Elucidating the underlying mechanisms of diabetic endotheliopathy using patient specific induced pluripotent stem cells

Abstract

Diabetic endotheliopathy is the main cause for impaired angiogenesis and reduced neovascularization that lead to microvascular injury and vascular complications. The pathogenesis for vascular complications arising from diabetes is complex. Elucidation of key underlying mechanisms will help the development of novel therapies and the discovery of potential biomarkers. The ability to generate functional endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) is a novel and powerful tool for cell-based therapies. Human iPSC-derived ECs (iPS-ECs) have a broad range of clinical applications including cell-based therapy, disease modelling and drug screening; they can be used in mechanistic studies towards the development of novel therapies and in the discovery of new biomarkers to be applied in regenerative medicine and treatment of diabetic vasculopathy. Here we utilize transcriptomic and proteomic technologies to assess patient-specific iPS-ECs from diabetic (DiPS-ECs) and non-diabetic (NiPS-ECS) donors in order to investigate the mechanisms driving endotheliopathy in diabetes.

Our in vitro and in vivo models recapitulate the effects of hyperglycaemia on the vasculature in the clinical setting. RNA-seq data showed differential expression of genes and proteins involved in EC function and angiogenesis were significantly downregulated in DiPS-ECs in comparison to NDiPS-ECs. Moreover, factors involved endothelial-to-mesenchymal transition were increased in DiPS-ECs. DiPS-ECs had significantly reduced impaired tube formation and barrier stability in vitro. DiPS-ECs displayed impaired angiogenic function demonstrated by poor post-transplant engraftment and decreased blood flow recovery (BFR) when injected to the hindlimb of mice following femoral artery ligation.

Proteomic and transcriptomic analysis confirmed imbalances in several pathways involved in endothelial function. Pathways involved in metabolism, homeostasis and stress response were found to be underrepresented in DiPS-ECs. RNA-seq revealed differences in the transcriptome of DiPS-ECs. EndMT related gene ontology was enriched in DiPS-ECs. Endothelial specific Roundabout protein 4 (ROBO4) is highly involved in pathways related to angiogenesis, barrier stability and endothelial health. Expression of ROBO4 was found to be impaired in DiPS-ECs and transcriptomic analysis along with in vitro and in vivo studies revealed its importance in vascular specification and angiogenesis. ROBO4 improved ECs function of DiPS-ECs and recovered their lost angiogenic function. Our data suggest that DiPS-ECs carry an imprint of the diabetic milieu which is reflected in their dysfunction. An unregulated EndMT process is proposed to cause a phenotype drift and poor angiogenic functionality observed in DiPS-ECs. ROBO4 seems to act as a fine tuning factor of this process. To the best of our knowledge, we aim to identify and report a novel disease-specific signature in diabetic iPS-ECs. Therefore, our human iPS-ECs model may serve as a valuable tool to study biological pathways and identify new treatments for diabetes-induced endotheliopathy.

Date of Award	Dec 2021
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Andriana Margariti (Supervisor) & Alan Stitt (Supervisor)