Delayed branching of endothelial capillary-like cords in glycated collagenI is mediated by early induction of PAI-1

Development of micro- and macrovascular disease in diabetes mellitus (DM) w arrants a thorough investigation into the repertoire of endothelial cell (E C) responses to diabetic environmental cues. Using human umbilical vein EC (HUVEC) cultured in three-dimensional (3-D) native collagen I (NC) or glyca ted collagen I (GC), we observed capillary cord formation that showed a sig nificant reduction in branching when cells were cultured in GC. To gain ins ight into the molecular determinants of this phenomenon, HUVEC subjected to GC vs. NC were studied using a PCR-selected subtraction approach. Nine dif ferent genes were identified as up- or downregulated in response to GC; amo ng those, plasminogen activator inhibitor-1 (PAI-1) mRNA was found to be up regulated by GC. Western blot analysis of HUVEC cultured on GC showed an in crease in PAI-1 expression. The addition of a neutralizing anti-PAI-1 antib ody to HUVEC cultured in GC restored the branching pattern of formed capill ary cords. In contrast, supplementation of culture medium with the constitu tively active PAI-1 reproduced defective branching patterns in HUVEC cultur ed in NC. Ex vivo capillary sprouting in GC was unaffected in PAI-1 knockou t mice but was inhibited in wild-type mice. This difference persisted in di abetic mice. In conclusion, the PCR-selected subtraction technique identifi ed PAI-1 as one of the genes characterizing an early response of HUVEC to t he diabetic-like interstitial environment modeled by GC and responsible for the defective branching of endothelial cells. We propose that an upregulat ion of PAI-1 is causatively linked to the defective formation of capillary networks during wound healing and eventual vascular dropout characteristic of diabetic nephropathy.