GLP-1 promotes angiogenesis in human endothelial cells in a dose-dependent manner, through the Akt, Src and PKC pathways

Abstract

Introduction: Novel anti-diabetic medications that mimic or augment the physiological actions of GLP-1 improve cardiovascular risk factors in diabetics and GLP-1 has been proposed to have a beneficial role in the cardiovascular system. GLP-1 may have a direct cardioprotective role by decreasing infarct size and protecting from ischemia-reperfusion injury while prolonging survival in rodent models. The mechanisms underlying these observations remain largely unknown. In vitro studies suggest that GLP-1 may promote endothelial cell proliferation, but no study to date has evaluated a potential direct effect of GLP-1 on angiogenesis.

Specific aim: To evaluate whether GLP-1 affects angiogenesis in humans and to elucidate underlying molecular mechanisms.

Material and methods: We utilized a 3D culture system where spherules of human umbilical vein endothelial cells (HUVECs) embedded in a collagen scaffold were treated with escalating doses of human recombinant GLP-1 (50-2000 nmol/L) and the formation of new vessels was observed and quantified. Signaling inhibitors were utilized to identify molecular pathways through which GLP-1 promotes angiogenesis.

Results: We demonstrate that GLP-1 promotes angiogenesis in a dose-dependent manner. The maximum effect on angiogenesis was observed at a GLP-1 dose of 500 nmol/L, while increased angiogenesis occurred in response to doses ranging from 200 nmol/L to 1000 nmol/L. Pre-treatment of the system with Akt inhibitor IV, Bisindolylmaleimide (PKC inhibitor) and src inhibitor I resulted in a significant decrease of the GLP-1 induced angiogenesis.

Conclusions: This is the first study to demonstrate that GLP-1 promotes angiogenesis in a HUVEC three dimensional in vitro model. This effect requires pharmacological doses and is mediated through the Akt, PKC and src pathways.

Keywords: 5' adenosine monophosphate (5’AMP) -activated protein kinase; AMPK; Akt; Angiogenesis; ELISA; Endothelial Nitric Oxide Synthase; Enzyme Linked Immunosorbent Assay; FAK; FMD; Flow-Mediated Dilatation; Focal Adhesion Kinase; GLP-1; Glucagon-Like Peptide 1; HCAECs; HUVECs; Human Coronary Artery Endothelial Cells; Human Umbilical Vein Endothelial Cells; LVEF; Left Ventricular Ejection Fraction; NOS; Nitric Oxide Synthase; OD; Optical Density; PI3K; PKA; PKC; Phosphatidylinositide 3-kinase; Protein Kinase A; Protein Kinase C; STAT-3; Signal transducer and activator of transcription 3; VEGF; Vascular Endothelial Growth Factor; eNOS; src.

Figure 1

A. Angiogenic effect of GLP-1 expressed as cumulative length of spurs in pixels. Error bars represent standard errors of the means. Star (*) represents statistically significant difference from the negative control. Negative CTRL: negative control; Positive CTRL: positive control (VEGF 20ng/mL); B. Phase contrast microscope images of HUVEC spherules treated with GLP-1 500nM; C. NC: Phase contrast microscope images of HUVEC spherules treated negative control; D. PC: Phase contrast microscope pictures of HUVEC spherules treated with positive control.

Figure 2

Angiogenic effect of GLP-1 expressed as cumulative length of spurs in pixels and inhibitor pre-treatment. Error bars represent standard errors of the means. Columns with the same letter are not statistically different between each other. NC: negative control, PC: positive control, GLP-1: non-pretreated 500nM GLP-1 treated cells, AG490: STAT-3 inhibitor, Compound-C: AMPK inhibitor, PD98059: erk inhibitor, SB203580: jnk inhibitor, SP600125: p38 inhibitor, AKT-INH-4: Akt inhibitor 4, Bisindolylmaleimide: PKC inhibitor); FAK-INH-14: focal adhesion kinase inhibitor 14; SRC-INH-I: src inhibitor 1.