Effect of insulin and heparin on glucose-induced vascular damage in cell culture.
BACKGROUND
Clinical trials have shown that tight glycemic control reduces the risk of diabetic microvascular complications, namely retinopathy, nephropathy, and neuropathy. The mechanism of these microvascular complications is not yet fully elucidated. The present study describes the effect of different concentrations of glucose on vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in cell culture. Our objective was to shed some light through this biological study on the mechanism and prevention of diabetic microvascular complications.


METHODS
ECs and VSMCs were treated with 5 mmol/L (90 mg/dL) or 30 mmol/L (540 mg/dL) D-glucose or D-glucose plus insulin or D-glucose plus insulin and heparin in culture. ECs were studied with light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) for surface changes. The cultured ECs were treated with vimentin antibodies and VSMCs with actin antibodies for immunoflourescence microscopy (IFM) study. Endothelin-1 (ET-1) assay was done on ECs culture medium using enzyme-linked immunosorbent assay (ELISA).


RESULTS
LM, SEM, and TEM of ECs treated with a physiological concentration (90 mg/dL) of D-glucose appeared the same as control. However, LM and SEM of ECs treated with a high concentration of D-glucose (540 mg/dL) showed pronounced intercellular gaps. This finding was further confirmed by TEM study. These gaps were minimally or not at all discernible when insulin, heparin, or a combination of both was added to the culture medium. IFM showed increased vimentin expression with a high concentration of D-glucose. Vimentin expression was attenuated with the addition of insulin or heparin in the medium and more markedly with combined insulin and heparin. Significant correlations were obtained between glucose levels, vimentin expression, and ET-1 levels. The higher the glucose level, the higher is the ET-1 production and the greater vimentin expression in ECs. Cultured VSMCs treated with a high concentration of D-glucose showed enhanced actin expression. Actin expression was blunted with the addition of insulin or heparin in the culture medium.


CONCLUSIONS
These biological findings indicate the salutary effect of insulin or insulin and heparin in the mitigation of vascular disorganization caused by a high concentration of D-glucose.
Mandal
A K
AK
Division of Nephrology, Department of Medicine, Veterans Affairs Medical Center and Wright State University, Dayton, Ohio, USA. tfischer@med-spec.com
Puchalski
J T
JT
Lemley-Gillespie
S
S
Taylor
C A
CA
Kohno
M
M
D002460:Cell Line; D019332:Endothelin-1 / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009994:Osmolar Concentration; D014652:Vascular Diseases / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology
D019332:Endothelin-1; D007328:Insulin; D006493:Heparin; D005947:Glucose