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Introduction and aim of thesis: Type 2 diabetes (T2D) is a major cause of disability due to the occurrence of micro and macrovascular complications. Patients who experience vascular complications are not only at very risk of mortality, but they are also exposed to a concrete risk of social and mental effects (i.e., depression, anxiety) which further dampen quality of life. Among the constellation of cardiovascular comorbidities, diabetes shows the highest association with peripheral artery disease (PAD), a condition characterized by atherosclerotic lesions, reduced blood supply and chronic ischemia of the lower extremities. T2D is highly prevalent in patients with PAD and has been co-diagnosed in nearly 40% of all PAD patients. Despite advances in revascularization strategies, the rate of limb amputation due to chronic limb ischemia remains high and associates with cardiovascular morbidity in diabetic patients. In this perspective, strategies that promote vascularization can be considered as a novel therapeutic option in diabetic patients with PAD. Epigenetic changes, defined as plastic chemical modifications of DNA/histone complexes – have shown to modulate gene activity by modifying chromatin accessibility to transcription factors. Whether epigenetic modifications affect angiogenesis and post-ischemic vascularization remains to be elucidated. The mammalian methyltransferase SETD7 has emerged as a pivotal modulator of gene expression by its ability to induce histone methylation. Chromatin changes induced by SETD7 foster transcriptional programs implicated in endothelial inflammation and oxidative stress. By this background, in the present thesis I investigate whether epigenetic signals induced by SETD7 affect angiogenic properties of endothelial cells in the setting of diabetes. Main research tasks: To investigate whether SETD7-dependent chromatin modifications modulate angiogenic properties of human aortic endothelial cells (HAECs) cultured in growth factor-free media and exposed to normal glucose (NG) and high glucose (HG) concentrations, a well-established in vitro model mimicking diabetic ischemia. To investigate whether pharmacological blockade of SETD7 restores angiogenic properties of endothelial cells (ECs) while boosting post-ischemic vascularization of diabetic mice with hindlimb ischemia. To investigate whether our experimental findings hold true in the human setting. To this end, we investigate whether the SETD7-dependent signaling is dysregulated in muscular specimens obtained from diabetic patients with PAD. 2 Methods: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results: RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HGtreated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycaemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7- dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA3G). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls. Conclusion: Targeting SETD7 represents a novel epigenetic-based therapy to boost eovascularization in diabetic patients with PAD.