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A common haplotype on 10q26 influences the risk of age-related macular degeneration (AMD) and encompasses two genes, LOC387715 and HTRA1. Recent data have suggested that loss of LOC387715, mediated by an insertion/deletion (in/del) that destabilizes its message, is causally related with the disorder. Here we show that loss of LOC387715 is insufficient to explain AMD susceptibility, since a nonsense mutation (R38X) in this gene that leads to loss of its message resides in a protective haplotype. At the same time, the common disease haplotype tagged by the in/del and rs11200638 has an effect on the transcriptional upregulation of the adjacent gene, HTRA1. These data implicate increased HTRA1 expression in the pathogenesis of AMD and highlight the importance of exploring multiple functional consequences of alleles in haplotypes that confer susceptibility to complex traits., Competing Interests: The authors have declared that no competing interests exist.

Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. [ABSTRACT FROM AUTHOR]

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165–induced migration, tube formation and permeability in vitro and VEGF-165–stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak. [ABSTRACT FROM AUTHOR]

Nerves and blood vessels often follow parallel trajectories as they course through the body to their distal targets. Proteins that regulate the process of axon guidance have likewise been shown to play a crucial role in blood vessel migration. With the recent description of the endothelial tip cell as an analog of the axonal growth cone, the nerve–vessel analogy seems complete. Notwithstanding these considerable similarities, one crucial difference remains between neural and vascular guidance. While a navigating axon is but a single cell, a sprouting vessel is composed of multiple cells that must be co-ordinately regulated. Recent studies of the Dll4-Notch1 signaling pathway have provided valuable insight into how the vasculature accomplishes this crucial task. [Copyright &y& Elsevier]

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity. [ABSTRACT FROM AUTHOR]

Axonal guidance and vascular patterning share several guidance cues, including proteins in the netrin family. We demonstrate that netrins stimulate proliferation, migration, and tube formation of human endothelial cells in vitro and that this stimulation is independent of known netrin receptors. Suppression of netrinla messenger RNA in zebrafish inhibits vascular sprouting, implying a proangiogenic role for netrins during vertebrate development. We also show that netrins accelerate neovascularization in an in vivo model of ischemia and that they reverse neuropathy and vasculopathy in a diabetic murine model. We propose that the attractive vascular and neural guidance functions of netrins offer a unique therapeutic potential. [ABSTRACT FROM AUTHOR]

Supravalvular aortic stenosis is an autosomal-dominant disease of elastin (Eln) insufficiency caused by loss-of-function mutations or gene deletion. Recently, we have modeled this disease in mice (Eln[SUP+/-]) and found that Eln haploinsufficiency results in unexpected changes in cardiovascular hemodynamics and arterial wall structure. Eln[SUP+/-] animals were found to be stably hypertensive from birth, with a mean arterial pressure 20-30 mmHg higher than their wild-type counterparts. The animals have only moderate cardiac hypertrophy and live a normal life span with no overt signs of degenerative vascular disease. Examination of arterial mechanical properties showed that the inner diameters of Eln[SUP+/-] arteries were generally smaller than wild-type arteries at any given intravascular pressure. Because the Eln[SUP+/-] mouse is hypertensive, however, the effective arterial working diameter is comparable to that of the normotensive wild-type animal. Physiological studies indicate a role for the reninangiotensin system in maintaining the hypertensive state. The association of hypertension with elastin haploinsufficiency in humans and mice strongly suggests that elastin and other proteins of the elastic fiber should be considered as causal genes for essential hypertension. [ABSTRACT FROM AUTHOR]

A hallmark of vascular smooth muscle cells (VSMCs) is their dynamic ability to assemble and disassemble contractile proteins into sarcomeric units depending upon their phenotypic state. This phenotypic plasticity plays an important role during vascular development and in obstructive vascular disease. Previously, we showed that the Elastin gene product, tropoelastin, activates myofibrillar organization of VSMCs. Recently, others have suggested that elastin does not have a direct signaling role but rather binds to and alters the interactions of other matrix proteins with their cognate receptors or disrupts the binding of growth factors and cytokines. In contrast, we provide evidence that tropoelastin directly regulates contractile organization of VSMCs. First, we show that a discrete domain within tropoelastin, VGVAPG, induces myofibrillogenesis in a time- and dose-dependent fashion. We confirm specificity using a closely related control peptide that fails to stimulate actin stress fiber formation. Second, the activity of VGVAPG is not affected by the presence or absence of other serum or matrix components. Third, both the elastin hexapeptide and tropoelastin stimulate actin polymerization through a common pertussis toxin-sensitive G protein pathway that activates RhoA-GTPase and results in the conversion of G to F actin. Collectively, these data support a model whereby the elastin gene product, signaling through the VGVAPG domain, directly induces VSMC myofibrillogenesis. [Copyright &y& Elsevier]

Guidance and patterning of axons are orchestrated by cell-surface receptors and ligands that provide directional cues. Interactions between the Robo receptor and Slit ligand families of proteins initiate signaling cascades that repel axonal outgrowth. Although the vascular and nervous systems grow as parallel networks, the mechanisms by which the vascular endothelial cells are guided to their appropriate positions remain obscure. We have identified a putative Robo homologue, Robo4, based on its differential expression in mutant mice with defects in vascular sprouting. In contrast to known neuronal Robo family members, the arrangement of the extracellular domains of Robo4 diverges significantly from that of all other Robo family members. Moreover, Robo4 is specifically expressed in the vascular endothelium during murine embryonic development. We show that Robo4 binds Slit and inhibits cellular migration in a heterologous expression system, analogous to the role of known Robo receptors in the nervous system. Immunoprecipitation studies indicate that Robo4 binds to Mena, a known effector of Robo-Slit signaling. Finally, we show that Robo4 is the only Robo family member expressed in primary endothelial cells and that application of Slit inhibits their migration. These data demonstrate that Robo4 is a bona fide member of the Robo family and may provide a repulsive cue to migrating endothelial cells during vascular development. [Copyright &y& Elsevier]