| Background:Endothelial progenitor cells (EPCs) have recently emerged as a new cell population in the circulation of mammals expressing CD133, CD31, CD34 and VEGFR-2. Increasing evidence indicates that they play a critical role in repair of vascular injury and pathological neovascularization. EPCs dysfunction has been linked to many cardiovascular disorders, including pulmonary arterial hypertension (PAH) in humans. Plexiform lesions are a hallmark of severe PAH, and are thought to signify a poor prognosis of PAH. More recent studies showed that patients with severe PAH had increased EPCs in blood, and that EPCs within plexiform lesions were originated from circulating EPCs, indicating that these cells are involved in the angioproliferative vascular remodelling. However, the underlying mechanisms remain unclear. PAH in broiler shares overlapping histological features with human patients, including medial hypertrophy, intimal proliferation and occlusive plexiform lesions in the pulmonary vasculature, and is thus thought to be an excellent model of human PAH. Furthermore, persistent inflammation and increased expression of hepatocyte growth factor (HGF) were found in plexiform lesions, but the mechanism involved is not yet fully understood. The plexiform lesion is characterized by round and slit-like vascular spaces lined by prominent endothelial cell-like cells termed as EPCs. The lesion is surrounded by chronic inflammatory cells including T and B lymphocytes and macrophages. Additionally, the inflammation was limited to the arterial lesions. Previous study demonstrated that tumour necrosis factor-α, a cytokine released by lymphocyte and macrophages, reduced proliferation, migration, adhesion and tube formation capacity of EPCs. We hypothesize that co-culture with lymphocyte may lead to EPCs dysfunction, which may explain why the EPCs at the site of pexiform lesions do not differentiate to endothelial cells.The overall purpose of present work was to study the features of EPCs in an avian species, and also evaluate the regulatory effect of HGF on EPC functions under inflammatory condition, with the emphasis on how EPCs contribute to the development of plexifrom lesions in PAH. No such studies have been carried out so far with regard to methods for culture of EPCs from chicken bone marrow, phenotypic characterization and In vitro angiogenic potential of PBMMNCs and BMMNCs from avian species and particularly regulatory effect of HGF on EPCs function under pathological conditions. This study was conducted in three parts:Experiment one was carried out to study, the differential characteristics of chicken bone marrow derived monocytes on EGM-2 and DMEM media. The aim was to compare the biological behaviors of EPCs derived from these two medium and also evaluate the effects on in vitro angiogenic potential. Bone Marrow derived MNCs retained their constitutive spindle-like morphology throughout the study period and expressed CD133, CD31 and VEGFR-2. In fact, there was no significant difference in number of Dil-ac-LDL/FITC- UEA-1, dual posit ive cells amongst groups. However, BMMNCs cultured on EGM-2 formed tube-like structures, which were not morphologically comparable to those developed by the cells cultured on DMEM media. Interestingly, EGM-2 proved to be better for EPCs induction as it increased cell number ex vivo, helped in endothelial differentiation, migration and also promoted in vitro vasculogenic potential of cells. However, DMEM enhanced early differentiation of cells morphologically, but did not support in endothelial differentiation, migration and also vasculogenic activity of cells. Keeping in view diversified effects of different medium on chicken marrow derived EPCs; we investigated phenotypes and in vitro angiogenic capacity of EPCs derived from BM and PB from chicken. PBMNCs developed typical EPC appearances, with initial spindle shape followed by a cobblestone appearance, whereas BMMNC-derived cells retained their constitutive spindle-like morphology throughout the experiment. Cells derived from both sources expressed CD133, CD31 and VEGFR-2, while PBMNCs-derived cells had decreased CD133 expression. Nevertheless, the number of Dil-ac-LDL/lectin dual-positive cells was not different between groups. Adipogenic and osteogenic lineages were verified merely in BMMNC- derived cells. PBMNC-derived cells formed tubular networks on Matrigel. However, BMMNC-derived cells formed few tube-like structures which were not morphologically comparable to those developed by their counterparts. Our results suggest that the so called "EPCs" derived from BMMNCs are not "true" EPCs, supporting previous findings on mammals that BM may not serve as an optimal isolation source of EPCs.We further extended our study on, In vitro effect of hepatocyte growth factor on the functionality of endothelial progenitor cells exposed to lymphocytes. Cells were confirmed as EPCs by ac-LDL uptake and lectin binding, a typical feature of endothelial lineage cells. At the end of treatments with HGF and/or lymphocytes, EPCs were detached with 0.25% trypsin-EDTA and used for the experiment. The exogenous effect of HGF on lymphocytes challenged peripheral blood derived endothelial progenitor cells (EPCs) was examined. Small colony clusters composed of spindle-like cells (5-7days) differentiated into monolayer of cobblestone-like cells around day 10. The proliferation of EPCs exposed to lymphocytes alone did not show significant change as compared to normal cultures, suggesting that exposure to lymphocytes alone does not affect the proliferation of EPCs. Interestingly, the proliferative capacity of EPCs was significantly inhibited by HGF alone at 5 or 10 ng/mL, but was markedly enhanced by 20 ng/mL HGF. About 17.2% of the EPCs cultured in normal medium underwent apoptosis, exposure to HGF at a concentration of 5,10 or 20 ng/mL significantly inhibited EPC death. A marked decline in cell apoptosis was observed as well when EPCs were treated with lymphocytes alone, indicating that HGF and lymphocytes individually produce significant suppression of EPC apoptosis. Combinations of HGF and lymphocytes were more effective than HGF alone in reducing apoptosis of EPCs. In fact, maximal suppression of cell apoptosis was observed with 5 ng/mL HGF, in EPCs already exposed to lymphocytes. HGF at a higher concentration (20ng/mL), significantly promoted migration and was in parallel with proliferation results. Whereas 10ng/mL HGF promoted cell migration in cells exposed to lymphocytes. Overall, lymphocytes exposure significantly enhanced migration in all groups. Likewise lowest HGF concentration at 5ng/mL exhibited higher migration rate again suggesting a quite possible synergistic effects at minimal dose. Overall, it was demonstrated that HGF stimulate the proliferation, migration, angiogenic capacity of lymphocytes exposed endothelial progenitor cells derived from chicken. Furthermore, HGF significantly reduced apoptosis, thus increase cell survival capability of lymphocytes exposed EPCs. It was concluded that increased number, and more mobilization, reduced cell death, significant tube formation consequently lead to over accumulation of EPCs at the site of vascular injury and development of plexiform lesion. |
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