The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues

Abstract

Diabetes is associated with poor outcomes following acute vascular occlusive events. This results in part from a failure to form adequate compensatory microvasculature in response to ischemia. Since vascular endothelial growth factor (VEGF) is an essential mediator of neovascularization, we examined whether hypoxic up-regulation of VEGF was impaired in diabetes. Both fibroblasts isolated from type 2 diabetic patients, and normal fibroblasts exposed chronically to high glucose, were defective in their capacity to up-regulate VEGF in response to hypoxia. In vivo, diabetic animals demonstrated an impaired ability to increase VEGF production in response to soft tissue ischemia. This resulted from a high glucose-induced decrease in transactivation by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates hypoxia-stimulated VEGF expression. Decreased HIF-1alpha functional activity was specifically caused by impaired HIF-1alpha binding to the coactivator p300. We identify covalent modification of p300 by the dicarbonyl metabolite methylglyoxal as being responsible for this decreased association. Administration of deferoxamine abrogated methylglyoxal conjugation, normalizing both HIF-1alpha/p300 interaction and transactivation by HIF-1alpha. In diabetic mice, deferoxamine promoted neovascularization and enhanced wound healing. These findings define molecular defects that underlie impaired VEGF production in diabetic tissues and offer a promising direction for therapeutic intervention.

Fig. 1.

Diabetes and prolonged exposure to high glucose impair hypoxic induction of VEGF expression. Cells were placed in hypoxia (0.5% O₂) or normoxia (21% O₂), and levels of VEGF secretion were measured by ELISA. (A) Dermal fibroblasts isolated from diabetic patients produced less VEGF. (B) Fibroblasts from normal human foreskins were cultured chronically (4 weeks) in low glucose (LG) or high glucose (HG). VEGF protein expression was significantly attenuated in cells grown in HG and exposed to hypoxia. (C) Schematic depicting murine ischemic model used. Tissue oxygen tensions are highest in the most proximal (cranial) part of the tissue bed [A] and decrease in a gradient to the most distal (caudal) part [C]. (D) VEGF levels in ischemic skin segments of normal mice measured 24 and 72 h after ischemic induction. (E) VEGF protein levels in ischemic skin tissue from db/db mice. All values represent mean ± SEM. *, P < 0.05 vs. baseline. ‡, P < 0.05 vs. zone A. ¶, P < 0.05 vs. 24 h. n = 6 for all panels. Nl, normal skin.

Fig. 2.

Hyperglycemia-induced impairment in HIF-1 transactivation occurs as a result of decreased HIF-1α binding to p300. (A) Plasmids containing a full-length VEGF-A promoter (pVEGF-kpnI-luc) or the hypoxia response element for the enolase gene (p2.1), both linked to firefly luciferase, were transfected into cells and subsequently exposed to hypoxia. HG exposure decreased HIF-1 transactivation with use of either plasmid. (B) HAECs were treated for 5 days with LG, HG, or HG after infection of GLO1 adenovirus (HG/GLO1). After exposure to hypoxia for 18 h, cell lysates were collected for immunoprecipitation (IP) of p300 and subsequently immunoblotted (IB) for p300, methylglyoxal (MG), or HIF-1α. β-actin was used as an input control (shown in red typeface). (C) Western blot quantitation of panel B. (D) HAECs were transfected with plasmids containing the HIF-1α-CAD and the p300-CH1 domain together with a reporter plasmid containing a Gal4-binding motif upstream of a firefly luciferase reporter gene. Cells were grown in LG or HG and exposed to normoxia or hypoxia, and the level of luciferase reporter activity subsequently measured. Impaired HIF-1α/p300 binding in HG was completely reversed with GLO1 treatment. *, P < 0.05 vs. LG group. n = 3.

Fig. 3.

High glucose-induced methylglyoxal modification of p300 at R354 decreases association of p300 with HIF-1α. (A) HAECs were transfected with either Gal4(AD) wild-type p300 or p300 single-mutant 354(Q), followed by treatment for 5 days in LG or HG, and subsequent exposure to 18 h of hypoxia. Cell lysates were collected for IP of Gal4(p300) and IB for Gal4(p300) and methylglyoxal (MG). (B) Western blot quantitation of panel A. (C) HAECs were treated as in panel A. Lysates were collected for IP of HIF-1α and IB for Gal4(p300) and HIF-1α. (D) Western blot quantitation of panel C. Immunoprecipitation inputs (shown in red typeface) were used as loading controls. *, P < 0.05 vs. LG group. n = 3. AD, activation domain.

Fig. 4.

DFO corrects HIF-1α/p300 binding and augments HIF-1 transactivation and VEGF expression in high glucose culture. (A) HAECs cultured with LG, HG, or HG with DFO (HG/DFO) were exposed to hypoxia and evaluated by mammalian 2-hybrid assay for HIF-1α/p300 binding. DFO treatment augmented HIF-1α/p300 binding in cells exposed to HG. (B) IP/WB of p300 and HIF-1α following treatment described in panel A. β-actin was used as an input control (shown in red typeface). (C) Western blot quantitation of panel B. (D) HIF-1 transactivation analyzed with the 5× hypoxia response element construct described in the Results section. DFO increased HIF-1-mediated luciferase activity in cells exposed to HG in mouse embryonic fibroblasts (MEFs). (E) VEGF ELISA demonstrating DFO's augmentation of VEGF expression by MEFs cultured in HG. *, P < 0.05 vs. HG/DFO group. n = 3.

Fig. 5.

DFO decreases ROS generation, prevents methylglyoxal modification of p300, and increases VEGF expression in high glucose culture. HAECs were treated for 5 days in LG, HG, HG with DFO (HG/DFO), HG with DMOG (HG/DMOG), or HG with DFO and DMOG (HG/DFO/DMOG). The following evaluations were then performed: (A) ROS generation, (B) IP/WB of p300 modification by methylglyoxal (MG), and (C) VEGF mRNA expression. *, P < 0.05 vs. LG group. n = 3.

Fig. 6.

DFO enhances wound healing and neovascularization in diabetic mice. Full-thickness skin wounds on diabetic mice were treated with every other day topical applications of 500 μM DFO, 1,000 μM DFO, or PBS. (A) Photographs of representative wounds for mice treated with PBS or 1,000 μM DFO. Wound resurfacing occurred at day 13 in the DFO-treated group versus day 23 in PBS-treated controls. (B) Graphical depiction of wound area as a function of time. DFO accelerated wound healing in a dose-dependent manner. (C) H&E stains of wound tissue at 7 days post-wounding showing increased granulation tissue in the wounds of DFO-treated animals. White borders delineate initial wound edges, and yellow borders indicate wound edges at day 7. (D) DFO- and PBS-treated mice were analyzed at 7 days post-wounding for vascularity (as assessed by CD31 immunohistochemistry). (E) Numbers of CD31-positive vessels per HPF in DFO-treated (1,000 μM) and PBS-treated mice at days 7, 13, and 21 post-wounding. (F) Wound tissue VEGF concentration at 7 days post-wounding for DFO-treated (1,000 μM) and PBS-treated mice. *, P < 0.05 vs. PBS group. n = 8.