Università degli Studi di Milano

The World Health Organization estimated that the incidence of the diabetes is projected to rise from 120 million in 1996 to 366 million in 2030. Diabetes mellitus refers to a metabolic disorder of multiple etiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long–term damage, dysfunction and failure of various organs. Nowadays, the main issues for diabetic patients are no longer those associated with survival but those associated with the chronic complications of the diabetes. In this context, diabetic foot ulcers represent a serious medical and socioeconomic issue worldwide. To date, the best predictors of clinical outcomes are the presence or the absence of ischemia, infection, footwear, pressure relief, and overall glycaemic control. None of them, however, reflects the activity of endogenous repair mechanisms. Endothelial progenitor cell (EPCs) availability and functionality are relevant to vascular repair. It was demonstrated in patients with diabetes, the availability of EPCs is reduced. Different mechanisms may potentially be involved, including impaired mobilization, proliferation, and apoptosis. Based on this consideration, the goal of my thesis is to determine the additive value of circulating EPCs in predicting major end-points such as mortality, amputation and post-angioplasty restenosis in a cohort of 120 type 2 diabetic patients with Critical Limb Ischemia (CLI). Moreover, we aimed to obtain mechanistic insights into diabetic progenitor cells impairment and to provide the first characterization of the EPC-associated miRNA with special respect to those potentially involved in the control of angiogenesis. To perform functional and molecular analysis normal criteria were not available so we decided to include in the study two different control groups: a groups of not diabetic CLI subjects and a group of healthy volunteer. To assess the first objective of this project we have to wait the end of 12 months follow-up (FU) so we reported an ad interim analysis of the data. To date, about 60% of patients have reached one year FU visit and we have registered 37 restenosis, 10 major amputations and 7 death events. We did not observe any statistical difference in the number of antigenically defined EPCs between complicated (with at least one event) and not complicated (no event) diabetic patients. Also the migration capacity analysis did not show difference between the two groups. Interestingly, our preliminary data showed a significant increase in the basal migration of EPC in patients with event compared with patients with no event, so we propose that the unspecific motility associated with altered directed migration may be indicative of the incapacity of the cells to find their way to injured tissues thereby compromising the healing process. This preliminary finding need to be confirmed after the end of the study. Second, to assess the other objective of the study we analyzed the diabetic EPC characteristics and functionality. We first measured by flow cytometry the percentage of antigenically-selected MNCs subpopulations with pro-angiogenic potential in all of the three groups of enrolled subject. We observed a decrease in CD34 pos and EPCs (CD45 dim ,CD34 pos ,CXCR4 pos ,KDR pos ) in ischemic patients as compared to healthy volunteer. Unexpectedly however, diabetic patients showed a higher percentage of progenitor cells compared with non diabetic controls. Functionally, migration assay showed an impairment of cell motility in chronic limb ischemic patients with or without diabetes compared to healthy subjects. Migration assay results showed an enrichment of EPCs in the controls only; these data are in line with those previously published on an altered migratory function of EPCs from diabetic patients. Mechanisms underlying the reduction of EPCs in diabetes are largely unknown. Weak bone marrow mobilization, impaired peripheral differentiation, and short survival in peripheral blood are all candidates and we therefore attempted to investigate the mechanisms involved in this impairment. Recently, miRNAs have been shown to regulate EC functions, including proliferation, migration and assembling in branched networks. To this aim, we performed a preliminary screening on EPCs from the patients enrolled and demonstrated that diabetes induces specific microRNAs de-regulation. Next, we selected two miRNAs, mir-15a and mir-16 based on their already known role in controlling migratory and apoptotic mechanisms in cancer. To investigate the role of mir-15a and mir-16, we transfected these miRNAs into healthy EPCs. We then analyzed cell functionality in migration and angiogenic assays and we observed that only co-expression of mir-15a/16 blocks EPCs migration and induce apoptosis. Next, we investigated if down-regulation of mir-15a/16 could restore diabetic EPC migratory ability. We observed that inhibition of mir-15a/16 recover FBS and SDF-1 induced migration in diabetic EPCs but no effect was observed in apoptotic assay. Bionformatic analyses predict BCL2, VEGFA and AKT3 to be target genes of miRNA-15a and -16, which support an anti-angiogenic and pro-apoptotic role of these miRNAs. In diabetic EPCs and mir over-expressing EPCs, we found a downregulation of AKT3, a protein involved in many EPCs functions like cell cycle progression, migration and survival. This study demonstrates, for the first time, that the spontaneous migration ability of a well antigenically characterized MNCs subpopulation of cells with EPC phenotype is significantly increased in diabetic patients that manifest at least one major adverse event (restenosis, amputation, death). This is the first evidence generated in a clinical trial that EPC migration could be used as prognostic marker for diabetic vascular complications. At the end of the study, the analysis of the follow up data generated on the entire patient cohort will enable us to generate crucial data on the a new EPC-associated risk for diabetic CLI patients to develop life-threatening vascular complications. Second, in this work we show for the first time that mir-15a and mir-16 are key regulator of diabetic EPCs pro-angiogenic function. In the future we want to deepen the role of microRNA in diabetic dysfunction because we know that miRNAs have tremendous therapeutic potential for the treatment of vascular diseases associated with aberrant pathological angiogenesis. In conclusion, EPCs have recently generated considerable attention as potential novel prognostic biomarkers for vascular integrity, and therapeutic clinical approaches using these cells are ongoing. Although the role of EPCs in these processes is well established, the challenge for the next decade is to identify and evaluate methods that increase EPC homing and incorporation, thereby enabling targeted delivery of EPCs to a site of interest. This goal might be achieved through the continued characterization of EPCs in animals and humans, coupled with investigations of the long-term potential of EPCs in vivo.