In the present study our data generated from

In the present study, our data generated from the biochemical assays (Fig. 7) and functional studies (Figs. 2–5) indicated that the C-terminal PDZ motif of CXCR2 is important for EPC motility and angiogenic activity both in vitro and in vivo. Our findings suggested that a PDZ-based CXCR2 macromolecular complex might regulate EPC function during postnatal neovascularization (Fig. 5) as well as its incorporation into endothelial tubule structure (Fig. 4). Growing evidence suggested that PDZ scaffold/adaptor proteins, such as NHERF1, NHERF2, and PDZK1, nucleate the formation of compartmentalized multiprotein complexes that are critical to specific cellular signaling (Li et al., 2005, 2007, 2010; Naren et al., 2003). These PDZ adaptor proteins provide scaffolds for localization and organization of membrane receptors or ion channels and downstream effectors to be close to each other, leading to efficient signaling transduction. It was previously reported that EPC adhesion to activated endothelium was selectively inhibited by the antiserum directed against the PDZ motif of a cell adhesion molecule, shCD146, suggesting that this PDZ motif is involved in the EPC adhesion process (Kebir et al., 2010). We have recently shown that the NHERF1-scaffolded CXCR2–PLC-β complexes regulate CXCR2 signaling in neutrophils and pancreatic cancer N6022 (Wu et al., 2012; Wang et al., 2013). We also demonstrated the direct interaction between NHERF1 and CXCR2 as well as NHERF1 and PLC-β3 in the crystal structure studies (Lu et al., 2013; Jiang et al., 2013, 2014). It is therefore conceivable that CXCR2 could also mediate potential interaction with certain PDZ scaffold proteins in EPCs. This interaction might consequently cluster CXCR2 with other relevant signaling molecules into multiprotein macromolecular signaling complexes. It is important to note that the present study suggested the presence of an NHERF1-mediated CXCR2–PLC-β3 complex in EPCs (Fig. 7), implying a functional significance of this complex in EPC mobilization, homing, and incorporation into neovasculature. Finally, the present study suggested that targeting the PDZ-based CXCR2 complex might be an effective strategy for therapeutic intervention of angiogenesis-dependent diseases. EPCs participate in pathologic angiogenesis and contribute to tumor growth and airway remodeling in asthma (Jones et al., 2009; Imaoka et al., 2011; Goon et al., 2006; Yeager et al., 2011). Inhibiting EPC angiogenic activity might thus represent a promising approach to restrict vessel growth in those pathological conditions. We showed that blocking the PDZ-mediated CXCR2–NHERF1 interaction is sufficient to inhibit EPC angiogenic activity resulting in significantly reduced vessel density in the mouse Matrigel plug assay (Fig. 5). This result suggested that targeting this PDZ interaction might have therapeutic applications in the EPC-associated diseases. Recent findings supported the idea that PDZ domains might indeed be valuable drug targets. Blocking the PDZ interaction between the NMDA glutamate receptor and PSD-95 has been shown to result in selective inhibition of neuronal nitric oxide synthase activation, which is expected to reduce ischemic brain injury during stroke (Aarts et al., 2002). Recent evidence suggested that blocking the PDZ domains of NHERF1 might be an interesting therapeutic approach to inhibit tumorigenesis associated with breast cancer (Dai et al., 2004). In addition, the PDZ domain of PICK1, which binds the C terminus of AMPA-type ionotropic glutamate receptors, has recently been recognized as a putative target in the treatment of neuropathic pain, excitotoxicity, and cocaine addiction (Bell et al., 2009; Bellone and Luscher, 2006). Therefore, targeting the PDZ-based CXCR2 complex in EPCs could prove valuable in therapeutic intervention of pathologic angiogenesis (such as tumor angiogenesis).
Acknowledgments