Novel engineered, membrane-tethered VEGF-A variants promote formation of filopodia, proliferation, survival, and cord or tube formation by endothelial cells via persistent VEGFR2/ERK signaling and activation of CDC42/ROCK pathways.

Therapeutic angiogenesis would be clinically valuable in situations such as peripheral vascular disease in diabetic patients and tissue reperfusion following ischemia or injury, but approaches using traditional isoforms of vascular endothelial growth factor-A (VEGF) have had little success. The isoform VEGF165 is both soluble and matrix-associated, but can cause pathologic vascular changes. Freely diffusible VEGF121 is not associated with pathologic angiogenesis, but its failure to remain in the vicinity of the targeted area presents therapeutic challenges. In this study, we evaluate the cellular effects of engineered VEGF variants that tether extracellular VEGF121 to the cell membrane with the goal of activating VEGF receptor 2 (VEGFR2) in a sustained, autologous fashion in endothelial cells. When expressed by primary human retinal endothelial cells (hRECs), the engineered, membrane-tethered variants eVEGF-38 and eVEGF-53 provide a lasting VEGF signal that induces cell proliferation and survival, increases endothelial permeability, promotes the formation of a cord/tube network, and stimulates the formation of elongated filopodia on the endothelial cells. The engineered VEGF variants activate VEGFR2, MAPK/ERK, and the Rho GTPase mediators CDC42 and ROCK, activities that are required for the formation of the elongated filopodia. The sustained, pro-angiogenic activities induced by eVEGF-38 and eVEGF-53 support the potential of engineered VEGF variants-overexpressing endothelial cells as a novel combination of gene and cell-based therapeutic strategy for stimulating endothelial cell-autologous therapeutic angiogenesis.

Discovery of sterically-hindered phenol compounds with potent cytoprotective activities against ox-LDL-induced retinal pigment epithelial cell death as a potential pharmacotherapy.

Late-stage dry age-related macular degeneration (AMD) or geographic atrophy (GA) is an irreversible blinding condition characterized by degeneration of retinal pigment epithelium (RPE) and the associated photoreceptors. Clinical and genetic evidence supports a role for dysfunctional lipid processing and accumulation of harmful oxidized lipids in the pathogenesis of GA. Using an oxidized low-density lipoprotein (ox-LDL)-induced RPE death assay, we screened and identified sterically-hindered phenol compounds with potent protective activities for RPE. The phenol-containing PPARγ agonist, troglitazone, protected against ox-LDL-induced RPE cell death, whereas other more potent PPARγ agonists did not protect RPE cells. Knockdown of PPARγ did not affect the protective activity of troglitazone in RPE, confirming the protective function is not due to the thiazolidine (TZD) group of troglitazone. Prototypical hindered phenol trolox and its analogs potently protected against ox-LDL-induced RPE cell death whereas potent antioxidants without the phenol group failed to protect RPE. Hindered phenols preserved lysosomal integrity against ox-LDL-induced damage and FITC-labeled trolox was localized to the lysosomes in RPE cells. Analogs of trolox inhibited reactive oxygen species (ROS) formation induced by ox-LDL uptake in a dose-dependent fashion and were effective at sub-micromolar concentrations. Treatment with trolox analog 2,2,5,7,8-pentamethyl-6-chromanol (PMC) significantly induced the expression of the lysosomal protein NPC-1 and reduced intracellular cholesterol level upon ox-LDL uptake. Our data indicate that the lysosomal-localized hindered phenols are uniquely potent in protecting the RPE against the toxic effects of ox-LDL, and may represent a novel pharmacotherapy to preserve the vision in patients with GA.

Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization.

Many patients with wet age-related macular degeneration do not respond well to anti- vascular endothelial growth factor A (VEGFA) therapy for choroidal neovascularization (CNV), and the efficacy of anti-VEGFA decreases over time. We investigated the hypothesis that fibrotic changes, in particular via endothelial-to-mesenchymal transition (EndoMT), play a role in CNV and alter the therapeutic effects of VEGFA pathway blockage. Induction of EndoMT of primary human retinal endothelial cells led to a significantly reduced response to VEGFA at the level of gene expression, cellular proliferation, migration, and tube formation. Suppression of EndoMT restored cell responsiveness to VEGFA. In a mouse model of spontaneous CNV, fibrotic changes and EndoMT persisted as the CNV lesions became more established over time. VEGFA receptor-2 (VEGFR2) antagonism further induced fibrosis and EndoMT in the CNV. The combination of VEGFR2 antagonism and fibrosis/EndoMT inhibition was more effective than either individual treatment in reducing CNV. Our data indicate that fibrosis and EndoMT are involved in the progression of CNV, are exacerbated by VEGFR2 inhibition, and could provide an explanation for the reduced efficacy of anti-VEGFA treatment over time.

Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells.

Moringa oleifera seeds are used in alternative medicine to treat inflammation, tumors and bacterial and protozoan infections, for example. The seeds contain lectins, which are carbohydrate-binding proteins with several biological properties including cytotoxicity to cancer cells. In this work, we examined the cytotoxicity of the coagulant M. oleifera lectin (cMoL) on B16-F10 murine melanoma cells. cMoL cytotoxic effects were evaluated through trypan blue assay and flow cytometry analysis. Mitochondrial superoxide levels and activation of caspases 3, 8 and 9 were measured. cMoL (1.5-16µM) reduced viability and caused cell death of B16-F10 cells with an IC50 of 9.72µM. Flow cytometry analysis indicated induction of necrosis and suggested the presence of cells in late apoptosis. Specificity for tumor cells was observed since death of normal human fibroblasts (GN) was not higher than 20% in treatments with cMoL from 1.5 to 16µM. Microscopy images revealed rounded shape and reduction of volume in B16-F10 cells treated with cMoL. cMoL increased mitochondrial ROS production and promoted caspases 3, 8 and 9 activation in B16-F10 cells, indicating the activation of apoptosis-related pathway. In conclusion, this study demonstrates that cMoL is cytotoxic to B16-F10 cells, which stimulates more investigation on the anticancer potential of this lectin.

pCramoll and rCramoll lectins induce cell death in human prostate adenocarcinoma (PC-3) cells by impairment of mitochondrial homeostasis.

Lectins from Cratylia mollis seed have shown potential in vivo antitumor actions, however the mechanism have not yet been addressed. Here we evaluated the antitumor effects of native (pCramoll) and recombinant (rCramoll) lectins from C. mollis against human prostate adenocarcinoma (PC-3) cells. The viability of PC-3 cells was analyzed with the MTT assay and ANNEXIN V/propidium iodide staining. The actions of pCramoll or rCramoll on mitochondrial superoxide production, free cytosolic calcium concentration and mitochondrial membrane potential were evaluated using fluorescent probes (MitoSox Red, Fura 2-AM and safranin O, respectively). pCramoll and rCramoll reduced the viability of PC-3 cells in a dose-dependent manner. Both lectins increased the generation of mitochondrial superoxide as well as the concentration of cytosolic calcium. These changes led to a decrease in oxidative phosphorylation, which impaired the formation of ATP. The resulting cell death was not blocked by MPT (mitochondrial permeability transition) inhibitors (Debio 025 or bongkrekic acid). Thus pCramoll and rCramoll promote PC-3 cell death through calcium signaling, leading to mitochondrial collapse. This work provides more insights into the action of pCramoll and rCramoll against cancer cells. These lectins represent valuable tools for biomedical research.

Endocytosis of tight junctions caveolin nitrosylation dependent is improved by cocoa via opioid receptor on RPE cells in diabetic conditions.

PURPOSE: Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. METHODS: Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). RESULTS: Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. CONCLUSIONS: The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.