Evaluation of the in vitro and in vivo angiogenic effects of exendin-4

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Highlights

  • We investigated the effects of exendin-4 on the angiogenic process.

  • Exendin-4 increased migration, sprouting, and tube formation by HUVECs in in vitro.

  • Exendin-4 increased sprouts in aortic rings and induced new vessels in Matrigel in in vivo.

  • Exendin-4 may be of potential use for the treatment of vascular complications of diabetes.

Abstract

Exendin-4, an analog of glucagon-like peptide (GLP)-1, has beneficial effects on cardiovascular disease induced by diabetes mellitus (DM). Recently, exendin-4 was reported to induce the proliferation of endothelial cells. However, its angiogenic effect on endothelial cells has not been clearly evaluated. Therefore, we investigated the effects of exendin-4 on the angiogenic process with respect to migration, sprouting, and neovascularization using in vitro and in vivo assays. Treatment with exendin-4 increased the migration of human umbilical vein endothelial cells (HUVECs) in in vitro scratch wound assays, as well as the number of lumenized vessels sprouting from HUVECs in in vitro 3D bead assays. These responses were abolished by co-treatment with exendin (9–39), a GLP-1 receptor antagonist, which suggests that exendin-4 regulates endothelial cell migration and tube formation in a GLP-1 receptor-dependent manner. In an ex vivo assay, treatment of aortic rings with exendin-4 increased the sprouting of endothelial cells. Exendin-4 also significantly increased the number of new vessels and induced blood flow in Matrigel plugs in in vivo assays. Our results provide clear evidence for the angiogenic effect of exendin-4 in in vitro and in vivo assays and provide a mechanism underlying the cardioprotective effects of exendin-4.

Introduction

Angiogenesis is a complex process involving endothelial cells sprouting from preexisting vessels, migrating directionally toward angiogenic stimuli in the perivascular space, and then elongating new vessels [1]. Angiogenesis can be observed in various vascular diseases such as coronary artery disease (CAD) and peripheral artery disease (PAD) [2]. Diabetes mellitus (DM) is associated with an increased incidence of morbidity and mortality from atherosclerotic diseases, including CAD and PAD. Although there have been experimental attempts to rescue impaired angiogenesis by adding abundant amounts of angiogenic growth factors or cells, a clinically available and safe treatment is still required to treat the vascular complications of DM.

Glucagon-like peptide (GLP)-1 is an incretin hormone that plays an important role in the regulation of glucose homeostasis and has been used in clinical treatments [3], [4]. Exendin-4 is a stable analog of GLP-1 that was originally isolated from the saliva of the Gila monster (Heloderma suspectum) [5], [6]. Exendin-4 displays ligand-binding affinity for the GLP-1 receptor (GLP-1R) and resistance to degradation by dipeptidyl peptidase IV (DPP-IV) [7], [8], and is approved in both Europe and the United States for clinical treatment of type 2 diabetes. Endothelial cells express GLP-1R and acute administration of GLP-1 improves endothelial dysfunction in type 2 diabetes patients with CAD [9], demonstrating an important role of GLP-1 in endothelial function. Interestingly, exendin-4 stimulates the proliferation of human coronary artery endothelial cells through cAMP-dependent protein kinase (PKA)- and phosphoinositide 3-kinase (PI3K)/Akt-dependent pathways [10]. Although some molecular mechanisms of the effect of exendin-4 on the proliferation of endothelial cells have been investigated, the effects of exendin-4 on angiogenic processes such as migration, sprouting, and tube formation by endothelial cells have not been elucidated. In the present study, we evaluated the angiogenic role of exendin-4 with scratch migration assays, fibrin gel bead assays, aorta ring sprouting assays, and Matrigel plug assays for neovascularization.

Section snippets

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from the Yale Vascular Biology and Therapeutics Core. Both cell types were cultured at 37 °C in a 5% CO2 incubator. For HUVECs, the growth medium was EGM-2 (Lonza, Basel, Switzerland) containing 2% fetal bovine serum (FBS) (Lonza). For experimental treatments, HUVECs (passages 3–7) were grown to 80–90% confluence.

Scratch migration assay

HUVECs were incubated on 6-well plates in EGM-2 medium overnight. After starvation for 6 h with EGM-2 medium, monolayers of

Exendin-4 induces the migration of HUVECs in the wound migration assay

To exclude any possible interference by HUVEC proliferation, the effects of exendin-4 on HUVEC migration were observed for 9 h, less than the doubling time of HUVECs (∼24 h). In response to wounding, HUVECs migrated to the denuded area in a manner that mimicked the pattern of endothelial cell migration in vivo. Untreated HUVECs hardly migrated into the denuded area, while treatment with exendin-4 increased migration by 25%. Co-treatment with exendin (9–39) completely abrogated the effect of

Discussion

We evaluated the effect of exendin-4 on angiogenesis using in vitro and in vivo assays. Exendin-4 increased migration, sprouting, and tube formation by HUVECs in in vitro assays. In addition, it increased sprout outgrowth in aortic rings and induced the formation of new vessels in Matrigel in in vivo assays. Our data represent the first morphological evidence that exendin-4 may regulate endothelial cell migration, tube network formation, and neovascularization.

Exendin-4, a stable analog of

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2039164)

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