Arben Dedja

Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes

Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.

Human amniotic fluid stem cells protect rat lungs exposed to moderate hyperoxia.

Treatment of bronchopulmonary dysplasia (BPD) remains as yet an unmet clinical need and recently stem cells have been proposed as a therapeutic tool in animal models. We investigated the role of amniotic fluid stem cells (AFS) in an adult rat model of hyperoxia lung injury.

Ureteral Stenosis in HDAF Pig‐to‐Primate Renal Xenotransplantation: A Phenomenon Related to Immunological Events?

The aim of this study was to analyze the incidence of ureteral stenosis in a life‐supporting human decay‐accelerating factor (hDAF) transgenic pig‐to‐cynomolgus monkey kidney transplantation model and determine the role of possible immunological events in its pathogenesis.

Extracellular matrix graft for vascular reconstructive surgery: evidence of autologous regeneration of the neoaorta in a murine model

OBJECTIVES The study aimed to evaluate the efficacy of the porcine small intestine submucosa extracellular matrix (SIS-ECM) in a murine model, as a possible vascular patch for clinical use in reconstructive vascular and potentially cardiac surgery. METHODS Fifteen adult male Sprague Dawley rats and five green fluorescent protein (GFP) rats were enrolled in this study. The SIS-ECM graft (6 mm long, 4 mm wide) was implanted for patch plasty on the abdominal aorta of the animal, after excising part of its anterior wall. Histology and immunohistochemistry were used to evaluate the results at 15, 30, 90 and 180 days post-surgery. RESULTS Graft re-population was demonstrated 15 days after implantation. The luminal surface of the regenerating tissue was partially covered by endothelial cells, and intimal hyperplasia occurred in the central part of the graft. Complete re-endothelialization of the patch with smooth muscle cells colonizing the graft and acting as the neoaortic wall was observed after 30 days. Near complete absorption of the biomaterial was observed after 180 days. No inflammatory cell reaction occurred. All animals survived and no graft aneurysm was observed. CONCLUSIONS A SIS-ECM patch allowed the colonization of host endothelial and smooth muscle cells in the graft. This material may be an ideal substitute for reconstructive vascular surgery, and its use could be extended to surgical repair of cardiac defects.

Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch.

Muscle tissue engineering can provide support to large congenital skeletal muscle defects using scaffolds able to allow cell migration, proliferation and differentiation. Acellular extracellular matrix (ECM) scaffold can generate a positive inflammatory response through the activation of anti-inflammatory T-cell populations and M2 polarized macrophages that together lead to a local pro-regenerative environment. This immunoregulatory effect is maintained when acellular matrices are transplanted in a xenogeneic setting, but it remains unclear whether it can be therapeutic in a model of muscle diseases. We demonstrated here for the first time that orthotopic transplantation of a decellularized diaphragmatic muscle from wild animals promoted tissue functional recovery in an established atrophic mouse model. In particular, ECM supported a local immunoresponse activating a pro-regenerative environment and stimulating host muscle progenitor cell activation and migration. These results indicate that acellular scaffolds may represent a suitable regenerative medicine option for improving performance of diseased muscles.

Methotrexate for immunosuppression in life-supporting pig-to-cynomolgus monkey renal xenotransplantation.

Abstract: Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantation models, but the data generated so far with MTX in pig‐to‐baboon cardiac transplantation studies have been disappointing. The potential of this agent was consequently explored in a life‐supporting pig‐to‐primate renal model using the cynomolgus monkey as the recipient species. Introductory in vitro and in vivo pharmacokinetic and pharmacodynamic studies with MTX were conducted in three cynomolgus monkeys. Subsequently,10 cynomolgus monkey recipients of a life‐supporting kidney from human decay‐accelerating factor transgenic pigs were administered MTX intravenously according to three different regimens. All the animals also received cyclosporine A and steroids. In addition, mycophenolate sodium (MPS) was administered post‐operatively in two of the three groups of transplanted animals. At clinically relevant concentrations, MTX is able in vitro to inhibit the mixed lymphocyte reactions (MLR) in cynomolgus monkeys. After intravenous administration, moreover, exposure of cynomolgus monkeys to MTX appeared to be higher than had been previously reported in baboons. Graft function was observed in the transplanted animals, which survived from 0 to 41 days. All but two animals revealed acute humoral rejection in the explanted graft and developed diarrhea. Diarrhea was the cause of euthanasia in five cases. It was unrelated to the administration of MPS and associated with severe histopathological signs of enteritis. This study demonstrates that the pharmacokinetic and pharmacodynamic profiles of MTX vary substantially between non‐human primate species. In vitro, MTX has immunosuppressive properties in the cynomolgus monkey at clinically relevant concentrations. In vivo, MTX has a very narrow therapeutic window in cynomolgus monkeys, however, as it does in baboons. We conclude that MTX is scarcely effective as an immunosuppressant, be it for induction or maintenance, in pig‐to‐cynomolgus monkey renal xenotransplantation.

Postnatal Hyperoxia Exposure Differentially Affects Hepatocytes and Liver Haemopoietic Cells in Newborn Rats

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O2 (moderate hyperoxia), or 95% O2 (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.

Cyclosporine and hyperoxia-induced lung damage in neonatal rats ☆

Cyclosporine effects on hyperoxia-induced histopathological and functional changes in the rat adult lung are controversial and the newborn lung has not been studied. Thus, we evaluated the effects of cyclosporine in young rats after 60% hyperoxia exposure postnatally. Experimental categories included: (1) room air for the first 5 postnatal weeks with daily subcutaneous injections of saline from postnatal day (PN)15 to PN35; (2) room air with daily injections of cyclosporine from PN15 to PN35; (3) 60% oxygen from PN0 to PN14 and then daily saline injections during the following three weeks; (4) 60% oxygen from PN0 to PN14 followed by cyclosporine treatment from PN15 to PN35. Hyperoxia significantly reduced the number of secondary crests and microvessel density, and it increased the mean alveolar size and septa thickness. Cyclosporine treatment did not significantly modify the hyperoxia-induced changes. Conversely, in normoxia, cyclosporine reduced microvessel density and the number of secondary crests. In conclusion, cyclosporine did not modify alveolar and microvascular parameters in hyperoxia exposure, although it caused some changes in normoxia.

Cobalt protoporpyhrin reduces caspase-3,-7 enzyme activity in neonatal porcine islets, but does not inhibit cell death induced by TNF-alpha.

Apoptotic phenomena observed in vitro following isolation and following transplantation contribute significantly to islet graft loss. Strategies to reduce apoptosis of islet tissue prior to and posttransplantation may improve graft survival and function and reduce the amount of tissue necessary to achieve insulin independence. The expression of cytoprotective proteins is one such strategy that may prolong islet survival. In this light, heme-oxygenase 1 (HO-1) upregulation has been studied in both allo- and xenotransplantation models. In this study, the effect of HO-1 on apoptosis in neonatal porcine islet-like cell clusters (NPICC) was assessed. In in vitro assessments of NPICC apoptosis, NPICC showed a high sensitivity to apoptotic stimulation using a combination of TNF-α and cycloheximide. Stimulation with TNF-α alone was sufficient to induce reproducible apoptotic responses as demonstrated by caspase-3,-7 activation and subdiploid DNA analysis. Dose-dependent, high-level HO-1 protein expression was achieved following culture of NPICC in medium containing either cobalt protoporphyrin (CoPP) or cobalt mesoporphyrin (CoMP). CoPP treatment resulted in the reduction of caspase-3,-7 enzyme activity following TNF-α stimulation. However, such an effect was not associated with a reduction in the levels of cell death. Indeed, the inhibition of caspase enzyme activity resulted in decreased PARP-1 cleavage, which may lead to heightened levels of necrosis in treated NPICC cultures, possibly explaining the observed commitment of NPICC to the death pathway.

Histopathological findings in the gastrointestinal tract of primate recipients of porcine renal xenografts following different immunosuppressive regimens

Abstract:  Background:  Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction or maintenance protocols in a large variety of xenotransplantation models. Combining the use of transgenic porcine organs expressing human decay‐accelerating factor (hDAF) with immunosuppressive therapy that included the use of CYP or MTX, survival of primate recipients of life‐supporting renal xenografts has been prolonged. However, both drugs can cause significant systemic toxicity and, in particular, gastrointestinal (GI) toxicity. To date only limited data have been reported on the histopathological features deriving from the use of such agents in non‐human primates.