Endothelial cell injury by high glucose and heparanase is prevented by insulin, heparin and basic fibroblast growth factor

Abstract

Background: Uncontrolled hyperglycemia is the main risk factor in the development of diabetic vascular complications. The endothelial cells are the first cells targeted by hyperglycemia. The mechanism of endothelial injury by high glucose is still poorly understood. Heparanase production, induced by hyperglycemia, and subsequent degradation of heparan sulfate may contribute to endothelial injury. Little is known about endothelial injury by heparanase and possible means of preventing this injury.

Objectives: To determine if high glucose as well as heparanase cause endothelial cell injury and if insulin, heparin and bFGF protect cells from this injury.

Methods: Cultured porcine aortic endothelial cells were treated with high glucose (30 mM) and/or insulin (1 U/ml) and/or heparin (0.5 microg/ml) and /or basic fibroblast growth factor (bFGF) (1 ng/ml) for seven days. Cells were also treated with heparinase I (0.3 U/ml, the in vitro surrogate heparanase), plus insulin, heparin and bFGF for two days in serum free medium. Endothelial cell injury was evaluated by determining the number of live cells per culture and lactate dehydrogenase (LDH) release into medium expressed as percentage of control.

Results: A significant decrease in live cell number and increase in LDH release was found in endothelial cells treated with high glucose or heparinase I. Insulin and/or heparin and/or bFGF prevented these changes and thus protected cells from injury by high glucose or heparinase I. The protective ability of heparin and bFGF alone or in combination was more evident in cells damaged with heparinase I than high glucose.

Conclusion: Endothelial cells injured by high glucose or heparinase I are protected by a combination of insulin, heparin and bFGF, although protection by heparin and/or bFGF was variable.

Figure 1

PAECs Injured by High Glucose were Protected by a Combination of Heparin and Insulin. PAECs were exposed to glucose (30 mM), insulin (1 U/ml), heparin (0.5 μg/ml) and glucose plus heparin plus insulin for seven days. Cell medium was changed and fresh reagents were added every other day. Cells were counted and LDH release in medium was determined 48 hrs after the last addition of reagents. Results are expressed as mean +/- SE of three dishes per group. Significantly different than a, control; b, glucose; c, glucose+heparin+insulin (P < 0.01) (one-way ANOVA).

Figure 2

The Protective Effect of Insulin and/or Heparin on PAECs Injured by High Glucose. PAECs were treated with glucose (30 mM), glucose plus insulin (1 U/ml), glucose plus heparin (0.5 μg/ml) and glucose plus insulin plus heparin for seven days. Cell medium was changed and fresh reagents were added every other day. Cells were counted and LDH release to medium was determined 48 hrs after the last addition of reagents. Results are expressed as mean +/- SE of three dishes per group. Significantly different than a, control; b, glucose; c, glucose + heparin; d, glucose + insulin (P < 0.01) (one-way ANOVA).

Figure 3

Insulin and/or Heparin Protected PAECs from High Glucose Injury when bFGF was Present in Cell Medium. PAECs were treated with glucose (30 mM), glucose plus bFGF (1 ng/ml), glucose plus bFGF plus insulin (1 U/ml), glucose plus bFGF plus heparin (0.5 μg/ml) and glucose plus bFGF plus insulin plus heparin for seven days. Cell medium was changed and fresh reagents were added every other day. Cells were counted and medium LDH was determined 48 hrs after the last addition of reagents. Results are expressed as mean +/- SE of three dishes per group. Significantly different than a, control; b, glucose; c, glucose+bFGF; d, glucose+bFGF+heparin (P < 0.05) (one-way ANOVA).

Figure 4

Heparinase I Induced PAECs Injury was Prevented by Insulin and/or Heparin. PAECs were treated with heparinase I (0.3 U/ml) and/or insulin (1 U/ml) and/or heparin (0.5 μg/ml) for 48 hrs in serum free medium 199, then cells were counted and media LDH was determined. Insulin and/or heparin were added immediately after heparinase I addition. All reagents were only added once. Results are expressed as mean +/- SE of three dishes per group. Significantly different than a, control; b, heparinase I; c, heparinase I+heparin; d, heparinase I+insulin (P < 0.005) (one-way ANOVA).

Figure 5

The Protective Effect of Insulin and/or Heparin on PAECs Injured by Heparinase I when bFGF was Present in Cell Medium. PAECs were treated with heparinase I (0.3 U/ml), heparinase I plus bFGF (1 ng/ml), heparinase I plus insulin (1 U/ml) plus bFGF, heparinase I plus heparin (0.5 μg/ml) plus bFGF and heparinase I plus insulin plus heparin plus bFGF for 48 hrs in serum free medium 199. After 48 hrs, cells were counted and media LDH was determined. Insulin, heparin and bFGF were added immediately after heparinase I addition. Results are expressed as mean +/- SE of three culture dishes per group. Significantly different than a, control ; b, heparinase I; c, heparinase I+bFGF (P < 0.05) (one-way ANOVA).