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Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR., Integrin-linked kinase (ILK) is an important mediator of integrin signaling. Here Park et al. show that mice with endothelial-specific deletion of Ilk develop vascular defects that resemble familial exudative vitreoretinopathy, and identify mutations in ILK in patients with exudative vitreoretinopathy suggesting a potential role in human pathogenesis.

Bismuth vanadate (BiVO4) has been highlighted as the most efficient photoanode light absorber for the solar light-driven water oxidation reaction despite the lack of fundamental understanding of its poor performance. Though the BiVO4 performance has been significantly improved, the underlying reasons for this performance are not well understood. Here, anisotropically grown BiVO4 is designed to achieve band edge engineering induced by coupled crystal facets for hydrogen evolution reaction (HER), which is not possible with isotropic BiVO4 powder suspension. HER performances depending on the exposure area of the crystal facets in the anisotropic BiVO4 are investigated using electronic conduction and in situ X-ray absorption spectroscopy during HER. Our results reveal that the nature of the exposed area of the optimized coupled crystal facets in a BiVO4 photoelectrode critically dominates the surface charge density and conductivity, efficiently driving the HER. Sequentially, coupling between different crystal facets of BiVO4 photoelectrode led energy band edge to be upward. The results show that the energy band edge alignment tuned by coupled crystal facet can be applicable to other inorganic photocatalyst/photoelectrodes to promote efficient artificial photosynthetic reactions.

Summary Generation of induced oligodendrocyte progenitor cells (iOPCs) from somatic fibroblasts is a strategy for cell-based therapy of myelin diseases. However, iOPC generation is inefficient, and the resulting iOPCs exhibit limited expansion and differentiation competence. Here we overcome these limitations by transducing an optimized transcription factor combination into a permissive donor phenotype, the pericyte. Pericyte-derived iOPCs (PC-iOPCs) are stably expandable and functionally myelinogenic with high differentiation competence. Unexpectedly, however, we found that PC-iOPCs are metastable so that they can produce myelination-competent oligodendrocytes or revert to their original identity in a context-dependent fashion. Phenotypic reversion of PC-iOPCs is tightly linked to memory of their original transcriptome and epigenome. Phenotypic reversion can be disconnected from this donor cell memory effect, and in vivo myelination can eventually be achieved by transplantation of O4+ pre-oligodendrocytes. Our data show that donor cell source and memory can contribute to the fate and stability of directly converted cells.

Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation.

Tumor angiogenesis is essential for tumor invasive growth and metastasis, and generates abnormal vascular structures unlike developmental neovessel formation. To reduce tumor vascular abnormalities such as leakage and perivascular cell coverage deficiency that limit cancer therapy effectiveness, novel therapeutic approaches focus on vessel normalization. We have previously shown that Dickkopf-1 (DKK1), a Wnt antagonist, inhibits and its homolog DKK2 enhances, angiogenesis in normal tissues. In the present study, we investigated the effects of DKK1 and DKK2 on tumor growth and angiogenesis. Treatment of B16F10 melanoma-bearing mice with adenovirus expressing DKK1 significantly reduced tumor growth but DKK2 increased growth compared with controls. Similar pattern of tumor growth was observed in endothelial-specific DKK1 and DKK2 transgenic mice. Interestingly, tumor vascular density and perfusion were significantly decreased by DKK1 but increased by DKK2. Moreover, coverage of blood vessels by pericytes was reduced by DKK1, while DKK2 increased it. We further observed that DKK1 diminished retinal vessel density and increased avascular area in an in vivo murine model of oxygen-induced retinopathy, whereas DKK2 showed opposite results. These findings demonstrate that DKK1 and DKK2 have differential roles in normalization and functionality of tumor blood vessels, in addition to angiogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s10456-013-9390-5) contains supplementary material, which is available to authorized users.

The Wnt signaling pathway is involved in a wide range of developmental and physiological processes, such as cell fate specification, tissue morphogenesis, and homeostasis. Thus, its dysregulation has been found in multiple diseases, including some cardiovascular disorders. The loss or gain of function of Wnt pathway components results in abnormal vascular development and angiogenesis. Further study has revealed that Wnt signaling in endothelial cells appears to contribute to vascular morphogenesis and endothelial cell specification. Owing to the significance of Wnt signaling in angiogenesis, Wnt antagonists have been considered potential treatments for neovascular disorders. In line with this, members of the Dkk protein family (Dkks), well-known Wnt antagonists, have been recently found to regulate angiogenesis. This review summarizes our present knowledge of the roles of Wnt signaling and Wnt antagonists, particularly Dkks, in angiogenic regulation and explores the therapeutic potential of Wnt antagonists.

Aims Although increased levels of myocardial receptor activator of nuclear factor (NF)-κB ligand (RANKL) have been reported in heart failure, the role of this pathway in mediating activation of inflammatory pathways during myocardial remodelling is less well understood. This study sought to determine the role of myocardial RANKL in regulating cytokine expression. Methods and results A marked increase in RANKL expression occurred as early as 6h following transverse aortic constriction (TAC) in mouse hearts and persisted at 3 and 17 days. An increase in tumour necrosis factor-α (TNF-α), interleukin (IL)-1α, and IL-1β was observed in the hypertrophied hearts only at 3 or 17 days after TAC. Treatment with losartan significantly attenuated TAC-induced cardiac hypertrophy, in parallel with decreased expression of RANKL, TNF-α, IL-1α, and IL-1β. Furthermore, injection of a RANKL-neutralizing monoclonal antibody attenuated RANKL-induced cytokine expression. RANKL stimulated expression of TNF-α, IL-1α, and IL-1β in neonatal rat cardiomyocytes via activation of NF-κB. RANKL-induced NF-κB activation and expression of these cytokines were both attenuated when RANK, receptor for RANKL, or TRAF2 or TRAF6, adaptors for RANK, was silenced by siRNA. Furthermore, inhibitors of phospholipase C (PLC), protein kinase C (PKC), and inhibitor of κB kinase also significantly inhibited RANKL-induced cellular activities, but inhibitors of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase, or p38 mitogen-activated protein kinase were without effect. Conclusion Our data demonstrate for the first time that the pressure-overloaded myocardium generates RANKL, which induces TNF-α, IL-1α, and IL-1β production via a RANK–TRAF2/TRAF6–PLC–PKC–NF-κB-mediated autocrine mechanism.

Neovessel formation is a complex process governed by the orchestrated action of multiple factors that regulate EC specification and dynamics within a growing vascular tree. These factors have been widely exploited to develop therapies for angiogenesis-related diseases such as diabetic retinopathy and tumor growth and metastasis. WNT signaling has been implicated in the regulation and development of the vascular system, but the detailed mechanism of this process remains unclear. Here, we report that Dickkopf1 (DKK1) and Dickkopf2 (DKK2), originally known as WNT antagonists, play opposite functional roles in regulating angiogenesis. DKK2 induced during EC morphogenesis promoted angiogenesis in cultured human endothelial cells and in in vivo assays using mice. Its structural homolog, DKK1, suppressed angiogenesis and was repressed upon induction of morphogenesis. Importantly, local injection of DKK2 protein significantly improved tissue repair, with enhanced neovascularization in animal models of both hind limb ischemia and myocardial infarction. We further showed that DKK2 stimulated filopodial dynamics and angiogenic sprouting of ECs via a signaling cascade involving LRP6-mediated APC/Asef2/Cdc42 activation. Thus, our findings demonstrate the distinct functions of DKK1 and DKK2 in controlling angiogenesis and suggest that DKK2 may be a viable therapeutic target in the treatment of ischemic vascular diseases.

Interleukin-33 (IL-33), a member of the IL-1 cytokine family, is emerging as a new regulator of immune responses and inflammatory vascular diseases. Although IL-33 and its cognate receptor ST2 appear to be expressed in vascular cells, the precise role of IL-33 in the vasculature has not been determined. In this study, we report a novel role of IL-33 as a potent endothelial activator, promoting both angiogenesis and vascular permeability. IL-33 increased proliferation, migration, and morphologic differentiation of human endothelial cells, consistently with increased angiogenesis in vivo. IL-33 also increased endothelial permeability with reduced vascular endothelial–cadherin-facilitated cell–cell junctions in vitro and induced vascular leakage in mouse skin. These effects of IL-33 were blocked by knockdown of ST2. Ligation of IL-33 with ST2 rapidly increased endothelial nitric oxide (NO) production through TRAF6-mediated activation of phosphoinoside-3-kinase, Akt, and endothelial NO synthase. Moreover, pharmacologic or genetic blockage of endothelial NO generation resulted in the inhibition of angiogenesis and vascular hyperpermeability induced by IL-33. These data demonstrate that IL-33 promotes angiogenesis and vascular leakage by stimulating endothelial NO production via the ST2/TRAF6-Akt-eNOS signaling pathway. These findings open new perspectives for the role of IL-33 in the pathogenesis of angiogenesis-dependent and inflammatory vascular diseases.

Angiogenesis is regulated by the dynamic interaction between endothelial cells (ECs). Hippo-Yes-associated protein (YAP) signalling has emerged as a key pathway that controls organ size and tissue growth by mediating cell contact inhibition. However, the role of YAP in EC has not been defined yet. Here, we show expression of YAP in the developing front of mouse retinal vessels. YAP subcellular localization, phosphorylation and activity are regulated by VE-cadherin-mediated-EC contacts. This VE-cadherin-dependent YAP phosphorylation requires phosphoinositide 3-kinase-Akt activation. We further identify angiopoietin-2 (ANG-2) as a potential transcriptional target of YAP in regulating angiogenic activity of EC in vitro and in vivo. Overexpression of YAP-active form in EC enhances angiogenic sprouting, and this effect is blocked by ANG-2 depletion or soluble Tie-2 treatment. These findings implicate YAP as a critical regulator in angiogenesis and provide new insights into the mechanism coordinating junctional stability and angiogenic activation of ECs.

Developing cartilage serves as a template for long-bone development during endochondral ossification. Although the coupling of cartilage and bone development with angiogenesis is an important regulatory step for endochondral ossification, the molecular mechanisms are poorly understood. One possible mechanism involves the action of Dickkopf (DKK), which is a family of soluble canonical Wnt antagonists with four members (DKK1–4). We initially observed opposite expression patterns of Dkk1 and Dkk2 during angiogenesis and chondrocyte differentiation: downregulation of Dkk1 and upregulation of Dkk2. We examined the in vivo role of Dkk1 and Dkk2 in linking cartilage/bone development and angiogenesis by generating transgenic (TG) mice that specifically express Dkk1 or Dkk2 in chondrocytes, hypertrophic chondrocytes, or endothelial cells. Despite specific expression pattern during cartilage development, chondrocyte- and hypertrophic chondrocyte-specific Dkk1 and Dkk2 TG mice showed normal developmental phenotypes. However, Dkk1 misexpression in endothelial cells resulted in defects of endochondral ossification and reduced skeletal size. The defects are caused by the inhibition of angiogenesis in developing bone and subsequent inhibition of apoptosis of hypertrophic chondrocytes and cartilage resorption. © 2012 American Society for Bone and Mineral Research.

Recent studies have demonstrated that the cell adhesion molecule, L1, is expressed in several malignant tumor types and its expression correlates with tumor progression and metastasis. However, the role of L1 in gallbladder carcinoma (GBC) remains unclear. Here, we demonstrate that L1 is expressed in GBC cells and plays an important role in the growth, motility, invasiveness, and adhesiveness of GBC cells. Specific depletion or overexpression of L1 in the GBC cell lines JCRB1033 and SNU-308, respectively, was achieved by lentivirus-mediated transduction and expression of an L1 mRNA-specific short hairpin RNA or full-length human L1. Stable depletion of L1 led to a significant decrease in GBC cell proliferation, migration and invasion, as well as decreased intracellular signaling through AKT and FAK. Overexpression of L1 in GBC cells enhanced these cellular activities. In a GBC xenograft nude mouse model, suppression of L1 markedly reduced tumor growth and increased the survival of tumor-bearing mice whereas L1 overexpression stimulated tumorigenicity. Taken together, these results suggest that L1 plays a crucial role in GBC progression and may be a novel therapeutic target in GBC treatment.