Studies Of Regulating Autophagy Of Endothelial Progenitor Cells Or Venous Thrombus Recanalization And Resolution

Deep venous thrombosis is commonly seen in vascular department,which may lead to deep venous valve insufficiency, pain, repeated swelling, varicosity of superficial veins,pigmentation, ulceration, necrosis and lethal complications like pulmonary embolism.Current therapies including medical anti-coagulation or thrombolysis and surgical thrombectomy may result in the decrease of coagulative function, hemorrhage inclination of digestive tract, incision complication and so on. Moreover, the effects of recanalization in late phase are not satisfactory. Therefore, how to treat DVT(Deep Veinous Thrombosis)more effectively is an interest-attracting study direction for vascular surgeons.BMEPCS(Bone marrow-derived endothelial progenitor cells) could differentiate into vascular endothelial cells and endocrine various factors promoting angiogenesis in ischemic issues. Relative studies demonstrate that EPCs(endothelial progenitor cells)could orientate to the site of DVT and enhance the organization and recanalization of thrombosis. The environment of ischemia and hypoxia, however, may depress the proliferation, migration and angiogenesis of EPCs. So it is a key step to improve the functions of EPCs in hypoxic environment for the application of EPCs in the treatment of DVT.Autophage is a highly conservative protection mechanism and correlates closely with cellular growth, proliferation and carcinogenesis. In addition, it is an adaptive response to decrease the toxicity of exogenous stimulations like hunger, hypoxia, infection, mediation,growth factor and oxidative stress. Recent studies exhibit the close relationship between autophage and cellular function. For example, the enhancement of autophage could inhibit cellular necrosis and apoptosis. Up-regulation of autophage may activate the phosphorylation of AKT signaling pathway and boost the migration and angiogenesis of bull artery endothelial cells. Likewise, down-regulation of autophage could inhibit the functions of endothelial cells. Duration of autophage, however, may induce cellularnecrosis and apoptosis. After reviewing literature, we recognise that there is little knowledge about the effects of regulating autophage on EPCs function, especially in the field of recanalization for DVT.The objective of current study is to explore the effects of autophage on EPCs and the promotion of recanalization for DVT and to provide a novel thought for the treatment of deep venous thrombosis. This study was divided into four parts and main methods and results are as follows.Part I Separation, Culture and Identification of Rat BMEPCsObjective: To separate, culture and identify rat bone-marrowed endothelial progenitor cells(BMEPCs)..separated by density gradient centrifugation and cultured about 14 to 21 days in EGM-2medium of endothelial cell culture system for directional induction and differentiation.Cellular growth was observed. CD133, marker of hemopoietic stem cells and VEGFR,v WF, markers of endothelial cells were detected by techniques like immunohistochemistry,immunofluorescence and flow cytometry. Matrigel tube experiment and Di I-ac-LDL uptake experiment were utilized to examine angiogenesis and swallow functions respectively.Results: Newly separated BMMNCs were round and adhered to plates 24 to 48 hours in vitro. Cells began to fuse and cover the entire plates in 10 to 12 days in vitro, exhibiting cobblestone appearance. Di I-ac-LDL uptake experiment showed that cultured cells possessed the ability to swallow Di I-ac-LDL like endothelial cells. Matrigel tube experiment exhibited that VEGFR, v WF were over-expressed on the surface of cultured cells, while markers of hemopoietic stem cells like CD133 were little, or even rarely expressed.Conclusion: The system was established to separate, culture, induce and identify BMEPCs. EPCs were obtained from BMMNCs of SD rats using EGM-2 medium of endothelial cell culture system. EPCs 14 to 21 days in vitro demonstrated the phenotype of late phage.Part II The Effects of Autophagy on EPCs Functions in Hypoxic Environment in vitroObjective: To explore the effects of autophagy on the proliferation, migration and angiogenesis of EPCs and investigate the potential molecular mechanisms.Methods: Cultured EPCs were divided into three groups: 3-MA group, ATG5 si RNA group and ATG5 group. EPCs in 3-MA group were added with 3-MA or not. EPCs in ATG5 si RNA group were transfected with ATG5 si RNA or not. EPCs in ATG5 group were transfected with ATG5 plasmid(ATG5-pc DNA 3.1) or only pc DNA. EPCs of all groups were cultured in hypoxic incubator(1% O2, 5% CO2 and 94% N2, 37℃) for 4hours. RT-RCR and Western Blot were employed to detect the expression of ATG5 and Beclin-1 in EPCs of each group. MTT method was utilized to examine the effects of autophagy on EPCs proliferation. Transwell assay was applied to evaluate the effects of autophagy on the migration of EPCs. Matrigel tube formation assay was used to detect the effects of autophag Y on angiogenesis of EPCs. Western Blot was utilized to examine the level of phosphorylated or dephosphorylated AKT.Results: RT-PCR and Western Blot showed that 3-MA or ATG5-si RNA, inhibitor of autophagy could down-regulate the expression of ATG5 and Beclin-1 on EPCs, while ATG5-pc DNA 3.1 may up-regulate the expression to large extent. MTT assay, transwell assay illustrated and matrigel tube formation assay showed that up-regulation of autophagy could promote the proliferation, migration and angiogenesis of EPCs in hypoxic environment, yet down-regulation may inhibit those functions. Moreover, the molecular mechanism of the promotion of EPCs by up-regulating autophagy is the activation of AKT.Conclusion: Up-regulation of autophagy could enhance the proliferation, migration and angiogenesis of EPCs in hypoxic environment.Part III The Construction of ATG5 Lentiviral Expression Vector and Its Stable Expression in EPCsObjective: EPCs were transfected with ATG5 lentiviral expression vector to developEPCs with stable expression of ATG5. Q-PCR was applied to validate the improvement of ATG5 expression in EPCs. Foundations to explore the effects of autophagy on the role played by EPCs in DVT recanalization in vivo.Methods: Pre-ATG5, the precursor sequence of ATG5 acquired by PCR was combined with p Ubi-MCS-EGFR after enzyme digestion to produce p Ubi-MCSEGFP-ATG5. Virus which was packaged in 293 T cells by p Ubi-MCS-EGFP-ATG5 and packaging vector was used to infect EPCs. Q-PCR was employed to detect the expression of ATG5 in infected EPCs.Results: The construction of ATG5 lentiviral expression vector was satisfactory and the infection of EPCs by virus could increase the expression of ATG5 stably.Conclusion: It is feasible to construct p Ubi-MCS-EGFP-ATG5 lentiviral expression vector and EPCs cell line with stable expression of ATG5.Part IV The Effects of Autophagy on The Role Played by EPCs in DVT RecanalizationObjective: To inquire the effects of autophagy on the role played by EPCs in DVT recanalization.Methods: EPCs infected by pUbi-MSC-EGFP/pUbi-MCS-EGFP-ATG5 were transplanted into the DVT models developed by the ligation of venae cava inferior of rats.Thrombosis was acquired after sacrifice of rats in 7 or 14 days after the transplantation.The weight of thrombosis was measured to observe thrombolysis. Frozen sections of fluorescent tracer were utilize to detect homing of EPCs. HE staining and CD34 immunohistochemistry were employed to examine the effects of EPCs in the organization and recanalization of DVT respectively.Results: The comparison of GFP-positive cells number was: EPCs/ATG5 group >EPCs/vector group > controlled group, demonstrating that transplanted EPCs could orientate to the site of DVT and up-regulation of ATG5 in EPCs could boost homing. The comparison of thrombosis weight of each group was: Controlled group > EPCs/vector group>EPCs/ATG5 group, showing that transplanted EPCs could promote thrombolysis and up-regulation of ATG5 in EPCs may enhance its own competence of thrombolysis. HEstaining and CD34 immunohistochemistry indicated that transplatation of EPCs could promote the organization and recanalization of DVT and up-regulation of ATG5 in EPCs may increase the its own competence to facilitate organization and recanalization of DVT.Conclusion: Transplanted EPCs could orientate to the site of DVT to promote thrombolysis, organization and recanalization of thrombosis. Up-regulation of ATG5 may activate autophagy to boost homing of EPCs and enhance the role of EPCs in organization and recanalization of DVT. Activation of EPCs autophagy presents a promising clinical therapy for the treatment of DVT.