Abstract

Objective - To establish a method for isolation and culture of subcutaneous microvascular endothelial cells (MVEC) from small human tissue biopsies to compare gene- and protein expression of insulin signaling molecules in MVEC from insulin-resistant and healthy control subjects.

Research and Design Methods - Stromavascular cells from subcutaneous needle biopsies of type 2 diabetic (T2D) and control subjects were expanded in culture and the endothelial cells selected with magnetic immune separation. Western blots and RT-PCR were used for protein- and gene expression assays.

Results - At least 99% of the expanded primary MVEC could be characterized as endothelial cells. The expression of insulin receptors (IR) was low but insulin increased tyrosine phosphorylation of both the IR and IRS-1 and activated PKB. The IRS-1 protein expression was reduced and the serine phosphorylation of PKB in response to insulin attenuated while basal and insulin-stimulated phosphorylation of ERK1/2 was increased in T2 MVEC. Endothelin-1 mRNA levels were significantly higher in T2D cells. The addition of endothelin-1 increased the phosphorylation of MAPK, an effect antagonized by the MEK-1 inhibitor PD98059. Furthermore, the endothelin ET_A and ET_B receptor antagonists BQ123 and BQ788 decreased basal MAPK activity in T2 MVEC and prevented the endothelin-1-induced activation.

Conclusions - We developed a system for isolation and culture of human MVEC from small needle biopsies. Our observations support the concept of “selective” insulin resistance, involving IRS-1 and the PI3kinase pathway, as an underlying factor for a dysregulated microvascular endothelium in T2D. Our data also support a role of endothelin-1 for the increased MAPK activity seen in non-stimulated T2D MVEC.