Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2

Bioactive 4‑Oxoheptanedioic Monoamide Derivatives of Proteins and Ethanolaminephospholipids: Products of Docosahexaenoate Oxidation

Oxidative stress causes lipid-derived oxidative modification of biomolecules that has been implicated in many pathological states. Phospholipids containing polyunsaturated fatty acids are major targets of free radical-initiated oxidation. Phospholipids that incorporate docosahexaenoate (DHA) are highly enriched in important neural structures including the brain and retina, where DHA comprises 40% and 60% of total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-<i>sn</i>-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine, HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole (CEP) derivatives were detected immunologically with anti-CEP antibodies in human tumors, retina, and blood. Now, we generated an anti-OHdiA antibody to test the hypothesis that KOHA adducts, which incorporate the primary amino groups of proteins or ethanolamine phospholipids in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in vivo. However, whereas the anti-CEP antibody is highly specific and does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA monoamide antibody cross-reacted with CEP epitopes making it of little value as an analytical tool for OHdiA monoamides but suggesting the possibility that OHdiA monoamides would exhibit receptor-mediated biological activity similar to that of CEP. An LC-MS/MS method was developed that allows quantification of OHdiA derivatives in biological samples. We now find that KOHA-PC forms OHdiA monoamide adducts of proteins and ethanolamine phospholipids and that OHdiA-protein levels are significantly higher than OHdiA-ethanloamine phospholipid levels in blood from healthy human subjects, 0.45 μM and 0.18 μM, respectively (<i>n</i> = 3, and <i>p</i> = 0.027). OHdiA monoamide epitopes are angiogenic, causing TLR2-dependent adhesion and tube formation by human umbilical vein endothelial cells. OHdiA monoamide epitopes are only slightly less potent than CEP epitopes that contribute to the pathological angiogenesis of age-related macular degeneration and tumor growth

Oxidative Modifications of Extracellular Matrix Promote the Second Wave of Inflammation via B\u3csub\u3e2\u3c/sub\u3e Integrins

Early stages of inflammation are characterized by extensive oxidative insult by recruited and activated neutrophils. Secretion of peroxidases, including the main enzyme, myeloperoxidase, leads to the generation of reactive oxygen species. We show that this oxidative insult leads to polyunsaturated fatty acid (eg, docosahexaenoate), oxidation, and accumulation of its product 2-(v-carboxyethyl)pyrrole (CEP), which, in turn, is capable of protein modifications. In vivo CEP is generated predominantly at the inflammatory sites in macrophage-rich areas. During thioglycollate-induced inflammation, neutralization of CEP adducts dramatically reduced macrophage accumulation in the inflamed peritoneal cavity while exhibiting no effect on the early recruitment of neutrophils, suggesting a role in the second wave of inflammation. CEP modifications were abundantly deposited along the path of neutrophils migrating through the 3-dimensional fibrin matrix in vitro. Neutrophil-mediated CEP formation was markedly inhibited by the myeloperoxidase inhibitor, 4-ABH, and significantly reduced in myeloperoxidase-deficient mice. On macrophages, CEP adducts were recognized by cell adhesion receptors, integrin aMb2 and aDb2. Macrophage migration through CEP-fibrin gel was dramatically augmented when compared with fibrin alone, and was reduced by b2-integrin deficiency. Thus, neutrophil-mediated oxidation of abundant polyunsaturated fatty acids leads to the transformation of existing proteins into stronger adhesive ligands for aMb2- and aDb2-dependent macrophage migration. The presence of a carboxyl group rather than a pyrrole moiety on these adducts, resembling characteristics of bacterial and/or immobilized ligands, is critical for recognition by macrophages. Therefore, specific oxidation-dependent modification of extracellular matrix, aided by neutrophils, promotes subsequent aMb2- and aDb2-mediated migration/retention of macrophages during inflammation

Inflammation-Dependent Oxidative Stress Metabolites as a Hallmark of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, with poor prognosis and no cure. Substantial evidence implicates inflammation and associated oxidative stress as a potential mechanism for ALS, especially in patients carrying the SOD1 mutation and, therefore, lacking anti-oxidant defense. The brain is particularly vulnerable to oxidation due to the abundance of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), which can give rise to several oxidized metabolites. Accumulation of a DHA peroxidation product, CarboxyEthylPyrrole (CEP) is dependent on activated inflammatory cells and myeloperoxidase (MPO), and thus marks areas of inflammation-associated oxidative stress. At the same time, generation of an alternative inactive DHA peroxidation product, ethylpyrrole, does not require cell activation and MPO activity. While absent in normal brain tissues, CEP is accumulated in the central nervous system (CNS) of ALS patients, reaching particularly high levels in individuals carrying a SOD1 mutation. ALS brains are characterized by high levels of MPO and lowered anti-oxidant activity (due to the SOD1 mutation), thereby aiding CEP generation and accumulation. Due to DHA oxidation within the membranes, CEP marks cells with the highest oxidative damage. In all ALS cases CEP is present in nearly all astrocytes and microglia, however, only in individuals carrying a SOD1 mutation CEP marks \u3e90% of neurons, thereby emphasizing an importance of CEP accumulation as a potential hallmark of oxidative damage in neurodegenerative diseases

The pY747 peptide has no effect on β3^−/− or DiYF mice.

<p><b>a</b>) pY747 peptide does not inhibit VEGF-induced aortic ring growth from β3^−/− mice. Mouse aortic rings were embedded in matrigel in the presence of 40 ng/mL of VEGF and 40 µM of peptides as indicated. <b>b</b>) Quantification of aortic ring assay as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031071#pone-0031071-g004" target="_blank">Fig. 4a</a>. <b>c</b>) pY747 could not inhibit bFGF-induced aortic ring growth, Mouse aortic rings were isolated from wild type (WT), β3^−/−, and DiYF mice and embedded in matrigel in the presence of 40 ng/mL of VEGF, 20 ng/mL of bFGF or pY747 peptides as indicated. Aortic rings were incubated for 3 days for wild type and β3^−/− aortic rings and 4 days for DiYF aortic rings (longer incubation was used to obtain visible aortic sprouting which is diminished in these mice). <b>d</b>) pY747 peptide does not inhibit angiogenesis in DiYF mice. Peptides' effect on <i>in vivo</i> angiogenesis in wild type mice and DiYF mice was tested as described. <b>e</b>) Quantification of blood vessels in matrigel plus assay as indicated.</p

Structural statistics for the 15 final NMR structures of VpepB.

a)<p>Ordered residues (residues with sum of phi and psi order parameters <1.8) are considered for r.m.s.d. calculations and Ramachandran statistics.</p