Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.     

Axl is essential for VEGF-A-dependent activation of PI3K/Akt

Herein, we report that vascular endothelial growth factor A (VEGF-A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF-A-dependent activation of VEGF receptor-2 (VEGFR-2), and subsequent TSAd-mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand-independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF-A-mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF-A- but not bFGF-dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF-A-induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF-A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF-A-driven processes.     

Osteopontin inhibition of miR-129-3p enhances IL-17 expression and monocyte migration in rheumatoid arthritis

BackgroundOsteopontin (OPN) is an important proinflammatory cytokine in rheumatoid arthritis (RA). Levels of OPN have been shown to be significantly correlated with interleukin-17 (IL-17) production and expression of Th17 cells in the synovial fluid of RA patients. Here, we investigated the role of OPN in monocyte migration, IL-17 production and osteoblasts.MethodsOPN and IL-17 expression profiles in osteoarthritis (OA) and RA synovial fluid were determined by enzyme-linked immunosorbent assay (ELISA). The expression of the microRNA, miR-129-3p, in osteoblasts was analyzed by real-time quantitative polymerase chain reaction (qPCR). Immunoreactive proteins were spotted by Western blotting. We used the collagen-induced arthritis (CIA) mouse model to investigate the role of OPN in monocyte migration during RA.ResultsOPN and IL-17 expression were higher in RA synovial fluid as compared to OA samples. We also found that OPN promotes IL-17 expression in osteoblasts and thereby enhances monocyte migration via the Syk/PI3K/Akt signaling pathway. miR-129-3p expression was found to be negatively regulated by OPN via the Syk/PI3K/Akt signal cascade. In contrast, lentiviral vectors expressing short hairpin RNA inhibited OPN expression and ameliorated articular swelling, cartilage erosion and monocyte infiltration in the ankle joints of CIA mice.ConclusionTo our knowledge, our study is the first to describe how OPN promotes monocyte migration by upregulating IL-17 expression in osteoblasts in RA disease.SignificanceThese findings indicate that OPN could serve as a potential therapeutic target for the treatment of RA.     

Microparticles stimulate angiogenesis by inducing ELR+ CXC‐chemokines in synovial fibroblasts

Microparticles (MPs) are small membrane‐vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro‐inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co‐cultured with increasing numbers of MPs. The effects of supernatants from co‐cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans‐well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro‐angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co‐cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio‐chamber assay, supernatants from RASFs co‐cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro‐angiogenic ELR⁺ chemokines. These pro‐angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.     

CXCL13/CXCR5 axis facilitates endothelial progenitor cell homing and angiogenesis during rheumatoid arthritis progression

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. The chemokine (C-X-C motif) ligand 13 (CXCL13) plays important roles in several cellular functions such as infiltration, migration, and motility. We report significantly higher levels of CXCL13 expression in collagen-induced arthritis (CIA) mice compared with controls and also in synovial fluid from RA patients compared with human osteoarthritis (OA) samples. RA synovial fluid increased endothelial progenitor cell (EPC) homing and angiogenesis, which was blocked by the CXCL13 antibody. By interacting with the CXCR5 receptor, CXCL13 facilitated vascular endothelial growth factor (VEGF) expression and angiogenesis in EPC through the PLC, MEK, and AP-1 signaling pathways. Importantly, infection with CXCL13 short hairpin RNA (shRNA) mitigated EPC homing and angiogenesis, articular swelling, and cartilage erosion in ankle joints of mice with CIA. CXCL13 is therefore a novel therapeutic target for RA.Subject terms: miRNAs, Rheumatoid arthritis     

Fibroblast growth factor-2 determines severity of joint disease in adjuvant-induced arthritis in rats.

Rheumatoid arthritis (RA), a systemic inflammatory disease of unknown etiology, mainly affects synovial joints. Although angiogenic growth factors, including fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF), may play a critical role in the development and progression of RA joint disease, little information is now available regarding their exact role in initiation and/or progression of RA. In this study, we show that both polypeptides were up-regulated in the rat joint synovial tissue of an adjuvant-induced model of arthritis (AIA), as well as human subjects with RA. FGF-2 overexpression via Sendai virus-mediated gene transfer significantly worsened clinical symptoms and signs of rat AIA, including hind paw swelling and radiological bone destruction, as well as histological findings based on inflammatory reaction, synovial angiogenesis, pannus formation, and osteocartilaginous destruction, associated with up-regulation of endogenous VEGF. FGF-2 gene transfer to non-AIA joints was without effect. These findings suggested that FGF-2 modulated disease progression, but did not affect initiation. Reverse experiments using anti-FGF-2-neutralizing rabbit IgG attenuated clinical symptoms and histopathological abnormalities of AIA joints. To our knowledge, this is the first report indicating direct in vivo evidence of disease-modulatory effects of FGF-2 in AIA, as probably associated with endogenous VEGF function. FGF-2 may prove to be a possible therapeutic target to treat subjects with RA.     

Toll-like receptor 2 induced angiogenesis and invasion is mediated through the Tie2 signalling pathway in rheumatoid arthritis

Background

Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo.

Methods

Microvascular endothelial cells (HMVEC) and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml). Angiopoietin 2 (Ang2), Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml) and matched IgG isotype control Ab (10 ug/ml).

Results

Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenenic tube formation (p<0.05), and upregulated Ang2 production in HMVEC (p<0.05) and RA synovial explants (p<0.05). Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI) to 617±103 (p<0.01). TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05). Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05).

Conclusion

TLR2 activation promotes angiogenesis, cell adhesion and invasion, effects that are in part mediated through the Tie2 signalling pathway, key mechanisms involved in the pathogenesis of RA.     

Roles of YAP in mediating endothelial cell junctional stability and vascular remodeling

Angiogenesis is a complex process involving dynamic interaction of various cell to cell interactions. Endothelial cell interactions regulated by growth factors, inflammatory cytokines, or hemodynamic stress are critical for balancing vascular quiescence and activation. Yes-associated protein (YAP), an effector of Hippo signaling, is known to play significant roles in maintaining cellular homeostasis. However, its role in endothelial cells for angiogenic regulation remains relatively unexplored. We demonstrated the critical role of YAP in vascular endothelial cells and elucidated the underlying molecular mechanisms involved in angiogenic regulation of YAP. YAP was expressed in active angiogenic regions where endothelial cell junctions were relatively loosened. Consistently, YAP subcellular localization and activity were regulated by VE-cadherin-mediated PI3K/Akt pathway. YAP thereby regulated endothelial sprouting via angiopoietin-2 expression. These results provide an insight into a model of coordinating endothelial junctional stability and angiogenic activation through YAP. [BMB Reports 2015; 48(8): 429-430]     

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function

Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial–mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1α expression and activation of Akt. After knockdown or inhibition of HIF-1α in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1α could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1α pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.     

Enhancement of placenta growth factor expression by oncostatin M in human rheumatoid arthritis synovial fibroblasts

Oncostatin M (OSM) belongs to IL‐6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of rheumatoid arthritis (RA) patients and it may arouse the inflammation responses in joints and eventually leads to bone erosion. Placenta growth factor (PLGF) is an angiogenic factor and highly homologous with vascular endothelial growth factor (VEGF). It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines including TNF‐α and IL‐6. Here, we demonstrated that OSM increased mRNA and protein levels of PLGF in a time‐ and concentration‐dependent manner in RA synovial fibroblasts. Inhibitors of JAK3 and PI3K antagonized OSM‐induced production of PLGF. OSM enhanced the phosphorylation of Tyr705‐STAT3, Ser727‐STAT3, Ser473‐Akt, and increased the nuclear translocation of phosphorylated STAT3 time‐dependently. Transfection of dominant negative Akt or application of PI3K inhibitorLY294002 significantly inhibited p‐Tyr705‐STAT3, p‐Ser727‐STAT3, and PLGF expression, indicating that Akt is involved in JAK3/STAT3/PLGF signaling cascade. To further examine whether STAT3 binds to the promoter region of PLGF, Chip assay was used and it was found that OSM could bind with PLGF promoter, which was inhibited by JAK3 and PI3K inhibitors. Accumulation of PLGF in the pannus may contribute to the inflammation, angiogenesis and joints destruction in RA patients. These findings demonstrated the important role of OSM in the pathology network of RA and provided novel therapeutic drug targets for RA treatment. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway

C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel^TM with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.     

Vascular endothelial growth factor signaling in VE-cadherin expression and tube-like formation by rheumatoid arthritic synovial fibroblast-like cells

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.     

Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis

The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. Fas ligand is critical to the homeostatic regulation of the immune response, but its role in the angiogenic process of RA remains to be defined. In this study, we investigated whether soluble Fas ligand (sFasL) induces synoviocyte apoptosis and regulates angiogenesis of endothelial cells in RA. The levels of sFasL were elevated in the synovial fluids of RA patients when compared to those of osteoarthritis (OA) patients, and they correlated inversely with vascular endothelial growth factor₁₆₅ (VEGF₁₆₅) concentrations. sFasL, ranging from 10 to 100 ng/ml, induced the apoptosis of RA fibroblast-like synoviocytes (FLS) in vitro, and thereby decreased VEGF₁₆₅ production. In addition, sFasL inhibited VEGF₁₆₅-induced migration and chemotaxis of endothelial cells to basal levels in a manner independent of the Fas-mediated cell death. sFasL dose-dependently suppressed the VEGF₁₆₅-stimulated increase in pAkt expression in endothelial cells, which might be associated with its anti-migratory effect on endothelial cells. Moreover, sFasL strongly inhibited neovascularization in the Matrigel plug in vivo. Our data suggest that sFasL shows anti-angiogenic activity within RA joints not only by inducing apoptosis of VEGF₁₆₅-producing cells but also by blocking VEGF₁₆₅-induced migration of endothelial cells, independent of Fas-mediated apoptosis.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.     

Proteomic studies of rat tibialis anterior muscle during postnatal growth and development

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.     

Over-expression of PDGFR-β promotes PDGF-induced proliferation, migration, and angiogenesis of EPCs through PI3K/Akt signaling pathway

The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes.     

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.