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.

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It has been suggested that clec14a may be involved in tumor angiogenesis. However, a molecular mechanism has not been clearly identified. In this study, we show for the first time that C-type lectin-like domain (CTLD) of clec14a may be important for regulating cell migration and filopodia formation. Using phage display technology, recombinant human antibodies specific to the CTLDs of human and mouse clec14a (clec14a-CTLD (immunoglobulin G) IgG) were selected. Functional assays using the antibodies showed that clec14a-CTLD IgGs specifically blocked endothelial cell migration and tube formation without affecting cell viability or activation. Further, clec14a-CTLD IgGs inhibited clec14a-mediated cell–cell contact by blocking interaction between CTLDs. Finally, clec14a cross-linking by the clec14a-CTLD IgGs significantly downregulated clec14a expression on the surface of endothelial cells. These results strongly suggest that the clec14a-CTLD may be a key domain in angiogenesis, and that clec14a-CTLD IgGs specifically inhibit angiogenesis by modulating CTLD-mediated cell interactions and clec14a expression on the surface of endothelial cells.

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Tumor blood vessels are leaky and immature, which causes inadequate blood supply to tumor tissues resulting in hypoxic microenvironment and promotes metastasis. Here we have explored tumor vessel modulating activity of Sac-1004, a recently developed molecule in our lab, which directly potentiates VE-cadherin-mediated endothelial cell junction. Sac-1004 could enhance vascular junction integrity in tumor vessels and thereby inhibit vascular leakage and enhance vascular perfusion. Improved perfusion enabled Sac-1004 to have synergistic anti-tumor effect on cisplatin-mediated apoptosis of tumor cells. Interestingly, characteristics of normalized blood vessels namely reduced hypoxia, improved pericyte coverage and decreased basement membrane thickness were readily observed in tumors treated with Sac-1004. Remarkably, Sac-1004 was also able to inhibit lung and lymph node metastasis in MMTV and B16BL6 tumor models. This was in correlation with a reduction in epithelial-to-mesenchymal transition of tumor cells with considerable diminution in expression of related transcription factors. Moreover, cancer stem cell population dropped substantially in Sac-1004 treated tumor tissues. Taken together, our results showed that direct restoration of vascular junction could be a significant strategy to induce normalization of tumor blood vessels and reduce metastasis.

Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling.

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