The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures 1 . The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system2 , 3; however, their role in the mammalian

METHODS Migration AssaysMigration assays were performed as described (3)(4)(5). Briefly, 16 h before the assay, 80% confluent 75 cm 2 flasks (Corning Costar) of human microvessel endothelial cells (HMVEC; Cambrex, Walkersville, MD), human coronary artery endothelial cells (HCAEC; Cambrex), human umbilical artery endothelial cells (HUAEC; Promocell, Heidelburg, Germany), or human umbilical vein endothelial cells (HUVEC; Promocell), were washed with Hank's Balanced Salt Solution (HBSS, Invitrogen) and serum-starved overnight in endothelial basal media (EBM-2, Cambrex) with 0.1% fatty-acid-free BSA (Sigma) and 0.5% fetal calf serum (FCS, Hyclone). The following day cells were lifted with Trypsin/EDTA solution (Promocell), mixed with an equal volume Trypsin Neutralization Solution (Promocell), and washed 3 times in migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS). Cells were resuspended at a density of 1.5×10 6 cells/ml and were allowed to recover for 1 h at 37°C (5% CO 2 ). 3.75 × 10 4 cells were plated into each well of a 48-well Boyden chamber apparatus (NeuroProbe, Cabin John, MD), and the wells were overlayed with an 8 μm pore polycarbonate membrane (NeuroProbe) that had been previously coated with 50 μg/ml human fibronectin (Biomedical Technologies, Inc., Stoughton, MA). Experiments performed with membranes coated with acetylated 1% gelatin from porcine skin (Sigma, St. Louis, MO) gave similar results. The apparatus was assembled and stored inverted at 37°C (5% CO 2 ) for 2 h. The apparatus was then re-inverted and 52 μl of purified chemoattractants [murine netrin-1 (R&D Systems, Minneapolis, MN), chicken netrin-2 (R&D Systems), murine netrin-4 (R&D Systems), murine netrin-G1a (R&D Systems), human VEGF 165 (R&D Systems), or control/ migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS) were added to the upper chambers, and the migration was allowed to proceed for 2 h at 37°C (5% CO 2 ). The membranes were then removed, fixed in methanol, stained with a Hema 3 stain set (Fisher Scientific, Pittsburgh, PA), and placed (migrated-side down) onto 50 × 75 mm glass slides. Before 90% mounting medium (in xylenes) and coverslips were applied, the non-migrated cells were removed from the exposed (non-migrated) side of the membrane with a moistened swab. Cells present on the migrated side of the membrane were manually counted (three random 200× fields per well), and data points for each experiment represent the average number of migrated cells from six separate wells (three 200× fields counted per well).Another method was employed in a separate laboratory to evaluate the effects of the netrins on mouse (MS1) endothelial cells (ATCC, Manassas, VA) using a modified Boyden chamber assay as described previously (6). Briefly, a 5 μm-polycarbonate filter (Poretics) was placed between upper and lower chamber. Cell suspensions (5×10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with serum-free medium containing

Fibroblast growth factors play important roles in angiogenesis, but their functions in lymphangiogenesis remain poorly understood. The homeodomain transcription factor Prox1 is essential for development of the lymphatic system by specifying lymphatic endothelial cell (LEC) fate. Here, we identify fibroblast growth factor (FGF) receptor (FGFR)-3 as a novel Prox1 target gene. Ectopic overexpression of Prox1 in blood vascular endothelial cells up-regulates FGFR-3. Prox1 induces the expression of the IIIc isoform, which we also found to be the major isoform of FGFR-3 expressed in LECs. This transcriptional activation is mediated by a direct binding of Prox1 to newly identified Prox1-response elements in the FGFR-3 promoter. Consistently, FGFR-3 is up-regulated in Prox1-positive newly formed lymphatic vessels during embryogenesis and its lymphatic-specific expression is maintained throughout development. We also found that FGF-1 and FGF-2 promote proliferation, migration, and survival of cultured LECs without involvement of vascular endothelial cell growth factor receptor-3. We show that FGF-2 binds to low-and high-affinity receptors on LECs and is efficiently internalized and processed. Moreover, functional inhibition of FGFR-3 using small interfering RNA represses LEC proliferation. Together, these results indicate that FGFR-3 is an initial target of Prox1 during the lymphatic cell fate specification and that FGF signaling may play an important role in lymphatic vessel development.

IntroductionThe lymphatic and blood vascular systems share many structural similarities, but with distinct functions. The lymphatic system is an open-ended network of endothelial cell-lined vessels working to maintain fluid homeostasis by unidirectionally transporting tissue fluid, extravasated plasma proteins, lipids, and cells from the interstitial space to the circulatory system via the thoracic duct. The lymphatic system has also been demonstrated to be a route for tumor metastasis. 1 A vast number of lymphangiogenic factors, some previously identified as regulators of blood vascular endothelium, 2 have been shown to induce physiologic and/or tumor lymphangiogenesis and tumor spreading. 1 Netrins are laminin-like secreted proteins, initially identified as axonal guidance molecules. 3 In mammals, the netrin family includes 5 ligands that act through 6 putative receptors, including deleted in colorectal cancer (DCC), neogenin, and the members of the Unc5 subfamily. 3 Like Netrin-1, Netrin-4 promotes neurite outgrowth 4 and regulates blood endothelial cell biology both positively and negatively. [5][6][7] Nothing is known currently about the roles of Netrins in the lymphatic vasculature.Here, we provide evidence that Netrin-4 functions as a pro-lymphangiogenic factor. We show that Netrin-4 induces proliferation, migration and survival of lymphatic endothelial cells through activation of p42/p44 MAPkinase, Akt/PI3kinase and mTor signaling pathways. We demonstrate that neogenin and Unc5b are expressed by lymphatic endothelial cells, yet their silencing does not suppress Netrin-4-induced biologic effects. Moreover, overexpression of Netrin-4 in mouse skin or in human breast tumors increases the density of lymphatics.Finally, we show that mice bearing Netrin-4-overexpressing tumors develop more metastases by an increased lymphatic permeability. Taken together, the data demonstrate that Netrin-4 functions as a pro-lymphangiogenic factor. MethodsRefer to supplemental Methods for details (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Animals and in vivo experimentsAnimal experiments were conducted with approval from the University of Utah Institutional Animal Care and Use Committee. In vivo experiments were performed as described 8,9 and in supplemental Methods. Cell culture and in vitro assaysLymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum. Human MCF7 breast cancer cells were a kind gift of Dr Alex Swarbrick (Garvan Institute, Australia) and were grown according to the ATCC recommendations. Mouse 66C14 mammary carcinoma line was provided by Dr Gary Sahagian (Tufts University). In vitro assays were performed as described 7,10 and in supplemental Methods. The online version of this ar...

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Development and stabilization of a vascular plexus requires the coordination of multiple signalling processes. Wnt planar cell polarity (PCP) signalling is critical in vertebrates for diverse morphogenesis events, which coordinate cell orientation within a tissue-specific plane. However, its functional role in vascular morphogenesis is not well understood. Here we identify PDZRN3, an ubiquitin ligase, and report that Pdzrn3 deficiency impairs embryonic angiogenic remodelling and postnatal retinal vascular patterning, with a loss of two-dimensional polarized orientation of the intermediate retinal plexus. Using in vitro and ex vivo Pdzrn3 loss-of-function and gain-of-function experiments, we demonstrate a key role of PDZRN3 in endothelial cell directional and coordinated extension. PDZRN3 ubiquitinates Dishevelled 3 (Dvl3), to promote endocytosis of the Frizzled/Dvl3 complex, for PCP signal transduction. These results highlight the role of PDZRN3 to direct Wnt PCP signalling, and broadly implicate this pathway in the planar orientation and highly branched organization of vascular plexuses.

Rationale: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves. Objective: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia. Methods and Results: We induced hindlimb ischemia in wild-type and Dhh –/– mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh –/– . By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle. Conclusions: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.

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