Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability

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.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Robo4 is a vascular-specific receptor that inhibits endothelial migration

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.     

Magic roundabout, a tumor endothelial marker: expression and signaling

Molecular signals that guide blood vessels to specific paths are not fully deciphered, but are thought to be similar to signals that mediate neuronal guidance. These cues are not only critical for normal blood vessel development, but may also play a major role in tumor angiogenesis. In this study, we have demonstrated the tumor endothelial specific expression of a Robo family member, magic roundabout (MRB), functionally characterized its role in endothelial cell migration and defined a signaling pathway that might mediate this function. We show that MRB is differentially over-expressed in tumor endothelial cells versus normal adult endothelial cells in numerous solid tumors. Moreover, over-expression of MRB in endothelial cells activates MRB in a ligand-independent fashion, and activation of MRB via Slit2, a putative ligand, results in inhibition of VEGF and FGF induced migration. We also demonstrate that MRB induced inhibition of endothelial migration is partially mediated by the Ras-Raf-Mek-Erk signaling pathway. We therefore hypothesize that expression of MRB is involved in regulating the migration of endothelial cells during tumor angiogenesis.     

Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling

abstract

The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.     

Robo4

During embryogenesis, neural and vascular networks undergo directed patterning, often following the same routes. Several guidance mechanisms, which evolved in order to connect the nervous system, have been adopted by blood vessels. One such system, Slit-Roundabout signaling was first identified by studying axonal growth cones. The identification of an endothelial-specific Roundabout family member, Robo4, led Jones and colleagues to an a priori hypothesis proposing that Robo4 plays a role in regulating vascular patterning. However, they were able to revise this presumptive hypothesis by identifying a role for Robo4 in maintaining vascular stability.     

Methylation of CLEC14A is associated with its expression and lung adenocarcinoma progression

Our main objective is probing the effect of methylation of CLEC14A on its expression and lung adenocarcinoma (LUAD) progression. Microarray analysis was utilized to screen out differentially downregulated genes with hypermethylation in LUAD tissues. The CLEC14A expression level was measured by western blot analysis and qRT-PCR. Methylation-specific-PCR was performed to evaluate methylation status of CLEC14A. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid (MTT) assay was used to check the relation between CLEC14A expression and cell proliferation. Cell cycle, cell apoptosis, migration, and invasion were respectively detected by the flow cytometry assay, wound healing assay, and transwell assay. Tumor xenograft models were established for investigating the effect of CLEC14A on tumor formation. CLEC14A expression in LUAD tissues was impaired compared with that in adjacent tissues, and CLEC14A promoter was highly methylated in LUAD. Overexpressing CLEC14A or inhibiting the methylation level of CLEC14A in A549 and LTEP-a-2 cells impeded the duplication of LUAD cells, promoted apoptosis, attenuated cell migration, and invasion ability, and arrested cell cycle at the G0/G1 phase. Overexpression of CLEC14A inhibited tumorigenesis of LUAD cells in nude mice. The promoter of CLEC14A is methylated in LUAD, leading to downregulation of CLEC14A in LUAD. CLEC14A acts as an antitumor role in LUAD by suppressing cell proliferation, migration, invasion, promoting cell apoptosis, and reducing tumorigenicity in nude mice. Thus, the inhibition of CLEC14A methylation is a novel strategy for the clinic treatment of LUAD.     

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

Background

Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro.

Results

Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells.

Conclusion

This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.     

Slit-Robo signaling mediates lymphangiogenesis and promotes tumor lymphatic metastasis

The Slit family of guidance cues binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit-Robo signaling had been reported to function as chemoattractive signal for vascular endothelial cells during angiogenesis. In this study, we found that Robo1 was expressed in lymphatic endothelial cells to mediate the migration and tube formation of these cells upon Slit2 stimulation, which were specifically inhibited by the function-blocking antibody R5 to Slit2/Robo1 interaction. To further explore the lymphangiogenic effect and significance mediated by Slit-Robo signaling, we intercrossed Slit2 transgenic mice with a non-metastatic RIP1-Tag2 mouse tumor model, and found that transgenic overexpression of Slit2 significantly enhanced tumor lymphangiogenesis and subsequently promoted mesenteric lymph node metastasis of pancreatic islet tumors. Taken together, our findings reveal that through interacting with Robo1, Slit2 is a novel and potent lymphangiogenic factor and contributes to tumor lymphatic metastasis.     

ROBO4 与缺血性脑血管病相关的研究进展

Roundabout(Robo)蛋白是神经轴突导向分子家族Slit蛋白的单次跨膜受体,属于一种神经细胞粘附分子。Robo蛋白在神经系统已被确认具有重要轴突导向功能。近年来研究发现,血管新生的内皮细胞表面只特异性地表达Robo4,且Robo4对内皮细胞迁移、病理性血管生成和血管完整性都具有调节作用。缺血性脑血管病是人类致残甚至死亡的主要疾病之一,由于短暂或持续的脑血流减少而造成脑细胞损伤,因此,恢复脑血流、促进血管再生对脑功能恢复至关重要。Robo4对血管方面的作用为我们进一步研究及了解其在血管生成中的机制提供重要依据,也为缺血性脑血管病的治疗提供新的发展方向。     

Endothelial Cell Surface Expressed Chemotaxis and Apoptosis Regulator (ECSCR) Regulates Lipolysis in White Adipocytes via the PTEN/AKT Signaling Pathway

Elevated plasma triglycerides are associated with increased susceptibility to heart disease and stroke, but the mechanisms behind this relationship are unclear. A clearer understanding of gene products which influence plasma triglycerides might help identify new therapeutic targets for these diseases. The Endothelial Cell Surface expressed Chemotaxis and apoptosis Regulator (ECSCR) was initially studied as an endothelial cell marker, but has recently been identified in white adipocytes, the primary storage cell type for triglycerides. Here we confirm ECSCR expression in white adipocytes and show that Ecscr knockout mice show elevated fasting plasma triglycerides. At a cellular level, cultured 3T3-L1 adipocytes silenced for Ecscr show a blunted Akt phosphorylation response. Additionally we show that the phosphatase and tensin homology containing (PTEN) lipid phosphatase association with ECSCR is increased by insulin stimulation. These data suggest a scenario by which ECSCR contributes to control of white adipocyte lipolysis. In this scenario, white adipocytes lacking Ecscr display elevated PTEN activity, thereby reducing AKT activation and impairing insulin-mediated suppression of lipolysis. Collectively, these results suggest that ECSCR plays a critical function in regulating lipolysis in white adipose tissue.     

The stability evaluation of mesenchymal stem cells differentiation toward endothelial cells by chemical and mechanical stimulation

Adipose-derived mesenchymal stem cells (ADSCs) are adult multipotent cells able to differentiate into several cell lineages. Vascular endothelial growth factor (VEGF) and the shear stress associated with blood flow are considered as the most important chemical and mechanical cues that play major roles in endothelial differentiation. However, the stability of endothelial-specific gene expression has not been completely addressed yet. ADSCs in passage 3 were cultured inside the tubular silicon tubes and then exposed to VEGF or shear stress produced in a perfusion bioreactor. To investigate the differentiation, the expression levels of Flk-1, von Willebrand factor (vWF), and vascular endothelial-cadherin (VE-cadherin) were studied using Real-Time PCR. For studying the endothelial differentiation stability, mRNA levels of the genes were evaluated in certain time intervals after completion of the tests so as to determine whether the expression level of each gene in different time points was stable and remained constant or not. Application of VEGF and shear stress caused an elevation in endothelial cells’ specific genes. Although there are some changes following the days after application of mechanical and chemical stimuli, the gene expression results depicted significantly higher gene expression between sequential chemically and mechanically incited groups. In conclusion, stress alone can be a differentiating factor, by itself. Our results verified the efficient stable differentiation ability of the chemical and mechanical factors.     

Robo4 Plays a Role in Bone Marrow Homing and Mobilization,but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4^−/− mice. Moreover, Robo4^−/− HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4^−/− HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.     

Signalling pathways in vascular endothelium activated by shear stress: relevance to atherosclerosis

Major advances in our understanding of how endothelial cells sense and respond to haemodynamic forces and, more specifically, to fluid shear stress have been achieved during the past 3 years. These include definition of potential shear stress receptors and multiple signalling pathways that mediate shear stress regulation of gene expression. A few studies have also pointed to the unique effects of complex shear stress on endothelial activation, thus leading to better understanding of the mechanisms that lead to the development of atherosclerosis.     

Trophoblast migration under flow is regulated by endothelial cells

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.     

Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion.     

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.