The Role And Mechanisms Of MicroRNA Let-7f In Regulating Endothelial Progenitor Cell Function In Type1 Diabetes

BackgroundEndothelial progenitor cells (EPCs) play an essential role in regulating angiogenesis. Recently, EPC therapy has become an attractive alternative strategy to current pharmacological, interventional, or surgical treatments for vascular complications characterized by impaired angiogenesis in diabetes, such as refractory wounds. However, several clinical trials of EPC therapy for ischemia revealed that autologous cell therapy for diabetic patients did not exert the same clinical efficacy as in non-diabetic patients. A major reason could be that EPCs from diabetic patients have extensive dysfunction in repairing tissue, consequently leading to inefficient therapy in the clinic. However, the mechanisms underlying EPC dysfunction in diabetes are incompletely understood. Recently, microRNAs (miRNAs) have been investigated to serve as novel regulators of angiogenesis. Among of them, let-7f has been found to exert a pro-angiogenic effect in mature endothelial cells. However, it is not clearly defined whether let-7f plays a critical role in regulating endothelial progenitor cell (EPC)-mediated angiogenesis, especially under diabetic conditions. This study aims to address the hypothesis that let-7f rescues EPC dysfunction in streptozotocin (STZ)-induced type 1 diabetes, and this beneficial effect contributes to AMP-activated protein kinase (AMPK) activation and p66shc inhibition. Objective1. To measure the endogenous expression level of let-7f in STZ-induced diabetic EPCs, and estimate the role of let-7f in regulating EPC in vitro angiogenic functions.2. To investigate the role and mechanism of AMPK in regulating STZ-induced diabetic EPCs functions.3. To investigate whether let-7f improves EPC functions through activating AMPK and inhibiting p66shc.Methods1. Animal model:Male C57BL/6 mice, aged 8 to 10 weeks, were injected intraperitoneally with citrate buffer (control) or streptozotocin (STZ) for 5 consecutive days. Mice with a blood glucose level> 280 mg/dL were considered diabetic and used for EPC isolation following 4 to 6 weeks of hyperglycemia.2. EPC culture and characterization:Bone-marrow derived EPCs were isolated and cultured for 7 days, and were characterized by DiI-acLDL and FITC-conjugated isolectin staining. To characterize the phenotype of the in vitro cultured EPCs, the expressions of Sca-1, Flk-1, CD34, VE-Cadherin (CD144), and CD11b were analyzed by flow cytometry and compared with freshly isolated bone marrow mononuclear cells.3. In vitro EPC function assays:In vitro angiogenic activity of EPCs was determined by Matrigel tube formation assay. The adhesion assay was performed to detect the ability of EPC adhesion to vitronectin. EPC migration was measured using Boyden chambers to migrate to vascular endothelial growth factor (VEGF).4. Real-time PCR:MiRNAs were isolated from EPCs using mirVana miRNA Isolation Kit. Quantitative (q) RT-PCR was performed with specific Taqman assays for miRNA and Taqman universal master mix. The relative miRNA expressions were normalized to internal control snoRNA55. Each reaction was performed in triplicate and differences of Ct values were calculated by the 2-ΔΔCT method. 5. Western Blot:The primary antibodies p-thrl72-AMPK,p-ser79-ACC,MnSOD,PP2A and p66shc were used. The blots were scanned with an Odyssey imager and band intensity was determined with Quantity One System.6. Adenovirus transfection in EPCs:EPCs after 7 days of culture were transfected by a dominant-negativeα2-AMPK mutant-expressing adenovirus (Ad-AMPK-DN) at the titer of 50 multiplicity of infection (MOI). For p66shc down-regulation, EPCs were transfected by recombinant adenovirus Ad-p66shcRNAi at the titer of 10MOI. Ad-β-gal was also used for internal control.7. Let-7f mimic/inhibitor and small interfering RNA transfection:Let-7f mimic,inhibitor,MnSODsiRNA,PP2AsiRNA and their scramble controls were transfected into EPCs with DharmaFECT Transfection Reagent 1 according to the manufacturer's protocol. After 72h of transfection, the cells were harvested for the following experiments.8. MnSOD activity assay:MnSOD activity in cultured EPCs was measured by using a commercial SOD Assay Kit according to the manufacturer's protocol.9. Detection of mitochondrial superoxide in EPCs:Measurements of mitochondrial superoxide anion formation in EPCs were performed using flow cytometry analysis by using a mitochondrial superoxide indicator MitoSOX. Results1. MiRNA let-7f expression was significantly decreased in diabetic EPCs and restored after controlling blood glucose by insulin. The expression level of let-7f was decreased by 65% in diabetic EPCs when compared with control EPCs. Let-7f expression was increased 4.5 fold after controlling blood glucose with insulin treatment. Let-7f expression in EPCs was not altered by advanced glycation endproducts (AGE), Ly83583 or H2O2 in vitro treatment. 2. Let-7f overexpression rescues the impaired EPC angiogenic functions in type 1 diabetes. Let-7f mimic transfection significantly rescued the impaired EPC functions, including tube formation, adhesion and migration, compared with scramble control transfection. In contrast, the abilities of normal EPC tube formation, adhesion, and migration were distinctly impaired by let-7f inhibitor transfection.3. AMPK activation rescues EPC functions in diabetic EPCs through increasing MnSOD level. AMPK activity was decreased in diabetic EPCs, and AMPK activation rescues EPC functions and restores MnSOD protein level and activity. Silencing MnSOD abolishes the improved diabetic EPC functions induced by AICAR treatment.4. Increased PP2A contributes to AMPK deficiency in diabetic EPCs. Diabetic EPCs showed a significantly higher level of PP2A protein, and inhibiting PP2A by siRNA or okadaic acid restored the deficient AMPK activation (phosphorylation of AMPK-Thr172 and ACC-Ser79) and consequently reversed MnSOD protein level in diabetic EPCs.5. Let-7f improves EPC functions in diabetic EPCs partly through AMPK. Let-7f overexpression promoted AMPK activation in diabetic EPCs, whereas let-7f inhibitor decreased the normal AMPK activity in EPCs. Ad-AMPK-DN transfection blunted the improvement of EPC functions resulting from let-7f overexpression in type 1 diabetes.6. Let-7f regulates EPC functions partly through suppressing p66shc level. EPCs from diabetic mice showed a much higher p66shc protein level, which was significantly diminished by let-7f overexpression. Ad-p66shcRNAi transfection rescued the impaired EPC functions in type 1 diabetes. In addition, Ad-p66shcRNAi transfection partly restored the impaired EPC functions induced by let-7f inhibition.Conclusion1. MiRNA let-7f plays a critical role in maintaining EPC functions in STZ-induced type 1 diabetes, and modulating let-7f expression may be a potential strategy in enhnacing the clinical effects of EPC cell therapy.2. AMPK activation rescues EPC functions in type 1 diabetes through up-regulating MnSOD and suppressing mitochondrial superoxide production.3. Let-7f plays a beneficial effect in protecting EPCs functions through promoting AMPK activation and inhibiting p66shc level in type 1 diabetes.