Focal adhesion kinase regulation of neovascularization

Abstract

In this review, we discuss the role of focal adhesion kinase (FAK), an intracellular tyrosine kinase, in endothelial cells in relation to neovascularization. Genetic and in vitro studies have identified critical factors, receptor systems, and their intracellular signaling components that regulate the neovasculogenic phenotypes of endothelial cells. Among these factors, FAK appears to regulate several aspects of endothelial cellular behavior, including migration, survival, cytoskeletal organization, as well as cell proliferation. Upon adhesion of endothelial cells to extracellular matrix (ECM) ligands, integrins cluster on the plane of plasma-membrane, while cytoplasmic domains of integrins interact with cytoskeletal proteins and signaling molecules including FAK. However, FAK not only serves as a critical component of integrin signaling, but is also a downstream element of the VEGF/VEGF–receptor and other ligand–receptor systems that regulate neovascularization. A complete understanding of FAK-mediated neovascularization, therefore, should address the molecular and cellular mechanisms that regulate the biology of FAK. Continued research on FAK may, therefore, yield novel therapies to improve treatment modalities for the pathological neovascularization associated with diseases.

Introduction

In mammals, the growth of blood vessels occurs mainly by vasculogenesis, angiogenesis, and lymphangiogenesis, together can be termed as neovascularization (Sabin, 1917, Sabine, 1920, Ausprunk and Folkman, 1997, Risau, 1997, Hanahan and Folkman, 1996). Neovascularization is a fundamental biological process that plays a critical role during the development and growth of an organism, and it is required for wound healing and tissue repair in adults (Ausprunk and Folkman, 1997, Hanahan and Folkman, 1996, Hanahan and Weinberg, 2000, Veikkola et al., 2000, Carmeliet, 2003). Angiogenesis is defined as the branching and spreading out of capillaries that are formed from a preformed vessels, primarily through the migration, recruitment, interconnection and lumenization of endothelial cells (Lubarsky and Krasnow, 2003, Kamei et al., 2006, Iruela-Arispe and Davis, 2009). Nevertheless, unwanted angiogenesis has been associated with the expansion of atherosclerotic lesions, diabetic retinopathy, psoriasis, and tumor progression (Wary, 2004, Kalluri et al., 2003, Carmeliet, 2003, Renault and Losordo, 2007, Adams and Alitalo, 2007). On the other hand, ischemic and cardiovascular diseases have been associated with insufficient angiogenesis (Renault and Losordo, 2007, Adams and Alitalo, 2007, Becker and D'Amato, 2007, Fukumura and Jain, 2007, Aird, 2008).

The vascular tree develops early in embryogenesis to nourish and provide oxygen to the developing organism (Risau, 1997, Watson and Cross, 2005, Robb and Elefanty, 1998, Hickey and Simon, 2006). In the mouse at embryonic day 7.5 (E7.5), the extraembryonic mesodermal cells of the yolk sac aggregate into clusters, representing the initial stage of blood island formation and hemoglobin accumulation (Sabine, 1920, Robb and Elefanty, 1998, Hickey and Simon, 2006, Palis et al., 1995, Gerber et al., 2002, Rossant and Hirashima, 2003, Coultas et al., 2005, Hasegawa et al., 2007). Soon after this stage, blood islands differentiate into an external layer of endothelial cells and an inner core of blood cells (Sabine, 1920, Risau, 1997). Simultaneously, embryonic cells of the proximal lateral mesoderm assemble into pre-endocardial tubes that connect anteriorly to generate cardiac endocardium around the anterior intestinal portal (Palis et al., 1995, Gerber et al., 2002, Rossant and Hirashima, 2003, Coultas et al., 2005). Posteriorly, angioblasts organize into paired dorsal aortas, which later assemble in the midline to form a single tube. Correspondingly, mesodermal cells in the allantois generate cords that produce the umbilical blood vessels (Gerber et al., 2002, Rossant and Hirashima, 2003, Coultas et al., 2005, Hasegawa et al., 2007). The allantois develops rapidly inside the exocoelomic cavity and fuses with the chorionic extraembryonic mesoderm (Palis et al., 1995, Gerber et al., 2002, Rossant and Hirashima, 2003, Coultas et al., 2005, Hasegawa et al., 2007). This fusion results in a signal that initiates the differentiation of the chorionic vasculature, which indirectly connects to the maternal vascular system of the placenta. These early events are described as “vasculogenesis”, which signifies the de novo formation of blood vessels from angiogenic precursor cells. In the mouse, vasculogenesis is usually complete by E7.5 days. Later in embryogenesis, the vascular tree grows by the sprouting, cell division, migration and assembly of endothelial cells derived from pre-existing vessels through a process termed angiogenesis. At the cellular level, Brachyury acts as a switch to induce formation of the mesodermal lineage. Mesodermally derived mesenchymal stem cells give rise to hemangioblasts (Fig. 1). Hemangioblasts are bipotential stem cells, which are characterized as CD133+/CD34+ and fetal liver kinase-1 (Flk1+) cells that can give rise to hematopoietic cells (HSCs) and angioblasts. The vascular tree grows by sprouting endothelial cells derived from angioblasts. Concurrent with VEGFR activation, cell–cell and cell–matrix interactions provide positional information related to when endothelial cells will elongate, interconnect, migrate, divide, and assemble into tube-like structures (Risau, 1997, Rossant and Hirashima, 2003, Coultas et al., 2005, Hasegawa et al., 2007). More recently, a subset of VE-cadherin+ and Runx-1+ endothelial cells, derived from dorsal aorta have been termed as “hemogenic endothelial cells”. Hemogenic endothelial cells originating from dorsal aorta between E8.5 and E11.5 days can give rise to hematopoietic cells that directly home into the liver (Chen et al., 2009, Swiers et al., 2010). Nevertheless, productive neovascularization requires not only endothelial cells, but also hematopoietic cells and the recruitment of pericytes and smooth muscle cells.

In vitro transgenic and knockout studies have provided evidence that vascular endothelial growth factor (VEGF) and the VEGF receptor-2 system (also known as fetal liver kinase (Flk-1) and kinase domain receptor (KDR)) play fundamental roles in the formation of blood vessels (Shima et al., 1996, Shalaby et al., 1997, Park et al., 2004). Flk-1 is one of earliest markers of endothelial cells and is first detected during mouse development in the extra-embryonic mesoderm of the yolk sac, where the blood islands form shortly after gastrulation, around day 7 postcoitum (Shima et al., 1996, Shalaby et al., 1997, Park et al., 2004, Lugus et al., 2009, Hellström et al., 2007). In the extraembryonic mesoderm, flk-1 appears shortly after gastrulation, followed sequentially by tie-2 and tie-1 over the next 24 h. In addition, flk-1 is initially observed in the extraembryonic mesoderm prior to its expression in the embryonic mesoderm (Shima et al., 1996, Shalaby et al., 1997, Park et al., 2004). Activation of the VEGFR-2/Flk1 receptor promotes endothelial elongation, motility and proliferation. Specifically, the activation of endothelial cells by VEGF induces expression of Delta like ligand-4 (Dll4) by one of the endothelial cells, which is referred to as the “tip cell”. Tip cells display a highly motile phenotype. Cell surface associated Dll4 expressed by the tip cell then ligate Notch receptor on the adjacent endothelial cell to induce intracellular signaling to activate the Hes and Hey transcriptional target genes (Siekmann and Lawson, 2007, Hofmann and Iruela-Arispe, 2007, Nakatsu et al., 2003, Benedito et al., 2009, Kume, 2009, Jakobsson et al., 2010). Activation of Hes and Hey promotes cell proliferation and these subsets of proliferating cells are termed as the “stalk cells” (Siekmann and Lawson, 2007, Hofmann and Iruela-Arispe, 2007, Nakatsu et al., 2003, Benedito et al., 2009, Kume, 2009, Jakobsson et al., 2010). Proliferative cells at some point receive a cell cycle “arrest” signal which induces morphogenic differentiation, lumenization and tube formation (Hofmann and Iruela-Arispe, 2007, Nakatsu et al., 2003, Benedito et al., 2009, Kume, 2009, Iruela-Arispe and Davis, 2009, Jakobsson et al., 2010).

All of these processes may involve FAK signaling, either directly or indirectly, in the context of key molecules and pathways controlling cell migration, VEGF production, cell proliferation and the formation of new vessels. Below, we review and discuss the structure, expression, and FAK regulation of vasculogenesis and angiogenesis in developmental settings and under pathological conditions.