Differentiation of mesothelial cells during their reparative regeneration

The investigation on regenerative processes of mesothelium of the parietal peritoneum was performed in 120 white mice under the effect of certain irritants producing lesions various in depth and intensity. Nuclear-cytoplasmic relations and ultramicroscopic cellular rearrangement were studied during the process of differentiation of the mesothelial regenerate. Two periods of the regenerative process are stated and it is demonstrated that rearrangement of the mesothelial cells and the mode of their division depend on intensity of the lesions. When the peritoneal lesion is severe, at the first stages of regeneration (the 1st period) rearrangement of cells towards their hypertrophy and increased functional activity is predominant in the mesothelium. Further (the 2d period), the number of mitotically dividing cells is increasing in the mesothelial regenerate and in rearrangement of the mesothelial cells the processes connected with a partial loss of their signs of specialization predominate. The transition from one period into another is gradual and duration of each depends on intensity of the lesion.     

Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat

Angiogenesis is important for adult tissue regeneration as well as normal development. Vascular endothelial growth factor (VEGF) is a unique potent angiogenic factor, and plays an essential role in regulating angiogenesis during embryonic development, normal tissue growth, and tissue regeneration. Recent evidence shows that nerve growth factor (NGF) also plays a role as an angiogenic regulator as well as a well-known neurotrophic factor. The aim of this study was to investigate whether thymus regeneration accompanies reparative angiogenesis and also to evaluate whether the thymic expression of VEGF is regulated by NGF in vivo and in vitro. Here, we show that high VEGF mRNA and protein levels are concomitant with reparative angiogenesis that occurs dramatically during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus. Fluorescent thymus angiography using FITC-dextran showed that thymic regeneration is associated with a much denser capillary network compared with normal control thymus. Furthermore, the expressions of NGF and TrkA were highly increased during thymic regeneration. We also show that NGF mediates thymic epithelial induction of VEGF expression in vitro and in vivo. Taken together, our results suggest that NGF-mediated VEGF up-regulation in thymic epithelial cells may contribute to reparative angiogenesis during thymic regeneration in adult.     

Neural differentiation of rat aorta pericyte cells

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain.     

Physiological and reparative regeneration of the mitochondria]

The ultrastructure of mitochondria of hepatocytes in normal and pathological conditions was studied. It was shown that the process of regeneration of the ultrastructure of swollen mitochondria with a lucent matrix up to the normal state was completed in hepatocytes of the rat and chick embryos within one day. It was established that one of the ways of intraorganoid regeneration of mitochondria in hepatocytes of chick embryos and of mice after injections of CCl4 twice a week for 5 months was clasmatosis of the destroyed mitochondria fragments and their removal through the partially disintegrated exterior membrane of mitochondria followed by the membrane restoration. The process of mitochondrial regeneration after clasmatosis of its fragments was shown to require two days in the chick embryo hepatocytes.     

The reparative regeneration of the endothelium of the mouse aorta during microsurgical interventions following local gamma irradiation (based on scanning electron microscopic data)

In experiments with rats, abdominal aorta was subjected to microsurgical anastomosis after local irradiation with doses of 40 and 50 Gy. Irrespective of the time interval between the operation and irradiation the iatrogenic defect was restored completely. With the operation performed 24 h after irradiation the platelet adhesion decreased, the proliferation was inhibited depending on radiation dose, and the pattern of the endotheliocyte migration changed. The above effects were absent with the operation performed one month after irradiation.     

Isolation and characterization of vasoformative endothelial cells from the rat aorta

Summary We describe here a modified nonenzymatic method for the isolation of rat aortic endothelial cells with vasoformative properties. Aortic rings placed on plastic or gelatin-coated surfaces generated outgrowths primarily composed of endothelial cells. Prompt removal of aortic explants after endothelial migration minimized fibroblast contamination. However, fibroblasts, because of their high proliferative rate tended to overgrow the endothelial cells even when present in small numbers. This potential pitfall was avoided by weeding out fibroblasts with the rounded tip of a bent glass pipette. Primary endothelial colonies free of fibroblasts were segregated in cloning rings, trypsin-treated, and transferred to gelatin-coated dishes. Endothelial cells were cultured in MCDB 131 growth medium containing 10% fetal bovine serum, endothelial cell growth supplement, and heparin. Using this technique, pure endothelial cell strains were obtained from single aortic rings. Confluent endothelial cells formed a contact-inhibited monolayer with typical cobblestone pattern. The endothelial cells were positive for Factor VIII-related antigen, took up DiI-Ac-LDL, and bound the Griffonia Simplicifolia-isolectin-B4. Endothelial cells cultured on collagen gel formed a polarized monolayer, produced basement membrane, displayed Weibel-Palade bodies and caveolae, and were connected by tight junctions. In addition, they reorganized into a network of microvascular cords and tubes when overlaid with a second layer of collagen and formed microvascular sprouts in response to fibroblast-conditioned medium. This isolation procedure yields stable strains of vasoformative endothelial cells, which can be used to study aortic endothelium-related angiogenesis and its mechanisms.     

The mechanisms of reparative regeneration of venous endothelium

The reaction of endothelial cells of the inferior vena cava in response to freezing-induced lesions has been analysed in the experiments on 34 young adult Kyoto-Wistar normotensive rats. First the de-endothelialized surface is covered with flattened platelets and then, three days after surgery, the endothelium is restored as a result of migration and proliferation of endotheliocytes. The migrating endothelial cells removed the adhered platelets from de-endothelialized surface. The young endothelium was presented by a single layer of strongly elongated endothelial cells whose axis was parallel to the flow of blood. An immature endothelium is characterized by an increased number of endotheliocytes. No essential differences in the reaction of venous and aortic endothelium have been revealed in response to freezing-induced lesions.     

Dietary L-arginine restores aspirin-induced endothelial dysfunction in rat aorta

L-Arginine induced elevation of the vascular prostanoid led us to think that the risk of coronary spasm may increase in L-arginine consumers when they are subjected to cyclooxygenase inhibitors and this limits the therapeutic value of aspirin. So the aim was to investigate the interaction of aspirin and dietary L-arginine in male rats. Animals were divided into four groups and fed with normal food. The first group received tap water while the second, third and fourth groups were subjected daily to aspirin (8.6 mg/kg), L-arginine (143 mg/kg) and aspirin + L-arginine combination in their drinking water respectively for 7 days. Vasomotor responses were recorded in the aortic rings suspended for isometric-force recordings. Aspirin treatment significantly reduced the dilation to acetylcholine and sodium nitroprusside. Attenuated phenylephrine contractility was associated with normal acetylcholine response in L-arginine group. Addition of L-arginine to aspirin treatment completely prevented aspirin-induced endothelial dysfunction but defective response to sodium nitroprusside persisted. Dietary L-arginine without affecting maximal dilation to acetylcholine significantly increased the share of dilator prostanoid which appears to resist aspirin. These results demonstrated that dietary L-arginine increases dilator prostaoid in rat aortic rings. Contrary to our expectation, co-administered L-arginine protected aspirin induced endothelial dysfunction and ruled out the limitation of aspirin use in L-arginine consumers.     

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (α-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.     

Vascular reactivity and endothelial NOS activity in rat thoracic aorta during and after hyperbaric oxygen exposure

Accumulating evidence suggests that hyperbaric oxygen (HBO) stimulates neuronal nitric oxide (NO) synthase (NOS) activity, but the influence on endothelial NOS (eNOS) activity and vascular NO bioavailability remains unclear. We used a bioassay employing rat aortic rings to evaluate vascular NO bioavailability. HBO exposure to 2.8 atm absolute (ATA) in vitro decreased ACh relaxation. This effect remained unchanged, despite treatment with SOD-polyethylene glycol and catalase-polyethylene glycol, suggesting that the reduction in endothelium-derived NO bioavailability was independent of superoxide production. In vitro HBO induced contraction of resting aortic rings with and without endothelium, and these contractions were reduced by the NOS inhibitor N(omega)-nitro-l-arginine. In addition, in vitro HBO attenuated the vascular contraction produced by norepinephrine, and this effect was reversed by N(omega)-nitro-l-arginine, but not by endothelial denudation. These findings indicate stimulation of extraendothelial NO production during HBO exposure. A radiochemical assay was used to assess NOS activity in rat aortic endothelial cells. Catalytic activity of eNOS in cell homogenates was not decreased by HBO, and in vivo HBO exposure to 2.8 ATA was without effect on eNOS activity and/or vascular NO bioavailability in vitro. We conclude that HBO reduces endothelium-derived NO bioavailability independent of superoxide production, and this effect seems to be unrelated to a decrease in eNOS catalytic activity. In addition, HBO increases the resting tone of rat aortic rings and attenuates the contractile response to norepinephrine by endothelium-independent mechanisms that involve extraendothelial NO production.     

Changes in the endothelium of the aorta of the rat during regeneration (according to the results of scanning electron microscopy)

In the experiments performed on 53 Kioto-Wistar normotensive rats, the reaction of endothelial cells of the abdominal aorta has been analysed in response to the lesion by freezing or by hypoosmotic effect. At first the deendothelized surface is covered with flatten thrombocytes, and then the continuity of the endothelium is restorted as a result of migration and proliferation of endotheliocytes. This is accompanied with a reconstruction of their cytoskeleton (structurization and redistribution). The immature endothelium is characterized by an increased numerical density of strongly elongated endothelial cells, decreased resistency to collagenase effect and a great adhesiveness to blood leucocytes. No essential differences in the reaction of endotheliocytes are revealed in dependence of deendothelization means.