Effects of high glucose on vascular endothelial growth factor synthesis and secretion in aortic vascular smooth muscle cells from obese and lean Zucker rats

Abstract

Type 1 diabetes is characterized by insulin deficiency, type 2 by both insulin deficiency and insulin resistance: in both conditions, hyperglycaemia is accompanied by an increased cardiovascular risk, due to increased atherosclerotic plaque formation/instabilization and impaired collateral vessel formation. An important factor in these phenomena is the Vascular Endothelial Growth Factor (VEGF), a molecule produced also by Vascular Smooth Muscle Cells (VSMC). We aimed at evaluating the role of high glucose on VEGF-A(164) synthesis and secretion in VSMC from lean insulin-sensitive and obese insulin-resistant Zucker rats (LZR and OZR). In cultured aortic VSMC from LZR and OZR incubated for 24 h with d-glucose (5.5, 15 and 25 mM) or with the osmotic controls l-glucose and mannitol, we measured VEGF-A(164) synthesis (western, blotting) and secretion (western blotting and ELISA). We observed that: (i) d-glucose dose-dependently increases VEGF-A(164) synthesis and secretion in VSMC from LZR and OZR (n = 6, ANOVA p = 0.002-0.0001); (ii) all the effects of 15 and 25 mM d-glucose are attenuated in VSMC from OZR vs. LZR (p = 0.0001); (iii) l-glucose and mannitol reproduce the VEGF-A(164) modulation induced by d-glucose in VSMC from both LZR and OZR. Thus, glucose increases via an osmotic mechanism VEGF synthesis and secretion in VSMC, an effect attenuated in the presence of insulin resistance.

Keywords: diabetes; glucose; insulin resistance; lean zucker rats; obese zucker rats; osmotic stress; vascular endothelial growth factor; vascular smooth muscle cells.

Figure 1

(A) Dose-dependent effects on Vascular Endothelial Growth Factor (VEGF)-A₁₆₄ synthesis (Western immunoblotting and its densitometric analysis) elicited by a 24 h of incubation with d-glucose (5.5, 15 and 25 mM) in Vascular Smooth Muscle Cells (VSMC) from lean insulin-sensitive Zucker fa/+ rats (LZR) and obese insulin-resistant Zucker fa/fa rats (OZR); (B) Role on VEGF-A₁₆₄ synthesis (Western immunoblotting and its densitometric analysis) elicited in VSMC from LZR and OZR by a 24 h of incubation with 19.5 mM l-glucose or mannitol, i.e., with a concentration iso-osmolar to 25 mM d-glucose, taking into account the presence of d-glucose 5.5 mM in the culture medium. Statistical analysis is described in the text.

Figure 2

(A) Dose-dependent effects on VEGF-A (ELISA) elicited by a 24 h incubation with d-glucose (5.5, 15 and 25 mM) in VSMC from LZR and OZR; (B) Role on VEGF-A secretion (ELISA) elicited in VSMC from LZR and OZR by a 24 h incubation with 19.5 mM l-glucose or mannitol, i.e., with a concentration iso-osmolar to 25 mM d-glucose, taking into account the presence of d-glucose 5.5 mM in the culture medium. Statistical analysis is described in the text.

Figure 3

(A) Dose-dependent effects on VEGF-A₁₆₄ secretion (Western immune blotting and its densitometric analysis) elicited by a 24 h of incubation with d-glucose (5.5, 15 and 25 mM) in VSMC from LZR and OZR.; (B) Effects on VEGF-A₁₆₄ secretion (Western immunoblotting and its densitometric analysis) elicited by a 24 h incubation with 19.5 mM l-glucose or mannitol in VSMC from LZR and OZR, i.e., with a concentration iso-osmolar to 25 mM d-glucose, taking into account the presence of d-glucose 5.5 mM in the culture medium. Statistical analysis is described in the text.

Figure 3

(A) Dose-dependent effects on VEGF-A₁₆₄ secretion (Western immune blotting and its densitometric analysis) elicited by a 24 h of incubation with d-glucose (5.5, 15 and 25 mM) in VSMC from LZR and OZR.; (B) Effects on VEGF-A₁₆₄ secretion (Western immunoblotting and its densitometric analysis) elicited by a 24 h incubation with 19.5 mM l-glucose or mannitol in VSMC from LZR and OZR, i.e., with a concentration iso-osmolar to 25 mM d-glucose, taking into account the presence of d-glucose 5.5 mM in the culture medium. Statistical analysis is described in the text.