A Novel Model of Long‐term Human Microvascular Endothelial Cell Serum Starvation

Abstract

Microvascular endothelial cell injury and progressive loss of microvasculature in the dermis, kidney, retina and perineural beds is caused by hyper‐and hypoglycemic insults in individuals with Diabetes mellitus (DM). The consequence of microvascular injuries occurring over years to decades is the onset of DM‐associated co‐morbidities such as delayed wound healing, renal failure, vision loss and peripheral neuropathy. The Diabetes Control and Complications Trial noted a correlation between baseline endogenous C‐peptide serum levels and the delay in onset of DM‐associated co‐morbidities. In immortalized human microvascular endothelial cells (HMEC) and primary human dermal microvascular endothelial cells treatment with 1.5nM C‐peptide and 0.5nM insulin prevented hyperglycemia‐induced oxidative injury. In studies of these cells using immunofluorescence to track C‐peptide and the C‐peptide receptor, G‐protein coupled receptor 146 (GPR146), endothelial cells repeatedly demonstrated cytosolic stores of C‐peptide. Fetal bovine serum, a component of almost all cell culture growth media was determined to have high concentrations of C‐peptide. The aim of this study was to develop a model of long term endothelial serum starvation that would result in depleted internal stores of C‐peptide, but would prevent endothelial cells transformation to smooth muscle cells or fibroblasts, as endothelial progenitor cells are capable of differentiating into one of these three cell types. We hypothesized that cytosolic C‐peptide would reduce over time with serum starvation. To test this hypothesis, HMEC were incubated for up to two months in MCDB13 media supplemented with human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, basic fibroblast growth factor, insulin‐like growth factor‐1, ascorbic acid, and L‐glutamine. Serum starved HMEC were harvested at various time points and stained for C‐peptide (MAB80561 R&D Systems clone 915612) and DAPI or lysed with lysis buffer with 2‐mercaptoethanol followed by RNA isolation, cDNA preparation and RT‐PCR evaluation of targets. Cytosolic C‐peptide fluorescent signal diminished over time in a linear manner but was never completely abolished. HMEC serum starved for 30 days retained endothelial markers CD31, CD34, VEGFR2, had no change in smooth muscle markers Smoothelin, or Smooth muscle actin, and similarly, had no change in expression Vimentin a fibroblast marker relative to un‐serum starved HMEC controls as determined by RT‐PCR. The ability to serum starve HMEC long‐term and substantially reduce internal C‐peptide stores has the potential to enhance research to elucidate the pathways employed by C‐peptide in the prevention of hyperglycemia‐induced microvascular injury.Support or Funding InformationNIDDK