Induction of angiogenesis by murine resistin: Putative role of PI3-kinase and NO-dependent pathways Stephanie A. Robertson, Colin J. Rae, Annette Graham ⁎ Vascular Biology Group, Department of Biological and Biomedical Sciences, Glasgow Caledonian University, UK abstract article info Article history: Received 24 April 2008 Received in revised form 17 July 2008 Accepted 24 July 2008 Available online 5 August 2008 Keywords: Obesity Adipocyte Angiogenesis Resistin Hyperplasia Hypertrophy Adipose tissue is a highly active endocrine organ, secreting bioactive molecules, adipokines, into the circulation. Obesity results in dysregulated adipokine secretion, contributing to pathophysiologies associated with this disorder, including insulin resistance and cardiovascular disease. Objectives: To establish whether resistin, a novel bioactive molecule produced by murine adipose tissue, and implicated in insulin resistance in rodents, can induce angiogenic responses in aortic tissues and endothelial cells in vitro, and to investigate the signal transduction pathways involved in these responses. Results: Recombinant murine resistin (5–100 ng ml - 1 ) induced sprouting of cellular networks and migration from murine aortic arch explants, primary aortic endothelial cells and in a ‘wound healing’ model utilising murine b.End5 endothelioma cells. The increased migration and sprouting of endothelial cells, due to resistin, were blocked by wortmannin (100 nM) and LY294002 (10 μM), inhibitors of phosphatidylinositol-3-kinase (PI3K), and accompanied by PI3K-dependent phosphorylation of Akt; moreover, while the changes were not associated with altered production of nitric oxide (NO), resistin-induced angiogenic responses were inhibited by IKK Inhibitor X (5 μM), an inhibitor of activation of nuclear factor (NF)-κB. Conclusions: Murine resistin induces endothelial cell migration and sprouting of cellular networks via a mechanism which appears dependent upon PI3K and NF-κB activity, but independent of altered NO production. Resistin may contribute to angiogenic responses sustaining adipose tissue expansion, or in arterial tissues distal to this site. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Increasing adiposity (overweight and obesity) is associated with insulin resistance and increased risk of type II diabetes and cardio- vascular disease (CVD). Adipose tissue is not only a highly active metabolic tissue, but also a dynamic endocrine organ, producing a number of bioactive proteins called adipocytokines, or adipokines [1], that play important roles in the development, or prevention, of insulin resistance, diabetes and atherosclerosis. Adipokines include tumour necrosis factor-α (TNFα), interleukin (IL)-6, IL-8, leptin, apelin, omentin, plasminogen activator inhibitor (PAI-I), adiponectin and resistin [2]; importantly, release of adipokines alters, both in amount and composition, as adipocytes undergo hypertrophy and hyperplasia [3]. Angiogenesis, the extension of existing vasculature by sprouting of capillaries from post-capillary venules, is an integral feature of adipogenesis, as continuous remodelling of the vascular network is required to sustain adipose tissue expansion [4]. Angiogenesis is a complex process, involving increases in vascular permeability, matrix degradation and migration and proliferation of endothelial cells, and regulated by ‘classical’ pro-angiogenic factors such as members of the vascular endothelial growth factor (VEGF) family [5]. However, adipokines also modulate angiogenic responses in endothelial cells [6–9], presumably to facilitate the expanding network of capillaries within adipose tissue. This has led to the suggestion that increased neovascularisation, induced by adipokines in the systemic circulation, may be one mechanism contributing to the established link between obesity and cardiovascular disease [10] or cancer [11]. Resistin belongs to a new gene family of small cysteine-rich secre- tory proteins, called resistin-like molecules (RELMs), originally discovered in a search for adipocyte-derived molecules linking obesity and insulin-resistance diabetes [12]. In rodents, resistin is derived largely from adipose tissue; mice deficient in resistin (Retn [-/-]) are protected from obesity-associated insulin resistance [13]. Members of the same family include mouse resistin-like alpha (Retnla), beta (Retnlb) and gamma (Retnlg) genes [14–16]. By contrast, human resistin shows significant sequence and tissue divergence from its murine ortholog [17], and only two human genes are recognised within this family of proteins, RETN and RETNLB [18]. Indeed, it has been suggested that murine Retnlg and human resistin (RETN) display a greater degree of similarity than retn and Regulatory Peptides 152 (2009) 41–47 ⁎ Corresponding author. Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK. Tel.: +44 141 331 3722; fax: +44 141 331 3208. E-mail address: Ann.Graham@gcal.ac.uk (A. Graham). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.07.008 Contents lists available at ScienceDirect Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep