Inhibitory effects of recombinant RTS-jerdostatin on integrin a1b1 function during adhesion, migration and proliferation of rat aortic smooth muscle cells and angiogenesis Gema Bolás a , Flávia Figueiredo de Rezende b , Carolina Lorente a , Libia Sanz a , Johannes A. Eble b , Juan J. Calvete a, * a Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain b Institute for Physiological Chemistry and Pathobiochemistry, University of Muenster, Waldeyerstr.15, 48149 Muenster, Germany article info Article history: Received 17 September 2013 Received in revised form 22 November 2013 Accepted 18 December 2013 Available online 11 January 2014 Keywords: Recombinant RTS disintegrin Jerdostatin a1b1 integrin inhibitor Cell adhesion Angiogenesis abstract Jerdostatin, a short RTS-disintegrin cloned from venom gland mRNA of Protobothrops jer- donii, selectively blocks the adhesion of a1b1 integrin to collagen IV. Integrin a1b1 is highly expressed in smooth muscle cells (SMC) surrounding small blood vessels and vascular endothelial cells. Vascular SMC adhesion, migration and proliferation are important pro- cesses during normal vascular development. Using recombinant jerdostatin we have investigated the role of the a1b1 integrin on the adhesion of vascular SMC to collagen IV, and the potential relevance of blocking this crucial component of focal adhesions as an anti-angiogenic strategy. Our results show that jerdostatin does not interact with canonical collagen-binding site on the isolated A-domain of the a1 integrin subunit. r-Jerdostatin inhibited the adhesion of RASMCs to immobilized CB3 fragment in a dose-dependent manner, triggering to round-up, retraction, and finally detachment of the cells. r-Jerdos- tatin did not affect the adhesion of human SMCs to CB3, presumably because the high expression of a2b1 integrin compensated for a1b1 integrin blockage by jerdostatin. r- Jerdostatin dose-dependently inhibited a1b1 integrin-dependent HUVEC tube formation. However, VEGF-driven tube formation in the matrigel assay was only completely abolished when binding of integrin a2b1 to collagen was also inhibited by the C-type lectin-like rhodocetin. As a whole, our work emphasizes the relevance of using specific inhibitors for dissecting the role of a1b1 integrin in physiological and pathological conditions. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Venoms of Viperidae snakes contain b1 and b3 integrin antagonists, the disintegrins, that represent a family of small (40–84 amino acids), cysteine-rich polypeptides (Calvete, 2010, 2013). The proper pairing of cysteines determines the conformation of a mobile loop, which protrudes 14–17 Å from the globular protein core and which harbors an integrin-recognition tripeptide motif at its apex. The active loop and the C-terminal tail exhibit concerted motions and form a conformational epitope involved in extensive in- teractions with both subunits of integrin receptors that ac- count for the specificity and selectivity of disintegrins towards b1 and b3 integrin receptors (Monleon et al., 2003; Calvete, 2010; Carbajo et al., 2011). Short (R/K)TS-dis- integrins that selectively target the a1b1 integrin (Calvete et al., 2007), such as obtustatin (Marcinkiewicz et al., 2003; Brown et al., 2008), viperistatin (Staniszewska et al., 2009; Momic et al., 2011), lebestatin (Olfa et al., 2005) and jerdostatin (Sanz et al., 2005), cluster into a distinct clade within the disintegrin family (Sanz-Soler et al., 2012). * Corresponding author. Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. Tel.: þ34 96 339 1778; fax: þ34 96 369 0800. E-mail address: jcalvete@ibv.csic.es (J.J. Calvete). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.12.006 Toxicon 79 (2014) 45–54