Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VEcadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.

Background-The ␣ v ␤ 3 integrin plays a critical role in cell proliferation and migration. We hypothesized that vascular cell proliferation, a hallmark of injury-induced remodeling, can be tracked by targeting ␣ v ␤ 3 integrin expression in vivo. Methods and Results-RP748, a novel 111 In-labeled ␣ v ␤ 3 -specific radiotracer, was evaluated for its cell-binding characteristics and ability to track injury-induced vascular proliferation in vivo. Three groups of experiments were performed. In cultured endothelial cells (ECs), TA145, a cy3-labeled homologue of RP748, localized to ␣ v ␤ 3 at focal contacts. Activation of ␣ v ␤ 3 by Mn 2ϩ led to increased EC binding of TA145. Left common carotid artery wire injury in apolipoprotein E Ϫ/Ϫ mice led to vascular wall expansion over a period of 4 weeks. RP748 (7.4 MBq) was injected into groups of 9 mice at 1, 3, or 4 weeks after left carotid injury, and carotids were harvested for autoradiography. Relative autographic intensity, defined as counts/pixel of the injured left carotid area divided by counts/pixel of the uninjured right carotid area, was higher at 1 and 3 weeks (1.8Ϯ0.1 and 1.9Ϯ0.2, respectively) and decreased significantly by 4 weeks after injury (1.4Ϯ0.1, PϽ0.05). Carotid ␣ v and ␤ 3 integrin expression was maximal at 1 week and decreased by 4 weeks after injury. The proliferation index, as determined by Ki67 staining, followed a temporal pattern similar to that of RP748 uptake. Dynamic gamma imaging demonstrated rapid renal clearance of RP748. Conclusions-RP748

Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is up-regulated in the neointima of remodeling arteries and modulates vascular smooth muscle cell (VSMC) growth. Platelet-derived growth factor (PDGF) is the prototypic growth factor for VSMCs and plays a key role in vascular remodeling. Here, we sought to further define ESDN function in primary human VSMCs. ESDN down-regulation by RNA interference significantly enhanced PDGF-induced VSMC DNA synthesis and migration. This was associated with increased ERK1/2, Src, and PDGF receptor (PDGFR)␤ phosphorylation, without altering total PDGFR␤ expression levels. In binding assays, ESDN down-regulation significantly increased 125 I-PDGF maximum binding (B max ) to PDGF receptors on VSMCs without altering the binding constant (K d ), raising the possibility that ESDN regulates PDGFR processing. ESDN down-regulation significantly reduced ligand-induced PDGFR␤ ubiquitination. This was associated with a significant reduction in the expression level of c-Cbl, an E3 ubiquitin ligase that ubiquitinylates PDGFR␤. Thus, ESDN modulates PDGF signaling in VSMCs via regulation of PDGFR surface levels. The ESDN effect is mediated, at least in part, through effects on PDGFR␤ ubiquitination. ESDN may serve as a target for regulating PDGFR␤ signaling in VSMCs.Vascular injury initiates a cascade of events that ultimately leads to vascular remodeling and often intimal hyperplasia. Vascular smooth muscle cell (VSMC) 2 proliferation and migration are key cellular events in this process. Platelet-derived growth factor (PDGF)-BB is released by platelets, endothelial cells, VSMCs, and inflammatory cells at the sites of vascular injury and is a particularly potent regulator of VSMC proliferation and migration (1). PDGF binding to PDGF receptor (PDGFR)␤ in VSMCs leads to receptor dimerization, autophosphorylation, and activation of downstream signaling pathways, including MAPK. The ligand-bound receptor is internalized through the endocytotic pathway and may either recycle to the membrane or undergo ubiquitination and lysosomal degradation (2). A number of endogenous stimulatory and inhibitory regulators, including the E3 ubiquitin ligase, c-Cbl (3), tightly regulate the mitogenic stimulus by modulating the duration and intensity of the signal.We have identified endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN, also called CLCP1 or DCBLD2) as a marker and regulator of cell proliferation in vascular remodeling (4). ESDN is a transmembrane protein with a domain structure similar to neuropilins (5, 6). ESDN can be induced by PDGF-BB and serum and is highly expressed in the neointima of injured rat (5), mouse (4), and human (4) arteries. ESDN expression parallels cell proliferation in the vessel wall in vivo (4). Furthermore, ESDN is up-regulated in proliferating VSMCs, and ESDN overexpression inhibits VSMC growth (4). Here, we expand the scope of our previous studies to demonstrate that ESDN regulates PDGF-induced VSMC migration and inhibits PDGF signa...