Klotho Improves EPCs Function And Injured Carotid Artery Repair

Background and aimsVascular injury diseases such as atherosclerosis can lead to heart failure, limb necrosis, paralysis and other consequences, is a serious threat to human health common and frequently occurring disease. Therefore, actively explore the research of vascular repair has great scientific significance and social economic benefits. The vessel wall is mainly composed of covering intimal endothelial cells and smooth muscle cells in the membrane formed, under physiological conditions maintain blood vessel structure and function of basic components of normal. Endothelial cell injury can lead to atherosclerotic plaque formation, proliferation of smooth muscle cells, is an important pathophysiological basis of vascular injury diseases occurrence, development. Is the mature endothelial cell terminal differentiation, regeneration ability is limited, not for the prevention and control strategy in situ effectively repair the injured vascular endothelial cells. Endothelial progenitor cells(EPCs) is a kind of precursor cell which could differentiate into mature endothelial cells. It is not only involved in the formation of human embryonic vascular involvement, and is involved in the formation of blood vessels after birth and re-endothelialization of vascular tissue injury repair process. When the body’s vascular damage occurs, EPCs will through proliferation, migration, adhesion to the injury site to quickly differentiate into vascular endothelial cells, play an active role in wound repair. Meanwhile EPCs cells can mobilization and homing feature, when a blood vessel or tissue damage. But the process of EPCs participate in re-endothelialization of vascular injury have that age-dependent increase with age, EPCs function will be damaged, leading to reduce the number of elderly patients with vascular endothelial damage repair capacity decline after endothelialization. Anti-aging Klotho protein is a key protein associated with human aging, clinical observations found that it play a vital role in the anti-aging process in humans. Klotho proteins or its metabolites with hormone effects may have a protective effect on vascular injury repair. In the Klotho gene mutation mice, the number of EPCs in vivo bone marrow and peripheral blood significantly decreased, suggesting that Klotho protein as one of the important anti-aging factors that may be involved in the EPCs function and repair of vascular injury. But the study of anti-aging Klotho protein specifically how to play a protective role in vascular injury and repair is still rare, and it is still a hot research topic. Therefore, the anti-aging Klotho protein and human endothelial progenitor cells(EPCs) are as the research object for the study, and research the Klotho protein on EPCs differentiation, proliferation, migration, adhesion and cell function paracrine effects; and build C57 BL / 6 mice neck artery injury model to reveal its ability to mobilize through EPCs homing promote vascular injury and repair.MethodsIsolated and induced culture EPCs, cell morphology was observed using their uptake of DIL-ac-LDL and UEA-1 binds specifically identified from human peripheral blood. EPCs isolated cells were added 0.1,1,10nmol/L anti-aging Klotho protein, and set the PBS control group, vascular endothelial growth factor VEGF-positive group(10nmol/L) and Akt signaling pathway inhibition group(while adding 10nmol/L Klotho protein and 1: 1000 Akt inhibitor) for continuous cultivation. The use of flow cytometry to detect different concentrations of Klotho induced cells in each group when the CD34+/CD133+ and CD31+/v WF+ positive rate of culture; Transwell method to detect migration in each group EPCs cells; cell adhesion experiments to detect the expression of adhesion ability; ELISA method to detect each group EPCs cells secrete factors change of SDF-1, bFGF, EGF; Western-blot method to detect the expression of VEGF and VEGFR-2 cells in each group EPCs.Set up the model of C57BL/6 carotid artery injury mice and identify, and they were injected with saline, Klotho protein, VEGF, Klotho + VEGF interference. The EPCs mobilization into peripheral blood in carotid injury mice were detected by flow cytometry when 3d; at the same time, the promoting effect of vascular repair factor, VEGF, SDF-1, EGF and b FGF secretion were detected by ELISA method; the ET-1 and NO content in different periods in peripheral blood in carotid artery injury mouse were detected; use the live cell fluorescence dye CM-Dil and Dio-perchlorate mark the EPCs cells treatment by Klotho protein induced, and injected via the tail vein to carotid artery injury mice, take out the injury segment of carotid artery after 3d and exposed endometrium surface, observe the homing EPCs lymphocytes with laser confocal microscope; using cy5-CD31 mark the homing EPCs differentiate into endothelial cells, the re-endothelialization of homing EPCs cells were detected in carotid artery injury mice at 14 d. And the impact of Klotho protein treatment on the neointimal hyperplasia of carotid artery injury mice were detected by H & E staining.ResultsThe project successfully from healthy human peripheral blood mononuclear cell layer separated and induced cultured EPCs cells, with the uptake of Dil-ac-LDL and UEA-1 binding specificity characteristics. Flow cytometry showed that 0.1、1、10nmol/L anti-aging Klotho protein can promote significantly positive rate of CD34+/CD133+ and CD31+/v WF+ in human peripheral blood mononuclear cells in the EPCs formation and differentiation into endothelial cells from cultured cells, and compare to the PBS control, P?0.05. Transwell, cell adhesion and ELISA experiments found that 0.1、1、10nmol/L Klotho protein could significantly promote the human EPCs cell migration, adhesion ability and the SDF-1, b FGF, EGF cytokines paracrine ability, compare to the PBS control, P?0.05; at the same time Western-blot assay showed that 0.1、1、10nmol/L Klotho protein can promote the expression of VEGF and VEGFR-2 in EPCs cells, compare to the PBS control, P?0.05. But the role of Klotho protein on EPCs cells were inhibited by the Akt signal pathway inhibitor(1:1000 Akt inhibitor). The C57BL/6 carotid artery mice model was established by wire injury mice. Flow cytometry showed that Klotho protein and VEGF can significantly promote the injury of carotid artery in mice EPCs cell mobilization from bone marrow into peripheral blood, P?0.05; and stimulate the blood vessel repair factor secretion contents of SDF-1, b FGF, EGF, VEGF to promote blood vessel repair, P?0.05; gradually improve the secretion of NO content and decrease the content of ET-1 in the peripheral blood of carotid artery injury mice, P?0.05; and could promote the EPCs homing to the injured vessel position, then differentiated into endothelial cells for the vascular repaired, and also reduce the injury of vascular intimal hyperplasia phenomenon.ConclusionsIn vitro, this research first directly reveal that the anti-aging Klotho protein has a promoting effect on EPCs differentiation, migration, adhesion and secretion ability of cell function, may play a role through the promotion of VEGF and VEGFR-2 expression, and related with Akt signaling pathway. In vivio, Meanwhile anti-aging Klotho protein can promote the body EPCs from the bone marrow of mice injured carotid mobilization to peripheral blood, stimulate VEGF, SDF-1, bFGF, EGF and other pro-angiogenic factors secreted repair, EPCs significantly increased cell homing to vascular injury and at the endothelium and inhibit the ability of neointimal hyperplasia, vascular injury and thus play a role in repair. This study demonstrated that Klotho protein as one of the old anti-factor can promote vascular repair cell function of EPCs seed cells, involved in the repair of blood vessels and play an important role for the clinical treatment of cardiovascular disease in vascular endothelial injury and repair of great value and significance.