Abstract-The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between ␤ 3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and ␤ 3 integrin appears to be synergistic, because VEGFR-2 activation induces ␤ 3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesionand growth factor-induced ␤ 3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent ␤ 3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and ␤ 3 integrin. Moreover, c-Src mediates growth factor-induced ␤ 3 integrin activation, ligand binding, ␤ 3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro-and antiangiogenic strategies. Key Words: angiogenesis Ⅲ endothelial cell Ⅲ ␤ 3 integrin signaling Ⅲ vascular endothelial growth factor receptor Ⅲ extracellular matrix proteins A ngiogenesis, the process of new blood vessel formation from preexisting vasculature, plays critical roles in tissue regeneration, postischemic tissue repair on myocardial infarction and stroke, and in the pathogenesis of cancer, rheumatoid arthritis, and diabetic microvascular disease. 1 Angiogenesis is triggered by angiogenic growth factors and their receptors in coordination with extracellular matrix (ECM) receptors known as integrins. 2 On integrin engagement, ECM triggers activation of numerous intracellular signaling pathways essential for endothelial cell (EC) survival, proliferation, migration, morphogenesis, and organization of ECs into blood vessels. 3 There are several manifestations of a tightly collaborative relationship between integrins and receptors for growth factors. 4,5 On ECs, engagement of ␣ v ␤ 3 integrin promotes phosphorylation and activation of vascular endothelial growth factor (VEGF) receptor (VEGFR)-2, thereby augmenting the mitogenic activity of VEGFs. 6 Among several integrins on ECs, ␣ v ␤ 3 is the most abundant and influential receptor regulating angiogenesis. 7 The upregulation of ␣ v ␤ 3 during angiogenesis suggests that this integrin might play a crucial role during this process. Indeed, antagonists of ␣ v ␤ 3 , including blocking monoclonal antibody (LM609) and RGD cyclic peptides, were shown ...

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.

Osteosarcoma (OS), which occurs most commonly in adolescents, is associated with a high degree of malignancy and poor prognosis. In order to develop an accurate treatment for OS, a deeper understanding of its complex tumor microenvironment (TME) is required. In the present study, tissues were isolated from six patients with OS, and then subjected to single-cell RNA sequencing (scRNA-seq) using a 10× Genomics platform. Multiplex immunofluorescence staining was subsequently used to validate the subsets identified by scRNA-seq. ScRNA-seq of six patients with OS was performed prior to neoadjuvant chemotherapy, and data were obtained on 29,278 cells. A total of nine major cell types were identified, and the single-cell transcriptional map of OS was subsequently revealed. Identified osteoblastic OS cells were divided into five subsets, and the subsets of those osteoblastic OS cells with significant prognostic correlation were determined using a deconvolution algorithm. Thereby, different transcription patterns in the cellular subtypes of osteoblastic OS cells were reported, and key transcription factors associated with survival prognosis were identified. Furthermore, the regulation of osteolysis by osteoblastic OS cells via receptor activator of nuclear factor kappa-B ligand was revealed. Furthermore, the role of osteoblastic OS cells in regulating angiogenesis through vascular endothelial growth factor-A was revealed. C3_TXNIP+ macrophages and C5_IFIT1+ macrophages were found to regulate regulatory T cells and participate in CD8+ T cell exhaustion, illustrating the possibility of immunotherapy that could target CD8+ T cells and macrophages. Our findings here show that the role of C1_osteoblastic OS cells in OS is to promote osteolysis and angiogenesis, and this is associated with survival prognosis. In addition, T cell depletion is an important feature of OS. More importantly, the present study provided a valuable resource for the in-depth study of the heterogeneity of the OS TME.