eEOC-mediated modulation of endothelial autophagy, senescence, and EnMT in murine diabetic nephropathy.

Diabetic nephropathy is the most frequent single cause of end-stage renal disease in our society. Microvascular damage is a key event in diabetes-associated organ malfunction. Early endothelial outgrowth cells (eEOCs) act protective in murine acute kidney injury. The aim of the present study was to analyze consequences of eEOC treatment of murine diabetic nephropathy with special attention on endothelial-to-mesenchymal transdifferentiation, autophagy, senescence, and apoptosis. Male C57/Bl6N mice (8-12 wk old) were treated with streptozotocin for 5 consecutive days. Animals were injected with untreated or bone morphogenetic protein (BMP)-5-pretreated syngeneic murine eEOCs on days 2 and 5 after the last streptozotocin administration. Four, eight, and twelve weeks later, animals were analyzed for renal function, proteinuria, interstitial fibrosis, endothelial-to-mesenchymal transition, endothelial autophagy, and senescence. In addition, cultured mature murine endothelial cells were investigated for autophagy, senescence, and apoptosis in the presence of glycated collagen. Diabetes-associated renal dysfunction (4 and 8 wk) and proteinuria (8 wk) were partly preserved by systemic cell treatment. At 8 wk, antiproteinuric effects were even more pronounced after the injection of BMP-5-pretreated cells. The latter also decreased mesenchymal transdifferentiation of the endothelium. At 8 wk, intrarenal endothelial autophagy (BMP-5-treated cells) and senescence (native and BMP-5-treated cells) were reduced. Autophagy and senescence in/of cultured mature endothelial cells were dramatically reduced by eEOC supernatant (native and BMP-5). Endothelial apoptosis decreased after incubation with eEOC medium (native and BMP-5). eEOCs act protective in diabetic nephropathy, and such effects are significantly stimulated by BMP-5. The cells modulate endothelial senescence, autophagy, and apoptosis in a protective manner. Thus, the renal endothelium could serve as a therapeutic target in diabetes-associated kidney dysfunction.

Identification of Endothelial Cells and Their Progenitors.

Blood vessel formation is a key feature in physiologic and pathologic processes. Once considered a homogeneous cell population that functions as a passive physical barrier between blood and tissue, endothelial cells (ECs) are now recognized to be quite "heterogeneous." While numerous attempts to enhance endothelial repair and replacement have been attempted using so called "endothelial progenitor cells" it is now clear that a better understanding of the origin, location, and activation of stem and progenitor cells of the resident vascular endothelium is required before attempting exogenous cell therapy approaches. This chapter provides an overview for performance of single-cell clonogenic studies of human umbilical cord blood circulating endothelial colony-forming cells (ECFC) that represent distinct precursors for the endothelial lineage with vessel forming potential.

Endothelial Progenitor Cells' Classification and Application in Neurological Diseases.

The therapeutic effects of endothelial progenitor cells (EPCs) on ischemic stroke have been extensively studied in recent years. However, the differences in early EPCs and endothelial outgrowth cells (EOCs) are still unclear. Clarifications of their respective properties and specific functioning characteristics contribute to better applications of EPCs in ischemic diseases. In this review, we discuss cellular origin, isolation, culture, surface markers of early EPCs and EOCs and relevant applications in neurological diseases. We conclude that EOCs possess all characteristics of true endothelial progenitors and have potent advantages in EPC-based therapies for ischemic diseases. A number of preclinical and clinical applications of EPCs in neurological diseases are under study. More studies are needed to determine the specific characteristics of EPCs and the relevant mechanisms of EPCs for neurological diseases.

Are endothelial outgrowth cells a potential source for future re-vascularization therapy?

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis after ischemic diseases. EPCs could have a potential for future therapies with the purpose of enhancing endothelial repair. However, due to the low amount of these cells in circulation they have to be expanded in vitro before administration into recipients. The purpose of this study was to analyse and evaluate possible changes in morphology and functionality as a result of in vitro ageing of a subtype of EPCs called endothelial outgrowth cells (EOCs), since such changes might compromise the cells' ability to participate in vasculogenesis. EOCs were isolated and grown from human umbilical cord blood using two methodologies with varying degree of cell purification. The changes between the two culture setups and the changes occurring in EOCs over time were traced by flow cytometry and assays for growth, tube formation, and beta-galactosidase production. The cells showed to be indistinguishable from each other during the first weeks of culture. The cells showed a changed morphology with bigger and more granular cells and the growth rate decreased with time. The cells also showed an increased Beta-galactosidase expression and decreased tube formation ability in late passage EOCs. Our data indicates that EOCs undergo senescence during long-term expansion and therefore time for cell harvest has to be validated in order to achieve functional cells still maintaining a therapeutic potential. A possible application in large animal or humans could be local injection of EOCs into affected areas and thereby reducing the need for long-term expansion of the cells.

In vivo bio-distribution and homing of endothelial outgrowth cells in a tumour model.

INTRODUCTION: Endothelial progenitor cells (EPCs) has been reported to have the potential for advancing revascularization of ischemic tissue. However, the heterogeneous nature of these cells calls for specification of the angiogenic potential of each subtype. The purpose of this study was to gain additional insight on the homing capacity of the EPC subtype, endothelial outgrowth cells (EOCs) in tumours using a well-established tumour model. METHODS: (111)Indium ((111)In) - and 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labelled EOCs derived from human umbilical cord blood were injected into mice with a C3H mammary carcinoma foot tumour. The subsequent capture of the EOCs was traced by estimation of activity in individual organs, autoradiography and fluorescence microscopy. RESULTS: (111)In activity was found in tumour and other organs. However, varying parts of the activity originated from free (111)In lost from EOCs. Autoradiography demonstrated accumulation of (111)In activity in the tumour rim. Microscopy proved that a least part of this radioactivity originated from the presence of human derived EOCs and that those EOCs were not located in the endothelial lining of vessels, in the tumour. CONCLUSION: The results demonstrated the presence of xenotransplanted EOCs in the rim of a C3H mammary carcinoma. They were, however, not located in the endothelial lining of the vessels, thus indicating that their effect in vasculogenesis might be mediated via paracrine mechanisms rather than differentiating into endothelial cells (ECs) in tumour vessels.

Endothelial Progenitor Cells' Classification and Application in Neurological Diseases

The therapeutic effects of endothelial progenitor cells (EPCs) on ischemic stroke have been extensively studied in recent years. However, the differences in early EPCs and endothelial outgrowth cells (EOCs) are still unclear. Clarifications of their respective properties and specific functioning characteristics contribute to better applications of EPCs in ischemic diseases. In this review, we discuss cellular origin, isolation, culture, surface markers of early EPCs and EOCs and relevant applications in neurological diseases. We conclude that EOCs possess all haracteristics of true endothelial progenitors and have potent advantages in EPC-based therapies for ischemic diseases. A number of preclinical and clinical applications of EPCs in neurological diseases are under study. More studies are needed to determine the specific characteristics of EPCs and the relevant mechanisms of EPCs for neurological diseases.

Effects of asymmetric dimethylarginine on apoptosis and expression of c-Jun N-terminal kinase in endothelial outgrowth cells / 天津医药

Objective To investigate the effects of asymmetric dimethylarginine (ADMA) on apoptosis and phosphorylation of c-jun N-terminal kinase (JNK) in endothelial outgrowth cells (EOCs). Methods The mononuclear cells were harvested from umbilical cord blood by ficoll density gradient centrifugation, and induced into EOCs and then expanded in vitro. The identified EOCs were treated with different concentrations of ADMA (0, 1, 5, 10, 20 μmol/L) for 48 h. The adherent cells were treated with 10 μmol/L ADMA,then different concentrations of JNK specific inhibitor SP600125 (0, 5,10,20 and 40 μmol/L) were added and incubated for 48 hours. Caspase-3 activity was measured by microplate reader. Apoptotic incidences of EOCs were quantitatively determined by flow cytometry. The expression of Caspase- 3 and phosphorylase-JNK (p-JNK) were detected by Western blot assay. Results The treatment of ADMA (1-20 μmol/L) significantly induced apoptosis in EOCs by enhancing Caspase-3 express and also induced phosphorylation of JNK (P<0.05). Meantime, the JNK specific inhibitor SP600125 could attenuate the apoptosis induced by ADMA during this process (F=6.733,P<0.05) and inhibit the expression of Caspase-3 and p-JNK. Conclusion ADMA can induce apoptosis in EOC, which may be achieved by activating JNK signal transduction pathway.