Oleic acid increases synthesis and secretion of VEGF in rat vascular smooth muscle cells: role of oxidative stress and impairment in obesity

Abstract

Obesity is characterized by poor collateral vessel formation, a process involving vascular endothelial growth factor (VEGF) action on vascular smooth muscle cells (VSMC). Free fatty acids are involved in the pathogenesis of obesity vascular complications, and we have aimed to clarify whether oleic acid (OA) enhances VEGF synthesis/secretion in VSMC, and whether this effect is impaired in obesity. In cultured aortic VSMC from lean and obese Zucker rats (LZR and OZR, respectively) we measured the influence of OA on VEGF-A synthesis/secretion, signaling molecules and reactive oxygen species (ROS). In VSMC from LZR we found the following: (a) OA increases VEGF-A synthesis/secretion by a mechanism blunted by inhibitors of Akt, mTOR, ERK-1/2, PKC-beta, NADPH-oxidase and mitochondrial electron transport chain complex; (b) OA activates the above mentioned signaling pathways and increases ROS; (c) OA-induced activation of PKC-beta enhances oxidative stress, which activates signaling pathways responsible for the increased VEGF synthesis/secretion. In VSMC from OZR, which present enhanced baseline oxidative stress, the above mentioned actions of OA on VEGF-A, signaling pathways and ROS are impaired: this impairment is reproduced in VSMC from LZR by incubation with hydrogen peroxide. Thus, in OZR chronically elevated oxidative stress causes a resistance to the action on VEGF that OA exerts in LZR by increasing ROS.

Figure 1

Time-dependent (4–24 h of incubation with 100 μM oleic acid) influence of oleic acid on VEGF-A mRNA transcription (Panel A); protein synthesis (Panel B) and secretion (Panel C) in VSMC from LZR.

Figure 2

Dose-dependent (24 h of incubation with 50–100 μM oleic acid) influence of oleic acid on VEGF-A mRNA transcription (Panel A); protein synthesis (Panel B) and secretion (Panel C) in VSMC from LZR.

Figure 3

Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A: (A) mRNA transcription; (B) protein synthesis and (C) secretion in VSMC from LZR and OZR.

Figure 4

Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panels A) and secretion (Panels B) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) Akt (LY294002); (ii) mTOR (Rapamycin); (iii) MEK (PD98059). All the inhibitors impair the oleic acid effects (n = 6, p = 0.04–0.02 vs. oleic acid alone).

Figure 5

Influence of a 24-h incubation with 100 μM oleic acid (on VEGF-A synthesis (Panels A) and secretion (Panels B) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of PKC (Staurosporine and LY379196).

Figure 6

Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panels A) and secretion (Panels B) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) NADPH oxidase (Apocynin); (ii) mitochondrial electron transport chain complex (Rotenone). All the inhibitors impair the oleic acid effects (n = 6, p = 0.04–0.02 vs. oleic acid alone).

Figure 7

Influence of a 6-h incubation with 100 μM oleic acid on phosphorylation of Akt (Panel A); p70S6K (Panel B); ERK-1/2 (Panel C) and PKC-beta (Panel D) in VSMC from LZR and OZR. Baseline values do not differ between LZR and OZR.

Figure 8

Influence of a 6-h incubation with 100 μM oleic acid on ERK-1/2 phosphorylation in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of PKC (Staurosporine and LY379196).

Figure 8

Influence of a 6-h incubation with 100 μM oleic acid on ERK-1/2 phosphorylation in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of PKC (Staurosporine and LY379196).

Figure 9

Influence of a 6-h incubation with 100 μM oleic acid on ERK-1/2 phosphorylation in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) NADPH oxidase (Apocynin); (ii) mitochondrial electron transport chain complex (Rotenone).

Figure 10

Time-dependent (15 min–24 h of incubation) influence of 100 μM oleic acid on superoxide anion in VSMC from LZR and OZR. Baseline values are greater in OZR than in LZR (n = 6, p = 0.002). Oleic acid effects are significant in LZR (n = 6, ANOVA, p = 0.0001 vs. baseline) but not in OZR.

Figure 11

Influence of a 24-h incubation with 100 μM oleic acid on superoxide anion in LZR without or with 1-h pre-incubation with inhibitors of: (i) PKC (Staurosporine and LY379196); (ii) NADPH oxidase (Apocynin); (iii) mitochondrial electron transport chain complex (Rotenone); (iv) xantine oxidase (Allopurinol). All the inhibitors, excepting Allopurinol, impair the oleic acid effects (n = 6, p = 0.03–0.0001 vs. oleic acid alone).

Figure 12

Influence of a 6-h incubation with 100 μM oleic acid on phosphorylation of Akt (Panel A); p70S6K (Panel B); ERK-1/2 (Panel C) and PKC-beta (Panel D) in VSMC from LZR without or with a 24-h pre-incubation with H₂O₂. H₂O₂ completely blunts the oleic acid effect on signaling molecules (n = 6, p = 0.001–0.0001 vs. oleic acid alone).

Figure 13

Influence of a 6-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panel A) and secretion (Panel B) in VSMC from LZR without or with a 24-h pre-incubation with H₂O₂. H₂O₂ completely blunts the above mentioned effects of oleic acid on VEGF-A (n = 6, p = 0.0001 vs. oleic acid alone).