| Background:Endothelial progenitor cells (EPCs) are specific mononuclear cells playing important roles in vascular repair and regeneration. They exert their functions in two predominant ways:by differentiating into mature endothelial cells (ECs) and by secreting cytokines. Their amount and viability are usually reduced in diseased individuals, thus are considered to be the reflection of endogenous angiogenic capacity and novel biomarkers to assess the severity of cardiovascular diseases. Cumulative experimental and clinical evidence suggests that transplantation of in vitro expanded EPCs has therapeutic effects in endothelium injury diseases including pulmonary arterial hypertension (PAH). Conversely, some researchers have reported inefficacy results of cell transplantation. EPCs have been related to inflammatory responses and arterial remodeling for participation in vasa vasorum and secretion pro-inflammatory factors. Pathological microenvironments in disease models are considered to play crucial roles in regulating outcomes of cell therapies. Control progenitor cells are beneficial to the PAH mice, whereas PAH ones may exacerbate endothelial injury. Increased plasma levels of soluble CD40ligand (sCD40L) are typical representatives of inflammatory related pathophysiological changes in PAH. CD40belongs to the superfamily of tumor necrosis factor receptors, and sCD40L is a member of the tumor necrosis factor superfamily. The engagement of sCD40L and its receptor CD40in cell surface can ignite and amplify inflammatory activities of vascular endothelium by regulating immune and non-immune cells. More recently, the system is gaining much attention for its role in the initiation and progression of PAH. But their effects on transplanted EPCs are yet to be further addressed. In this study we were prompted to investigate the potential effects of CD40pathway on regulating transplanted EPC functions and the application in PAH.Materials and Methods:(1) EPCs were isolated from femurs of male SD rats by density gradient centrifugation with ficoll. Some EPCs were incubated with recombinant murine sCD40L at0.1,0.5,1or2μg/ml for24h at day7.(2) Some EPCs were infected with Lv-shRNA for18h.(3) We observed the regulation of sCD40L on EPC migration, adhesion, proliferation, and in vitro vasculogenesis functions. EPC transmigration was assessed with the use of transwell24-well plate. Adhesion assay tested adherent abilities of EPCs. Proliferative function was assessed with the use of the Cell Counting Kit-8. Vasculogenesis assay was performed using an in Vitro Angiogenesis Assay Kit.(4) EPC condition medium (CM) was carefully collected and added to culture ECs instead of EBM-2for24h. ECs were collected and tested using in vitro Angiogenesis Assay Kit. The measurements of VEGF, IL-6, sICAM levels in cell culture medium were determined by ELISA kits.(5) PAH models were established by subcutaneously injected monocrotaline. Rat hemodynamic, vascular remodeling and serum levels of sCD40L were measured every week. (6) Control EPC group received transplantation of wide type EPCs infected with Lv-NC-shRNA while shRNA-CD40EPC group received Lv-shRNA-CD40infected EPCs. Both control and shRNA-CD40groups were respectively accepted EPCs at day7,14, and21. We compared the results within and between two groups.(7) CD31was identified by immunofluorescence stain. Transplanted cells with green fluorescent protein were observed under a confocal microscope. To quantitatively analyze the ratio of substitution, total lengths of green fluorescent and perimeter of vessels were measured.Results:(1) sCD40L dose-dependently impaired EPC migration, adhesion, proliferation, and in vitro vasculogenesis functions.(2) sCD40L could apparently increase the secretion of IL-6, VEGF, and sICAM of EPCs, and these effects appeared to be dose-dependent.(3) The blend of growth factors in EPC CM could obviously enhance EC in vitro tube formation function. On the contrary, after pretreatment with sCD40L, EPC CM seriously hampered it.(4)14days after MCT injection, we noticed significant changes in all targets tested. All EPC transplanted groups achieved therapeutic effects comparing to blank or vehicle groups. Control EPC efficacies gradually waned. By comparing the two types of EPCs, we found that shRNA-CD40EPCs played a more effective and enduring role than control ones injected at the same time.(5) More shRNA-CD40EPCs combined into endothelium, but less into adventitia and media, comparing to control EPCs injected at the same time Conclusion:In vitro, sCD40L dose-dependently impaired EPC migration, adhesion, proliferation, and in vitro vasculogenesis functions, but stimulated the strong paracrine ability of early outgrowth EPCs to release pro-inflammatory factors. The present study reaffirmed the application of EPC therapy and revealed the superiorities of CD40knockdown EPCs in PAH treatment. Therapeutically shutting down CD40pathway can achieve a better and longer lasting effect of EPC therapy in PAH. |
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