Delayed branching of endothelial capillary-like cords in glycated collagen I is mediated by early induction of PAI-1.
Development of micro- and macrovascular disease in diabetes mellitus (DM) warrants a thorough investigation into the repertoire of endothelial cell (EC) responses to diabetic environmental cues. Using human umbilical vein EC (HUVEC) cultured in three-dimensional (3-D) native collagen I (NC) or glycated collagen I (GC), we observed capillary cord formation that showed a significant reduction in branching when cells were cultured in GC. To gain insight into the molecular determinants of this phenomenon, HUVEC subjected to GC vs. NC were studied using a PCR-selected subtraction approach. Nine different genes were identified as up- or downregulated in response to GC; among those, plasminogen activator inhibitor-1 (PAI-1) mRNA was found to be upregulated by GC. Western blot analysis of HUVEC cultured on GC showed an increase in PAI-1 expression. The addition of a neutralizing anti-PAI-1 antibody to HUVEC cultured in GC restored the branching pattern of formed capillary cords. In contrast, supplementation of culture medium with the constitutively active PAI-1 reproduced defective branching patterns in HUVEC cultured in NC. Ex vivo capillary sprouting in GC was unaffected in PAI-1 knockout mice but was inhibited in wild-type mice. This difference persisted in diabetic mice. In conclusion, the PCR-selected subtraction technique identified PAI-1 as one of the genes characterizing an early response of HUVEC to the diabetic-like interstitial environment modeled by GC and responsible for the defective branching of endothelial cells. We propose that an upregulation of PAI-1 is causatively linked to the defective formation of capillary networks during wound healing and eventual vascular dropout characteristic of diabetic nephropathy.
Chen
J
J
Department of Medicine and Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8152, USA.
Brodsky
S
S
Li
H
H
Hampel
D J
DJ
Miyata
T
T
Weinstein
T
T
Gafter
U
U
Norman
J T
JT
Fine
L G
LG
Goligorsky
M S
MS
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001011:Aorta; D015152:Blotting, Northern; D015153:Blotting, Western; D002196:Capillaries / Q000502:physiology; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen / Q000031:analogs & derivatives; D018076:DNA, Complementary / Q000032:analysis; D003921:Diabetes Mellitus, Experimental / Q000503:physiopathology; D003928:Diabetic Nephropathies / Q000503:physiopathology; D004730:Endothelium, Vascular / Q000502:physiology* / Q000648:ultrastructure; D006031:Glycosylation; D051379:Mice; D018345:Mice, Knockout; D018919:Neovascularization, Physiologic; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013997:Time Factors; D014471:Umbilical Veins
D000906:Antibodies; D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D003094:Collagen