Abstract
Introduction
Despite advances in revascularization strategies, type 2 diabetic (T2D) patients with peripheral artery disease (PAD) continue to have a high risk of limb amputation. Hence, strategies that promote vascularization can be considered as a novel therapeutic option in T2D patients with PAD. Epigenetic modifications of histones and DNA have emerged as key modulators of gene expression. Mono-methylation of histone 3 at lysine 4 (H3K4m1) – a specific epigenetic signature induced by the methyltransferase SETD7 – favours a chromatin conformation enabling the transcription of genes involved in inflammation and oxidative stress.
Purpose
To investigate whether SETD7 modulates angiogenesis in experimental diabetes.
Methods
Human aortic endothelial cells (HAECs) were cultured in growth factor-free medium and exposed either to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) for 48 hours. SETD7 protein and H3K4me1 levels were investigated by Western blot and chromatin immunoprecipitation (ChIP). Knockdown of SETD7 was achieved by small interfering RNA (siRNA). Pharmacological blockade of SETD7 was performed by using the highly selective inhibitor (R)-PFI-2, while its inactive enantiomer, (S)-PFI-2, was used as a control. Scratch and tube formation assays were performed to investigate the impact of SETD7 on angiogenic properties of HAECs. RNA sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA) were employed to unveil putative genes regulated by SETD7 in HG-treated HAECs. SETD7 expression was also investigated in muscular specimens isolated from type 2 diabetic (db/db) mice and non-diabetic mice undergoing hindlimb ischemia for 21 days.
Results
HG exposure in HAECs led to a time-dependent increase of both SETD7 gene and protein expression, as compared to NG. SETD7 upregulation in HG-treated HAECs was associated with an increase of H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Of interest, both gene silencing and pharmacological blockade of SETD7 rescued hyperglycemia-induced impairment of angiogenic properties in HAECs. RNA-seq in HG-treated HAECs with and without SETD7 depletion unveiled an array of differentially expressed genes, which were mainly involved in blood vessel growth and angiogenic response, as assessed by IPA analysis. Among dysregulated genes, ChIP assays showed that SETD7-dependent chromatin changes enabled the transcription of Semaphorin 3G (SEMA-3G), a negative regulator of endothelial cell migration. Indeed, gene silencing of SETD7 blunted SEMA-3G expression in HAECs exposed to HG. Consistent with our in vitro observations, SETD7 was upregulated in adductor muscle specimens from db/db mice undergoing hindlimb ischemia as compared to non-diabetic animals.
Conclusions
Pharmacological blockade of SETD7 by (R)-PFI-2 may represent a potential therapeutic approach to boost post-ischemic vascularization in T2D patients with PAD.
Antagonistic effects of exogenous Slit2 on VEGF‑induced choroidal endothelial cell migration and tube formation
