VEGF signalling enhances lesion burden in KRIT1 deficient mice

The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial‐specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.     

ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis

Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis.     

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.     

KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3–0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function.In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells.Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.     

Vascular Endothelial Growth Factor-Receptor 1 Inhibition Aggravates Diabetic Nephropathy through eNOS Signaling Pathway in db/db Mice

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice.While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling.In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.     

Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-β1 Integrin Complex Formation,Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both β1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between β1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-β1 integrin complex formation identified using proximity ligation assays. Signaling downstream of β1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.     

Amplification of protease-activated receptors signaling in sporadic cerebral cavernous malformation endothelial cells

In the central nervous system, thrombin-mediated activation of protease-activated receptors (PARs) results in neuroinflammation and increased vascular permeability. These events have been linked to cancer and neurodegeneration. Endothelial cells (ECs) isolated from sporadic cerebral cavernous malformation (CCM) specimens showed dysregulation of genes involved in “thrombin-mediated PAR-1 activation” signaling. CCM is a vascular disease involving brain capillaries. In CCM, ECs show defective cell junctions. Oxidative stress and neuroinflammation play a key role in disease onset and progression. In order to confirm the possible role of thrombin pathway in sporadic CCM pathogenesis, we evaluated PARs expression in CCM-ECs. We found that sporadic CCM-ECs overexpress PAR1, PAR3 and PAR4, together with other coagulation factor encoding genes. Moreover, we investigated about expression of the three familial CCM genes (KRIT1, CCM2 and PDCD10) in human cerebral microvascular ECs, following thrombin exposure, as well as protein level. Thrombin exposure affects EC viability and results in dysregulation of CCM gene expression and, then, in decreased protein level. Our results confirm amplification of PAR pathway in CCM suggesting, for the first time, the possible role of PAR1-mediated thrombin signaling in sporadic CCM. Thrombin-mediated PARs over activation results in increased blood-brain barrier permeability due to loss of cell junction integrity and, in this context, also the three familial CCM genes may be involved.     

A novel snake venom vascular endothelial growth factor (VEGF) predominantly induces vascular permeability through preferential signaling via VEGF receptor-1

Vascular endothelial growth factor (VEGF)/vascular permeability factor induces both angiogenesis and vascular permeability mainly through VEGF receptor (VEGFR)-2 activation. VEGF binds VEGFR-1 as well, but the importance of VEGFR-1 signaling in vascular permeability has been largely neglected. Here, we report the purification and characterization of a novel VEGF-like protein from Trimeresurus flavoviridis Habu snake venom. The Habu snake has a venom-specific VEGF-like molecule, T. flavoviridis snake venom VEGF (TfsvVEGF), in addition to VEGF-A. TfsvVEGF has almost 10-fold less mitotic activity than VEGF(165), a predominant isoform of human VEGF-A, but a similar effect on vascular permeability. TfsvVEGF bound VEGFR-1 and induced its autophosphorylation to almost the same extent as VEGF(165), but bound VEGFR-2 weakly and induced its autophosphorylation almost 10-fold less effectively than VEGF(165). This unique binding affinity for VEGFR-1 and VEGFR-2 leads to the vascular permeability-dominant activity of TfsvVEGF. These results suggest that Habu snakes have acquired a highly purposive molecule for a toxin, which enhances the toxicity in envenomation without inducing effective angiogenesis and the following regeneration of damaged tissues, taking advantage of the difference in signaling properties involving VEGFR-1 and VEGFR-2 between vascular permeability and angiogenesis. TfsvVEGF is thus a potent inducing factor selective for vascular permeability through preferential signaling via VEGFR-1. These data strongly indicate the importance of VEGFR-1 signaling in vascular permeability.     

Structural Basis for the Disruption of the Cerebral Cavernous Malformations 2 (CCM2) Interaction with Krev Interaction Trapped 1 (KRIT1) by Disease-associated Mutations

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 Å co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1^NPX(Y/F)3, revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1^NPX(Y/F)3. We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.     

R-Ras Protein Inhibits Autophosphorylation of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells and Suppresses Receptor Activation in Tumor Vasculature

Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

Tumor Suppressor Lzap Suppresses Wnt/β-Catenin Signaling to Promote Zebrafish Embryonic Ventral Cell Fates via the Suppression of Inhibitory Phosphorylation of Glycogen Synthase Kinase 3

Wnt/β-catenin signaling controls various cell fates in metazoan development, and its dysregulation is often associated with cancer formation. However, regulations of this signaling pathway are not completely understood. Here, we report that Lzap, a tumor suppressor, controls nuclear translocation of β-catenin. In zebrafish embryos disruption of lzap increases the expression of chordin (chd), which encodes a bone morphogenetic protein (BMP) antagonist that is localized in prospective dorsal cells and promotes dorsal fates. Consistently, lzap-deficient embryos with attenuated BMP signaling are dorsalized, which can be rescued by overexpression of zebrafish lzap or bmp2b or human LZAP. The expansion of chd expression in embryos lacking lzap is due to the accumulation of nuclear β-catenin in ventral cells, in which β-catenin is usually degraded. Furthermore, the activity of GSK3, a master regulator of β-catenin degradation, is suppressed in lzap-deficient embryos via inhibitory phosphorylation. Finally, we also report that a similar regulatory axis is also likely to be present in a human tongue carcinoma cell line, SAS. Our results reveal that Lzap is a novel regulator of GSK3 for the maintenance of ventral cell properties and may prevent carcinogenesis via the regulation of β-catenin degradation.     

Pancreatic Endoplasmic Reticulum Kinase Activation Promotes Medulloblastoma Cell Migration and Invasion through Induction of Vascular Endothelial Growth Factor A

Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK) in response to endoplasmic reticulum (ER) stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A). Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.     

Virus-induced decline in soluble vascular endothelial growth receptor 2 is associated with plasma leakage in dengue hemorrhagic Fever

Some individuals infected with dengue virus develop dengue hemorrhagic fever (DHF), a viral hemorrhagic disease characterized by a transient period of localized plasma leakage. To determine the importance of vascular endothelial growth factor A (VEGF-A) in this syndrome, we compared plasma levels of VEGF-A and the soluble forms of its receptors in patients with DHF to patients with dengue fever (DF), a milder form of dengue virus infection without plasma leakage. We observed a rise in the plasma levels of free, but not total VEGF-A in DHF patients at the time of plasma leakage. This was associated with a decline in the soluble form of VEGF receptor 2 (VEGFR2) and VEGF-soluble VEGFR2 complexes, but not the soluble form of VEGFR1. The severity of plasma leakage in patients inversely correlated with plasma levels of soluble VEGFR2. In vitro, dengue virus suppressed soluble VEGFR2 production by endothelial cells but up-regulated surface VEGFR2 expression and promoted response to VEGF stimulation. In vivo, plasma viral load correlated with the degree of decline in plasma soluble VEGFR2. These results suggest that VEGF regulates vascular permeability and its activity is controlled by binding to soluble VEGFR2. Dengue virus-induced changes in surface and soluble VEGFR2 expression may be an important mechanism of plasma leakage in DHF.     

The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features

Background

Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2).

Methods

Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability.

Results

Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding.

Conclusions

VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability.

General significance

This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties.     

VEGF-induced vascular permeability is mediated by FAK

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.     

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.     

Integrin alpha x stimulates cancer angiogenesis through PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression in blood vessel endothelial cells

Integrin alpha x (ITGAX), a member of the integrin family, usually serves as a receptor of the extracellular matrix. Recently, accumulating evidence suggests that ITGAX may be involved in angiogenesis in dendritic cells. Herein, we report a direct role of ITGAX in angiogenesis during tumor development. Overexpression of ITGAX in human umbilical vein endothelial cells (HUVECs) enhanced their proliferation, migration, and tube formation and promoted xenograft ovarian tumor angiogenesis and growth. Further study showed that overexpression of ITGAX activated the PI3k/Akt pathway, leading to the enhanced expression of c-Myc, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor 2 (VEGFR2), whereas, the treatment of cells with PI3K inhibitor diminished these effects. Besides, c-Myc was observed to bind to the VEGF-A promoter. By Co-Immunoprecipitation (Co-IP) assay, we manifested the interaction between ITGAX and VEGFR2 or the phosphorylated VEGFR2. Immunostaining of human ovarian cancer specimens suggested that endothelial cells of micro–blood vessels displayed strong expression of VEGF-A, c-Myc, VEGFR2, and the PI3K signaling molecules. Also, overexpression of ITGAX in HUVECs could stimulate the spheroid formation of ovarian cancer cells. Our study uncovered that ITGAX stimulates angiogenesis through the PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression during cancer development.