| Background: Endothelial colony forming cells(ECFCs)is a subset of endothelial progenitor cells that contributes to reendothelialization and angiogenesis.Exploring the underlie mechanism between ECFCs dysfunction and angiogenesis could enhance the cognition of vascular regeneration disorders and provide novel therapy for vascular diseases.Decreased expression of micro RNA(mi R)-139-5p is involved in inhibiting the metastasis and progression of diverse malignancies,however,the role of miR-139-5p in ECFCs-induced vascular formation remains unclear.In chapter 1,we hypothesized that miR-139-5p regulates diabetic ECFCs dysfunction and mechanism in vascular regeneration.We aimed to clarify that miR-139-5p regulates ECFCs function and the underlie mechanism,and to provide novel target for diabetic vascular complication micro RNAs therapy and cell therapy.Hyaluronic acid(HA)is currently applied as a biomaterial in tissue engineering.Mesenchymal stem cells(MSCs)combined with ECFCs was reported to effectively promote vascular generation in vivo.However,the efficiency of HA combined with MSCs-ECFCs on angiogenesis is unclear.Based on the results of chapter1 that miR-139-5p regulates ECFCs function,in chapter 2,we hypothesized that miR-139-5p mediates HA induced MSC-ECFCs mixed cells angiogenesis.We aimed to provide practical therapy for vascular diseased.The present study demonstrated that miR-139-5p plays a vital part in stem(progenitor)cells-induced angiogenesis.Methods: In chapter 1,Peripheral blood ECFCs was collected from diabetic patients and healthy adults.The expression of miR-139-5p and the proliferation,migration and angiogenesis of ECFCs in vitro were detected,respectively.Up-regulated and down-regulated the expression of miR-139-5p in ECFCs by transfected with miR-139-5p mimic and inhibitor,and / or up-regulated and down-regulated the expression of c-Jun by transfected with plasmid or small interfering RNA(si RNA),respectively.And then tested effects of miR-139-5p and c-Jun on ECFCs function.The targeting effect of miR-139-5p on the activity of c-Jun promoter and promoter binding of c-Jun / VEGF was tested by dual luciferase reporter assay and Ch IP assay,respectively.Pretreated diabetic ECFCs with anti-VEGFR2 antibody and/or anti-PDGF antibody to detect whether the miR-139-5p downregulation promoted DM-ECFCs angiogenesis in vitro through VEGF and PDGF.The effects of miR-139-5p and ECFCs on angiogenesis in vivo were evaluated by in vivo Matrigel plug assays and diabetic hind limb ischemia models.In chapter 2,human umbilical cord blood derived ECFCs and umbilical cord derived MSCs were isolated.The in vitro proliferation and migration of cells were tested with / or without HA dilution,respectively.The in vivo angiogenesis of HA-MSC-ECFC was evaluated by in vivo Matrigel plug assays and mouse hindlimb ischemia models.The expression of CD44,a HA receptor,and micro RNA(mi R)-139-5p were detected in ischemic muscles and HA-treated cells,respectively.After transfection with si RNA to downregulate CD44 or transfection with plasmid to upregulate CD44,the expression of miR-139-5p was tested.CD31 + cells from MSC-ECFC co-culture system treated with / or without HA dilution were sorted by flow cytometry to clarify the effect of HA and miR-139-5p expression on co-cultured ECFCs.Finally,upregulate miR-139-5p expression in HA-treated MSCs-ECFCs to detect whether HA regulates the proliferation and migration of mixed cells through miR-139-5p.Results:Chapter 1: 1.Diabetic ECFCs showed significantly decreased proliferation,migration and tube formation ability.Meanwhile,the miR-139-5p expression was significantly elevated while c-Jun,VEGF and PDGF expression were significantly decreased.2.miR-139-5p mimics significantly inhibited tube formation,migration,proliferation,and downregulated expression of c-Jun,vascular endothelial growth factor(VEGF),and platelet-derived growth factor(PDGF)-B in healthy ECFCs;moreover,miR-139-5p inhibitors reversed the tendency in diabetic ECFCs.Further,gain-and-loss function experiments revealed that miR-139-5p regulated ECFCs function through c-Jun.3.Dual luciferase reporter assays revealed that miR-139-5p targeted 3’-Untranslated region of c-Jun.Ch IP assays revealed that miR-139-5p inhibited binding of c-Jun to VEGF promoter.4.Pretreated ECFCs with anti-VEGFR2 and / or anti-PDGF blocked the effect of miR-139-5p downregulation promoted diabetic ECFCs in vitro angiogenesis.5.Matrigel plug assays showed that miR-139-5p downregulation elevated in vivo angiogenesis of diabetic ECFCs,and miR-139-5p upregulation inhibited the angiogenesis of healthy ECFCs.6.miR-139-5p antagomi R combined with ECFCs injection effectively promote blood flow recovery and angiogenesis in diabetic hind limb ischemia mice.CM-Dil tracing showed that miR-139-5p antagomi R combined injection effectively promoted ECFCs local retention.Chapter 2: 7.HA promoted the migration of ECFCs and promoted the proliferation and migration of MSCs and MSCs-ECFCs in vitro,respectively.8.HA promoted in vivo angiogenesis of mono-or mixed-cells,respectively.The vascular density and diameter of HA-MSC-ECFC Matrigel plugs was significantly higher than that of other groups;9.Intramuscular injection of HA-MSC-ECFC effectively improved the recovery of ischemic blood flow in murine hind limbs.And mouse specific CD31 immunofluorescence showed significant increase of neo-vascular density in ischemic muscles.Laser confocal microscopy revealed that human-derived cells grew into the host vasculature and formed connections,as shown by mouse-specific CD31+/human-specific CD31+double staining;10.The expressions of CD44 was significantly increased while expression of miR-139-5p was significantly decreased in ischemic muscles in HA-MSC-ECFC group.11.HA treatment significantly increased the expression of CD44 while decreased the expression of miR-139-5p in ECFCs and MSCs-ECFCs,respectively.HA-treated MSCs only showed elevated CD44 expression,without change in miR-139-5p expression.12.Overexpression of CD44 in ECFCs significantly decreased the expression of miR-139-5p,and down-regulation of CD44 in HAtreated ECFCs significantly increased miR-139-5p expression.HA treatment significantly increased proportion of CD31 positive cells in MSCs-ECFCs co-cultured system.Besides,the expression of miR-139-5p was down-regulated in CD31 positive cells from HA-MSC-ECFC,and upregulated miR-139-5p expression in MSCs-ECFCs blocked HAincreased CD31 positive cells proportion.13.Upregulation of miR-139-5p blocked the proliferation and migration of MSCs-ECFCs promoted by HA in vitro.Conclusions: The present study confirms that upregulation of miR-139-5p mediates diabetic ECFCs dysfunction through inhibiting cJun/VEGF and PDGF pathways,revealing a novel mechanism in diabetic ECFCs dysfunction and provides a promising therapy of diabetic ischemic vascular complications.Besides,HA facilitates angiogenesis of MSCECFCs through CD44/miR-139-5p signaling pathways,providing a novel treatment approach for vascular diseases.In conclusion,miR-139-5p down-regulation promotes ECFCs function and angiogenesis. |
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