Vijayendra Agrawal

Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2

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.

Sac-1004, a novel vascular leakage blocker, enhances endothelial barrier through the cAMP/Rac/cortactin pathway

The maintenance of endothelial barrier is critical for the vascular homeostasis and is maintained by the interaction of adherens junction (AJ) and tight junction (TJ) proteins between adjacent cells. This interaction is stabilized by actin cytoskeleton forming cortical actin ring. Here, we developed a novel vascular leakage blocker, Sac-1004 and investigated its mechanism of action in endothelial cells (ECs). Sac-1004 inhibited endothelial hyperpermeability induced by vascular endothelial growth factor, histamine and thrombin via stabilization of cortical actin ring and AJ proteins at the cell-cell junction. Treatment of Sac-1004 in ECs increased cAMP levels and activated Rac, both of which are known to strengthen endothelial barrier. Furthermore, Sac-1004 induced phosphorylation of cortactin and its localization at cell membrane that is essential for the stabilization of cortical actin ring. These effects of Sac-1004 on ECs were significantly abrogated by dideoxyadenosine (adenylyl cyclase inhibitor) and NSC23766 (Rac inhibitor). Taken together, our findings indicate that Sac-1004 blocks vascular leakage by enhancing endothelial integrity via the cAMP/Rac/cortactin pathway and imply the potential usefulness of Sac-1004 in the development of therapeutic means for vascular leakage-related diseases.

Sac-0601 prevents retinal vascular leakage in a mouse model of diabetic retinopathy

Endothelium integrity is important for the normal functioning of vessels, the disruption of which can lead to disease. The blood-retinal barrier required for normal retinal function is compromised in diabetic retinopathy, causing retinal vascular leakage. Previously, we demonstrated the ability of Sac-0601[((2R,3S)-3-acetoxy-6-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-2H-pyran-2-yl)methyl acetate], a pseudo-sugar derivative of cholesterol, to increase survival of retinal endothelial cells. In the present study, we evaluated the ability of Sac-0601 to prevent retinal vascular leakages in vitro and in vivo. Sac-0601 treatment blocked VEGF-induced formation of actin stress fibers and stabilized the cortical actin ring in retinal endothelial cells. It also inhibited degradation of occludin, an important tight junction protein, and blocked VEGF-induced disruption of its linear pattern at the cell border. The [(14)C] sucrose permeability assay demonstrated that Sac-0601 was able to prevent VEGF-induced retinal endothelial permeability. The compound inhibited the vascular leakage in retina of mice intravitreally injected with VEGF. And it also significantly reduced the leakage in retina of diabetic retinopathy mice model. Taken together, our findings suggest the potential therapeutic usefulness of Sac-0601 for retinal vascular permeability diseases.

Hhip regulates tumor-stroma-mediated upregulation of tumor angiogenesis

Tumor growth is governed by the coordinated action of various types of cells that are present in the tumor environment. Fibroblasts, which constitute a major fraction of the stroma, participate actively in various signaling events and regulate tumor development and metastasis. The Hedgehog (Hh) pathway plays an important role in promoting tumor malignancy via fibroblasts; however, the role of hedgehog interacting protein (hhip; inhibitor of Hh pathway) in tumor growth is poorly understood. Here we implanted B16F10 tumors in hhip+/− mice to study the tumor growth characteristics and the vascular phenotype. Furthermore, the mechanism involved in the observed phenomena was explored to reveal the role of hhip in tumor growth. The tumors that were implanted in hhip+/− mice exhibited accelerated growth and increased tumor angiogenesis. Although we observed a decrease in hypoxia, blood vessels still had abnormal phenotype. We found that increased Hh signaling in tumor fibroblasts induced a high expression of vascular endothelial growth factor (VEGF), which subsequently resulted in an increased proliferation of endothelial cells. Thus, the heterozygous knockdown of hhip in mice could affect Hh signaling in tumor fibroblasts, which could cause the increased production of the growth factor VEGF. This signaling, via a paracrine effect on endothelial cells, increased tumor vascular density.

Glucal-conjugated sterols as novel vascular leakage blocker: Structure–activity relationship focusing on the C17-side chain

A series of glucal-conjugated sterols as novel vascular leakage blocker were identified through design, synthesis and biologically evaluation. In addition, the structure-activity relationship (SAR) of the glucal-conjugated sterols focusing on the C17-side chain was also established. The sterol analogs linked with the rigid C17-side chain side chains exhibited potent cell survival activities. In particular, analog 21l, which possesses a cyclopentyl oxime moiety, was shown to have excellent pharmacological effects on retinal vascular leakage in a diabetic mouse model.

Combined effect of vascular-leakage-blocker Sac-1004 and antiangiogenic drug sunitinib on tumor angiogenesis.

Tumor blood vessels are often leaky because of poor covering by mural cells and loose cell-to-cell contacts. Leaky vessels result in hemorrhage and limited vascular perfusion, which lead to hypoxic tumor microenvironment. Antiangiogenic agents have been shown to normalize the tumor blood vessels, albeit temporarily. Continued administration has been found to be associated with increased tumor hypoxia, a major driving force behind chemoresistance and metastasis. Sac-1004 was recently demonstrated to prevent vascular leakage, normalize tumor vessels and prevent metastasis in sustained manner. Here, we sought that combining antiangiogenic agent, sunitinib with Sac-1004 could have better inhibitory effect upon tumor growth. We found that B16F10 tumor growth was significantly reduced and tumor-bearing mice survival was increased upon combining sunitinib therapy with Sac-1004. In concordance with this observation, tumor vascular perfusion was substantially improved in tumors receiving combination therapy. In addition, tumor vascular leakage was reduced to higher extent in combination treatment group as compared to either therapy alone, an effect attributed to improved vascular junction. Interestingly, hypoxia in tumor environment was significantly reduced, when sunitinib was combined with Sac-1004. Taken together, our data demonstrates that combining antiangiogenic therapy with vascular-leakage inhibiting agent might be a beneficial strategy to combat cancer.