Integrin signaling is critical for pathological angiogenesis

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis

Inhibition of Integrin α\u3csub\u3ed\u3c/sub\u3eβ\u3csub\u3e2\u3c/sub\u3e-Mediated Macrophage Adhesion to End Product of Docosahexaenoic Acid (DHA) Oxidation Prevents Macrophage Accumulation During Inflammation

A critical step in the development of chronic inflammatory diseases is the accumulation of proinflammatory macrophages in the extracellular matrix (ECM) of peripheral tissues. The adhesion receptor integrin αDβ2 promotes the development of atherosclerosis and diabetes by supporting macrophage retention in inflamed tissue. We recently found that the end product of docosahexaenoic acid (DHA) oxidation, 2-(ω-carboxyethyl)- pyrrole (CEP), serves as a ligand forαDβ2.CEPadduct withECM is generated during inflammation-mediated lipid peroxidation. The goal of this project was to identify a specific inhibitor for αDβ2-CEP interaction that can prevent macrophage accumulation. Using a specially designed peptide library, Biacore-detected protein-protein interaction, and adhesion of integrin-transfected HEK 293 cells, we identified a sequence (called P5 peptide) that significantly and specifically inhibited αD-CEP binding. In the model of thioglycollate-induced peritoneal inflammation, the injection of cyclic P5 peptide reduced 3-fold the macrophage accumulation in WT mice but had no effect in αD-deficient mice. The tracking of adoptively transferred, fluorescently labeled WT and αD-/- monocytes in the model of peritoneal inflammation and in vitro two-dimensional and three-dimensional migration assays demonstrated thatP5peptide does not affectmonocytetransendothelial migration or macrophage efflux from the peritoneal cavity but regulates macrophage migration through the ECM. Moreover, the injection ofP5peptide intoWTmiceona high-fat diet prevents macrophage accumulation in adipose tissue in anαDβ2-dependent manner.Takentogether, these resultsdemonstratetheimportance of αDβ2-mediated macrophage adhesion for the accumulation of infiltrating macrophages in the inflamed ECM and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes

The angiogenic response is dictated by β3 integrin on bone marrow–derived cells

Angiogenesis is dependent on the coordinated action of numerous cell types. A key adhesion molecule expressed by these cells is the αvβ3 integrin. Here, we show that although this receptor is present on most vascular and blood cells, the key regulatory function in tumor and wound angiogenesis is performed by β3 integrin on bone marrow–derived cells (BMDCs) recruited to sites of neovascularization. Using knockin mice expressing functionally stunted β3 integrin, we show that bone marrow transplantation rescues impaired angiogenesis in these mice by normalizing BMDC recruitment. We demonstrate that αvβ3 integrin enhances BMDC recruitment and retention at angiogenic sites by mediating cellular adhesion and transmigration of BMDCs through the endothelial monolayer but not their release from the bone niche. Thus, β3 integrin has the potential to control processes such as tumor growth and wound healing by regulating BMDC recruitment to sites undergoing pathological and adaptive angiogenesis

Microglia Control Vascular Architecture via a TGFβ1 Dependent Paracrine Mechanism Linked to Tissue Mechanics

© 2020, The Author(s). Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFβ1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFβ1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFβ1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFβ1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFβ1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS

ERK1/2-Akt1 Crosstalk Regulates Arteriogenesis in Mice and Zebrafish

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis

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