Induced Pluripotent Stem Cell-Derived Endothelial Cells From Human Diabetic Donors Carry an Imprint of the Diabetic Milieu

Diabetic endotheliopathy is the main cause for impaired angiogenesis and reduced neovascularization that lead to microvascular injury and vascular complications. The pathogenic basis
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) from small
amounts of blood 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 1,2,3,4 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 that genes and proteins involved in angiogenesis and
EC function were significantly downregulated in DiPS-ECs in
comparison to NiPS-ECS (n=3, p<0.05). Specific epsins regulating VEGF-mediated angiogenesis were downregulated in
DiPS-ECs, leading to increased signalling VEGF pathway activation6. Moreover factors involved in E-cadherin signalling,
endothelial-to-mesenchymal transition and fibrosis were
increased in DiPS-ECs. We detected abnormal capillary permeability and barrier integrity in DiPS-ECs using xCELLigence®.
DiPS-ECs had significantly reduced barrier integrity and barrier recovery (n=3, p<0.001, ±SEM) and also displayed
impaired tube formation in vitro (n=3, ±SEM, p<0.05).
DiPS-ECs displayed impaired function demonstrated by decreased blood flow recovery (BFR) compared to NiPS ECs
(n=3) when injected to the hindlimb of mice following femoral artery ligation. Finally, our proteomic and transcriptomic
analysis confirmed imbalances in several angiogenic genes
including endothelial specific Roundabout protein 4 (ROBO4)
that is highly involved in pathways related to angiogenesis,
barrier stability and endothelial health 7. 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 development and angiogenesis. Our
data support the impaired angiogenic functionality of DiPSECs cells in vitro and in vivo and show that DiPS-ECs carry
an imprint of the diabetic milieu which is reflected in their
dysfunction. To the best of our knowledge, we have identified
a novel disease-specific signature in diabetic iPS-ECs, therefore
our human iPS-EC model may serve as a valuable tool to
study biological pathways and identify new treatments for diabetes-induced endotheliopathy.