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The current study tested the hypothesis of whether specific lipids may control angiogenic reactions. Using the chorioallantoic membrane assay of the chick embryo, new vessel formation was analyzed quantitatively by gas chromatography and mass spectrometry as well as bioinformatics tools including an angiogenesis analyzer. Our biochemical experiments showed that a specific lipid composition and stoichiometry determine the angiogenesis microenvironment to accelerate or inhibit vessel formation. Specific lipids of angiogenesis determinants in the vessel area and the non-vessel area were identified as nitrooleic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitic acid, oleic acid, linoleic acid, linolenic acid, epoxyoleic acid, lysophosphatidylcholine (LPC), cholesterol, 7-ketocholesterol, and docosahexaenoyl lysophosphatidylcholine (DHA-LPC). Vessel formation happens on the surface area of the hydrophilic membrane of the yolk. Our biochemical data demonstrated that angiogenesis was followed in the white lipid complex area to generate more branches, junctions, segments, and extremities. We analyzed lipid fragments in the vessel, non-vessel, and albumen area to show that each area contains a specific lipid composition and stoichiometry. Mass spectrometry data demonstrated that the vessel area has higher concentrations of nitrooleic acid, palmitic acid, stearic acid, LPC, lysophosphatidylethanolamine, cholesterol, oleic acid, linoleic acid, 7-ketocholesterol, and DHA-LPC; however, DHA and EPA were abundant in the hydrophobic non-vessel area. The purpose of vessel formation is to wrap up the yolk area to transport nutrients including specific fatty acids. Besides, angiogenesis requires aqueous albumen shown by distance-dependent vessel formation from albumen and oxygen. Higher concentrations of fatty acids are required for energy and carbon structure from the carbon-carbon bond, membrane building blocks, and amphiphilic detergent to solubilize a hydrophobic environment in the aqueous blood layer. The current study may guide that the uncovered hydrophobic or zwitterionic molecules such as DHA and DHA-LPC may control angiogenesis as antiangiogenic or proangiogenic molecules as potential drug targets for treating uncontrolled angiogenesis-related diseases, including diabetic retinopathy and age-related macular degeneration.
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Constant or intense light degenerates the retina and retinal pigment epithelial cells. Light generates reactive oxygen species and nitric oxide leading to initial reactions of retinal degeneration. Apoptosis is the primary mechanism of abnormal death of photoreceptors, retinal ganglion cells, or retinal pigment epithelium (RPE) in degenerative retinal diseases, including diabetic retinopathy and age-related macular degeneration. The current study evaluated the function of erythropoietin (EPO) on angiogenesis and apoptosis in the retina and RPE under oxidative stress. We determined the pro-angiogenic and antiapoptotic mechanism of EPO under stress conditions using a conditional EPO knockdown model using siRNA, EPO addition, proteomics, immunocytochemistry, and bioinformatic analysis. Our studies verified that EPO protected retinal cells from light-, hypoxia-, hyperoxia-, and hydrogen peroxide-induced apoptosis through caspase inhibition, whereas up-regulated angiogenic reactions through vascular endothelial growth factor (VEGF) and angiotensin pathway. We demonstrated that the EPO expression in the retina and subsequent serine/threonine/tyrosine kinase phosphorylations might be linked to oxidative stress response tightly to determining angiogenesis and apoptosis. Neuroprotective roles of EPO may involve the balance between antiapoptotic and pro-angiogenic signaling molecules, including BCL-xL, c-FOS, caspase-3, nitric oxide, angiotensin, and VEGF receptor. Our data indicate a new therapeutic application of EPO toward retinal degeneration based on the dual roles in apoptosis and angiogenesis at the molecular level under oxidative stress.
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The current study determined the natural angiogenic molecules using an unbiased metabolomics approach. A chick chorioallantoic membrane (CAM) model was used to examine pro- and antiangiogenic molecules, followed by gas chromatography-mass spectrometry (GCMS) analysis. Vessel formation was analyzed quantitatively using the angiogenic index (p < 0.05). At embryonic day one, a white streak or circle area was observed when vessel formation begins. GCMS analysis and database search demonstrated that angiogenesis may initiate when oleic, cholesterol, and linoleic acids increased in the area of angiogenic reactions. The gain of function study was conducted by the injection of cholesterol and oleic acid into a chick embryo to determine the role of each lipid in angiogenesis. We propose that oleic acid, cholesterol, and linoleic acid are natural molecules that set the platform for the initiation stage of angiogenesis before other proteins including the vascular endothelial growth factor, angiopoietin, angiotensin, and erythropoietin join as the angiome in sprout extension and vessel maturation.
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[This corrects the article DOI: 10.1007/s40200-020-00524-1.].
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PURPOSE: The current study aims to determine the molecular mechanisms of diabetic retinopathy (DR) using the protein-protein interactome and metabolome map. We examined the protein network of novel biomarkers of DR for direct (physical) and indirect (functional) interactions using clinical target proteins in different models. METHODS: We used proteomic tools including 2-dimensional gel electrophoresis, mass spectrometry analysis, and database search for biomarker identification using in vivo murine and human model of diabetic retinopathy and in vitro model of oxidative stress. For the protein interactome and metabolome mapping, various bioinformatic tools that include STRING and OmicsNet were used. RESULTS: We uncovered new diabetic biomarkers including prohibitin (PHB), dynamin 1, microtubule-actin crosslinking factor 1, Toll-like receptor (TLR 7), complement activation, as well as hypothetical proteins that include a disintegrin and metalloproteinase (ADAM18), vimentin III, and calcium-binding C2 domain-containing phospholipid-binding switch (CAC2PBS) using a proteomic approach. Proteome networks of protein interactions with diabetic biomarkers were established using known DR-related proteome data. DR metabolites were interconnected to establish the metabolome map. Our results showed that mitochondrial protein interactions were changed during hyperglycemic conditions in the streptozotocin-treated murine model and diabetic human tissue. CONCLUSIONS: Our interactome mapping suggests that mitochondrial dysfunction could be tightly linked to various phases of DR pathogenesis including altered visual cycle, cytoskeletal remodeling, altered lipid concentration, inflammation, PHB depletion, tubulin phosphorylation, and altered energy metabolism. The protein-metabolite interactions in the current network demonstrate the etiology of retinal degeneration and suggest the potential therapeutic approach to treat DR.