Vildagliptin stimulates endothelial cell network formation and ischemia-induced revascularization via an endothelial nitric-oxide synthase-dependent mechanism

Abstract

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production.

Keywords: Adiponectin; Angiogenesis; Endothelial Cell; Nitric-oxide Synthase; Signaling.

FIGURE 1.

Vildagliptin promotes revascularization following hindlimb ischemia in mice. A, representative images of LDBF for wild-type (WT) mice subjected to surgery-induced hind limb ischemia and treated with vildagliptin or vehicle. B, quantitative analysis of the ischemic/normal LDBF ratio in the vildagliptin or vehicle-treated mice on postoperative days 3, 7, 14, and 21 (n = 10 in each group). *, p < 0.05 versus vehicle control. C, microscopic photographs of capillary density in ischemic adductor muscles 21 days after surgery. Capillaries were immunostained with anti-CD31 antibody. Red, CD31; blue, DAPI. Scale bar, 50 μm. D, quantitative analysis of the capillary density of ischemic muscles in vildagliptin or vehicle-treated mice (n = 5). Results are presented as mean ± S.D. N.S, not significant.

FIGURE 2.

eNOS signaling is essential for vildagliptin-induced revascularization. A, immunoblotting with the indicated antibodies was performed on the ischemic adductor muscle of mice treated with vildagliptin or vehicle 3 days after surgery. Representative blots are shown. B, quantitative analysis of relative changes in phosphorylated Akt (left) and eNOS (right). Relative phosphorylation levels were normalized to total protein signal (n = 4). Results are presented as mean ± S.D. C, representative images of LDBF for WT and eNOS-KO mice treated with vildagliptin or vehicle control. D, quantitative analysis of the ischemic/normal LDBF ratio in vildagliptin or vehicle-treated WT and eNOS-KO mice on postoperative days 3, 7, 14, and 21 (n = 5 in each group). *, p < 0.05 versus vehicle controls (WT). Results are presented as mean ± S.D. N.S, not significant.

FIGURE 3.

Vildagliptin promotes endothelial cell differentiation. A, representative photomicrographs of network formation of HUVECs treated with 5 nm vildagliptin and/or 5 nm GLP-1. B, quantitative analyses of network tube length are shown (n = 3). *, p < 0.05. HUVECs were seeded on Matrigel-coated plates incubated with vildagliptin (1, 5, or 10 nm) and/or GLP-1 (1, 5, or 10 nm) at 37 °C for 6 h (n = 3). *, p < 0.05. C, the representative immunoblots in DPP-4 in the presence of siRNA targeting DPP-4 or unrelated siRNA in HUVECs. D, effect of knockdown of DPP-4 on the network tube length. Results are shown as the mean ± S.D. (n = 3). N.S, not significant.

FIGURE 4.

eNOS signaling participates in endothelial cell responses to vildagliptin. A and B, time-dependent changes in the phosphorylation of Akt and eNOS signaling in HUVECs treated with 5 nm vildagliptin and/or 5 nm GLP-1. Shown are quantitative analysis of relative changes in phosphorylated Akt and eNOS. Relative phosphorylation levels were normalized to each total protein signal (n = 3). *, p < 0.05 versus control (0 min). C, contribution of PI3K and eNOS to vildagliptin-mediated endothelial cell differentiation. HUVECs were pretreated with LY294002 (50 μmol/liter), l-NAME (1 mmol/liter), or vehicle (dimethyl sulfoxide), and treated with 5 nm vildagliptin and/or 5 nm GLP-1 at 37 °C for 6 h (n = 3). The Matrigel assay was performed. Quantitative analyses of the network tube length are shown. Results are shown as the mean ± S.D. (n = 5). *, p < 0.05; **, p < 0.01.

FIGURE 5.

Src kinase is involved in vildagliptin-stimulated endothelial cell differentiation. A, quantitative analyses of network tube length are shown. HUVECs were pretreated with Src kinase inhibitor PP2 (10 μm) for 30 min and treated with vildagliptin (5 nm) or GLP-1 (5 nm) at 37 °C for 6 h (n = 3). The Matrigel assay was performed. * p < 0.05. B, effects of PP2 on vildagliptin or GLP-1 stimulated phosphorylation of Akt and eNOS. HUVECs were pretreated with PP2 (10 μm) for 30 min and treated with vildagliptin (5 nm) or GLP-1 (5 nm) at 37 °C for 60 min. Relative phosphorylation levels were normalized to total protein signal (n = 4). Results are presented as mean ± S.D. N.S., not significant.

FIGURE 6.

Role of adiponectin in the vildagliptin-induced angiogenic response. A, representative images of LDBF in WT and adiponectin-KO (APN-KO) mice treated with vildagliptin or vehicle control. B, quantitative analysis of the ischemic/normal LDBF ratio in vildagliptin or vehicle-treated WT and APN-KO mice on postoperative days 3, 7, 14, and 21 (n = 5 in WT, n = 4 in APN-KO). *, p < 0.05 versus vehicle (WT); #, p < 0.05 versus vildagliptin treatment (APN-KO). Results are presented as mean ± S.D. C, quantitative analyses of network tube length are shown. HUVECs were treated with adiponectin (30 μg/ml) and/or GLP-1 (5 nm) at 37 °C for 6 h (n = 3). The Matrigel assay was performed. Results were expressed relative to the values compared with control. *, p < 0.05. D, effects of adiponectin on phosphorylation of Akt and eNOS. HUVECs were treated with adiponectin (30 μg/ml) and/or GLP-1 (5 nm) at 37 °C for 60 min. Relative phosphorylation levels were normalized to total protein signal (n = 4). Results are presented as mean ± S.D. *, p < 0.05. E and F, expression of adiponectin in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were incubated with vildagliptin (0, 1, or 100 nm) or GLP-1 (0, 1, or 100 nm) for 24 h, then adiponectin mRNA expression was determined by real-time RT-PCR (n = 3 in each group). Results are presented as mean ± S.D. N.S, not significant.

FIGURE 7.

Proposed scheme for the angiogenic actions of vildagliptin. The DPP-4 inhibitor, vildagliptin, stimulates revascularization through an eNOS-dependent mechanism. The angiogenic actions of vildagliptin are mediated by both the GLP-1-dependent effects and the direct actions of vildagliptin. The beneficial actions of GLP-1 on revascularization are mediated partly through its ability to increase adiponectin production.