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

SUMMARYFibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin 51 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development.We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/ N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N-and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor ␤ (PDGFR␤) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.[Keywords: PDGF-B; angiogenesis; heparan sulfate; pericyte; vascular development] Supplemental material is available at http://www.genesdev.org. Tissue morphogenesis depends on cell-cell interactions, controlling directed cell migration proliferation, differentiation, and cell survival. Specificity is often regulated at the level of selective ligand-receptor interaction. However, the spatial distribution and local concentration of the ligand determine the range of the signal, and, as exemplified by morphogens of the hedgehog, TGF␤, and Wnt family members, also the nature of the signal. Indeed, spatial restriction defines the activities of most peptide growth factors and many secreted neural guidance molecules. In vascular development, peptide growth factors of the VEGF and platelet-derived growth factor (PDGF) families regulate the migration and proliferation of endothelial cells and supporting mural cells; i.e., pericytes (PC) and vascular smooth muscle cells (vSMC). The longitudinal migration and proliferation of vSMC/PC depend on paracrine signaling of endothelial derived PDGF-B to PDGF receptor-␤ (PDGFR␤) expressed on vSMC/PC (Lindahl et al. 1997;Hellström et al. 1999). PDGF-B is secreted as a homodimer (PDGF-BB), which signals by mediating dimerization of its receptor. Conditional inactivation of Pdgf-b in the endothelium demonstrated that endothelial cells are the

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin a4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin b1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

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The apoA-I/C-III/A-IV gene cluster, like most intestinespecific genes, displays a specific pattern of expression along the intestinal cephalocaudal and crypt-to-villus axes. We have shown that this specific pattern of expression requires the distal apoA-IV promoter and the apoC-III enhancer. Using a new set of transgenic mice, we demonstrate here that the restriction of apoA-IV gene transcription to villus enterocytes requires a hormoneresponsive element (HRE) located within the apoA-IV distal promoter. We showed, using nuclear extracts from villus or crypt epithelial cells, that this HRE bound the transcription factor hepatic nuclear factor 4 (HNF-4). We also found that the HNF-4␥ isoform was produced only in the villus, whereas the HNF-4␣ isoform was produced along the entire length of the crypt-to-villus axis. Our results demonstrate that the HRE of the distal apoA-IV promoter is responsible for the restriction of gene expression to villus epithelial cells and that this HRE binds HNF-4 isoforms. The in vivo observation of parallel gradients for apoA-IV and HNF-4␥ gene expression raises questions concerning whether this transcription factor plays a specific role in the control of enterocyte differentiation.

In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4alpha and gamma. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4alpha repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4alpha and gamma functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial-cell functionality. In-vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose-dependent decrease in both the secretion of the vasoconstrictor agent endothelin-1 (ET-1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol´L 21 ) and pharmacological noncytotoxic (1.0 and 2.0 mmol´L 21 ) concentrations. Mean percentage variation from control for ET-1 production was 236.2^18.9% for 0.5 mmol´L 21 homocysteine and 241.5^26.8% for 1.0 mmol´L 21 homocysteine, after incubation for 8 h. Mean percentage variation from control for steady-state mRNA was 217.3^7.1% for 0.5 mmol´L 21 homocysteine and 246.0^10.1 for 1.0 mmol´L 21 homocysteine, after an incubation time of 2 h. ET-1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co-incubation of cells with homocysteine and the sulfhydryl inhibitor N-ethylmaleimide prevented the effect of homocysteine on ET-1 production, confirming a sulfhydryl-dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium-dependent vasodilation by primary inhibition of ET-1 production.