to existing optical tests, high frequency ultrasound presents the advantages of characterizing the mechanical properties of whole blood clotting. Ultrasound longitudinal wave velocity and integrated attenuation coefficient (IAC) were simultaneously assessed in a 10 to 30 MHz frequency range during the whole blood coagulation process in vitro in rats under anticoagulant therapy. Differences between humans and rats were also clearly emphasized in non-clotting blood and in clotting blood using specific criteria deduced from acoustic parameters (ultrasound velocity for non-clotting blood:=1574+/-2m/s for rats and 1583+/-3m/s for humans and IAC=2.25+/-0.14 dB/cm for rats and 1.5+/-0.23 dB/cm for humans). We also measured the coagulation time t(0) from the acoustic velocity (t(0) =11.15+/-7 min for control rat blood and 43.3+/-11.4 min for human blood). Different doses of heparin were administered to rats. The sensitivity of the ultrasound device to the effects of heparin was evaluated. Differences between non-treated rats and chronically and acutely treated rats were recorded and quantified. We particularly noted that the slope S and the amplitude I of the variations in acoustic velocity were linked to clot retraction, which is a good indicator of the platelet function. The amplitude of the variations in S was between (20+/-8) x1 0(-3) m/s(2) for control group rats, and (0.92+/-0.35) x 10(-3) m/s(2) for chronic heparin-treated group rats. The values of I were 15 times higher for control group rats than for chronic heparin-treated group rats.