Angiogenesis-inflammation cross-talk: vascular endothelial growth factor is secreted by activated T cells and induces Th1 polarization

Vascular endothelial growth factor (VEGF) and its receptors are critical in angiogenesis. The main player in the secretion and response to VEGF is the endothelial cell. We initiated this study to test whether T cells can secrete VEGF and are able to respond to it. Here we show that VEGF is secreted by T cells on stimulation by specific Ag or by IL-2 and by hypoxia; thus, activated T cells might enhance angiogenesis. Hypoxia also induced the expression in T cells of VEGFR2, suggesting that T cells might also respond to VEGF. Indeed, VEGF augmented IFN-gamma and inhibited IL-10 secretion by T cells responding to mitogen or Ag; thus, VEGF can enhance a Th1 phenotype. Encephalitogenic T cells stimulated in the presence of VEGF caused more severe and prolonged encephalomyelitis. Thus, T cells can play a role in angiogenesis by delivering VEGF to inflammatory sites, and VEGF can augment proinflammatory T cell differentiation.     

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary fibrosis in mice

Monocyte chemoattractant protein-1 (MCP-1) is a proinflammatory chemokine and may play an important role in the development of pulmonary fibrosis. We examined a new therapeutic strategy that comprises the transfection of the mutant MCP-1 gene into skeletal muscles as a biofactory for anti-MCP-1 therapy against bleomycin-induced pulmonary fibrosis in mice. Overexpression of the mutant MCP-1 gene at 10-14 days after intratracheal instillation of bleomycin resulted in decreased DNA damage, apoptosis, and pulmonary fibrosis at 14 days. However, overexpression of the mutant MCP-1 at 0-4 days after bleomycin instillation did not result in decreased pathological grade, DNA damage, or apoptosis at 7 and 14 days. Because, in this model, inflammatory cell infiltration begins at 3 days and is followed by interstitial fibrosis, it is likely that MCP-1 has an important role to play in the development of fibrogenesis but not in the development of early lung inflammation. This method does not require the use of viral vector or neutralizing antibody, and, as such, it is possible to avoid problems regarding the pathogenicity of the viral vector or immunocomplex. This new strategy may be a beneficial method of treating pulmonary fibrosis from the viewpoint of clinical application.     

Endogenous expression of angiogenesis-related factors in response to muscle injury

Gene therapy has developed a new strategy to treat a variety of ischemic diseases using angiogenic growth factors. However, the endogenous expression pattern of angiogenesis-related factors in response to muscle injury is not fully characterized. In the present study, we investigated the expression of angiogenesis-related factors, vascular endothelial growth factor, angiopoietin-1, -2, monocyte chemoattractant protein-1, and their receptors during muscle regeneration. Mice underwent freeze injury, and then the gastrocnemius muscles were isolated 1, 3, 5, 7, 10, 14, and 28 days after surgery. Generally, changes in gene expression were most dramatic during the early stage of muscle regeneration, and were attenuated as angiogenesis progressively developed and then returned to steady-state levels. VEGF mRNA began to increase from day 3 and peaked at day 5 after muscle injury. VEGF receptors, Flt-1, KDR/Flk-1, and neuropilin-1 mRNAs were increased from 3- to 9-fold at day 3 after muscle injury. At the same time, angiopoietin-1 and angiopoietin-2 mRNA were increased by 3- and 15-fold respectively, concomitantly with an increase in their receptors and Tie-2 mRNA. Finally, MCP-1 and CC-chemokine receptor 2 mRNAs were sharply up-regulated by 1600- and 100-fold, respectively, at day 3 after muscle injury. These results suggest that the molecular events implicated in angiogenesis occur at an early stage of muscle regeneration.     

Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.     

IGFBP‐4 enhances VEGF‐induced angiogenesis in a mouse model of myocardial infarction

Vascular endothelial growth factor (VEGF) is a well‐known angiogenic factor, however its ability in promoting therapeutic angiogenesis following myocardial infarction (MI) is limited. Here, we aimed to investigate whether dual treatment with insulin‐like growth factor binding protein‐4 (IGFBP‐4), an agent that protects against early oxidative damage, can be effective in enhancing the therapeutic effect of VEGF following MI. Combined treatment with IGFBP‐4 enhanced VEGF‐induced angiogenesis and prevented cell damage via enhancing the expression of a key angiogenic factor angiopoietin‐1. Dual treatment with the two agents synergistically decreased cardiac fibrosis markers collagen‐I and collagen‐III following MI. Importantly, while the protective action of IGFBP‐4 occurs at an early stage of ischemic injury, the action of VEGF occurs at a later stage, at the onset angiogenesis. Our findings demonstrate that VEGF treatment alone is often not enough to protect against oxidative stress and promote post‐ischemic angiogenesis, whereas the combined treatment with IGFBP4 and VEGF can utilize the dual roles of these agents to effectively protect against ischemic and oxidative injury, and promote angiogenesis. These findings provide important insights into the roles of these agents in the clinical setting, and suggest new strategies in the treatment of ischemic heart disease.     

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and 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.     

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions. gene expression; osteogenesis; angiogenesis     

Expression of hypoxia-inducible factors in the peri-implantation mouse uterus is regulated in a cell-specific and ovarian steroid hormone-dependent manner. Evidence for differential function of HIFs during early pregnancy

Increased uterine vascular permeability and angiogenesis are hallmarks of implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor (VEGF). We previously showed that VEGF isoforms and VEGF receptors are expressed in the uterus, suggesting the role of VEGF in uterine vascular permeability and angiogenesis required for implantation and decidualization. We have recently shown that estrogen promotes uterine vascular permeability but inhibits angiogenesis, whereas progesterone stimulates angiogenesis with little effect on vascular permeability. However, the mechanism of differential steroid hormonal regulation of uterine angiogenesis remains unresolved. Oxygen homeostasis is essential for cell survival and is primarily mediated by hypoxia-inducible factors (HIFs). These factors are intimately associated with vascular events and induce VEGF expression by binding to the hypoxia response element in the VEGF promoter. HIFalpha isoforms function by forming heterodimers with the aryl hydrocarbon nuclear translocator (ARNT) (HIF-beta) family members. There is very limited information on the relationship among HIFs, ARNTs, and VEGF in the uterus during early pregnancy, although the role of HIFs in regulating VEGF and angiogenesis in cancers is well documented. Using molecular and physiological approaches, we here show that uterine expression of HIFs and ARNTs does not correlate with VEGF expression during the preimplantation period (days 1-4) in mice. In contrast, their expression follows the localization of uterine VEGF expression with increasing angiogenesis during the postimplantation period (days 5-8). This disparate pattern of uterine HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests HIFs have multiple roles in addition to the regulation of angiogenesis during the peri-implantation period. Using pharmacological, molecular, and genetic approaches, we also observed that although progesterone primarily up-regulates uterine HIF-1alpha expression, estrogen transiently stimulates that of HIF-2alpha.     

Apelin-13 increases myocardial progenitor cells and improves repair postmyocardial infarction

Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and has beneficial effects against myocardial ischemia-reperfusion injury. Little is known about the role of apelin in the homing of vascular progenitor cells (PCs) and cardiac functional recovery postmyocardial infarction (post-MI). The present study investigated whether apelin affects PC homing to the infarcted myocardium, thereby mediating repair and functional recovery post-MI. Mice were infarcted by coronary artery ligation, and apelin-13 (1 mg·kg(-1)·day(-1)) was injected for 3 days before MI and for 14 days post-MI. Homing of vascular PCs [CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/stromal cell-derived factor (SDF)-1α(+), and CD133(+)/CXC chemokine receptor (CXCR)-4(+)] into the ischemic area was examined. Myocardial Akt, endothelial nitric oxide synthase (eNOS), VEGF, jagged1, notch3, SDF-1α, and CXCR-4 expression were assessed at 24 h and 14 days post-MI. Functional analyses were performed on day 14 post-MI. Mice that received apelin-13 treatment demonstrated upregulation of SDF-1α/CXCR-4 expression and dramatically increased the number of CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/SDF-1α(+), and c-Kit(+)/CXCR-4(+) cells in infarcted hearts. Apelin-13 also significantly increased Akt and eNOS phosphorylation and upregulated VEGF, jagged1, and notch3 expression in ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Furthermore, treatment with apelin-13 promoted myocardial angiogenesis and attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 14 days post-MI. Apelin-13 increases angiogenesis and improves cardiac repair post-MI by a mechanism involving the upregulation of SDF-1α/CXCR-4 and homing of vascular PCs.     

Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts

In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose- and time-dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk-1 and Flt-1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up-regulated by a common mediator such as VEGF.     

Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway

The biologically active factors known as adipocytokines are secreted primarily by adipose tissues and can act as modulators of angiogenesis. Visfatin, an adipocytokine that has recently been reported to have angiogenic properties, is upregulated in diabetes, cancer, and inflammatory diseases. Because maintenance of an angiogenic balance is critically important in the management of these diseases, understanding the molecular mechanism by which visfatin promotes angiogenesis is very important. In this report, we describe our findings demonstrating that visfatin stimulates the mammalian target of the rapamycin (mTOR) pathway, which plays important roles in angiogenesis. Visfatin induced the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) in human endothelial cells. Inhibition of the mTOR pathway by rapamycin eliminated the angiogenic and proliferative effects of visfatin. The visfatin-induced increase in VEGF expression was also eliminated by RNA interference-mediated knockdown of the 70-kDa ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR. Visfatin inactivated glycogen synthase kinase 3β (GSK3β) by phosphorylating it at Ser-9, leading to the nuclear translocation of β-catenin. Both rapamycin co-treatment and p70S6K knockdown inhibited visfatin-induced GSK3β phosphorylation at Ser-9 and nuclear translocation of β-catenin. Taken together, these results indicate that mTOR signaling is involved in visfatin-induced angiogenesis, and that this signaling leads to visfatin-induced VEGF expression and nuclear translocation of β-catenin.     

Rapamycin inhibits transforming growth factor β-induced peritoneal angiogenesis by blocking the secondary hypoxic response

Patients with end-stage kidney disease on peritoneal dialysis often develop progressive scarring of the peritoneal tissues. This manifests as submesothelial thickening and is associated with increased vascularization that leads to ultrafiltration dysfunction. Hypoxia induces a characteristic series of responses including angiogenesis and fibrosis. We investigated the role of hypoxia in peritoneal membrane damage. An adenovirus expressing transforming growth factor (TGF) β was used to induce peritoneal fibrosis. We evaluated the effect of the mTOR inhibitor rapamycin, which has been previously shown to block hypoxia-inducible factor (HIF) 1α. We also assessed the effect of HIF1α independently using an adenovirus expressing active HIF1α. To identify the TGFβ1-independent effects of HIF1α, we expressed HIF1α in the peritoneum of mice lacking the TGFβ signalling molecule Smad3. We demonstrate that TGFβ-induced fibroproliferative tissue is hypoxic. Rapamycin did not affect the early angiogenic response, but inhibited angiogenesis and submesothelial thickening 21 days after induction of fibrosis. In primary mesothelial cell culture, rapamycin had no effect on TGFβ-induced vascular endothelial growth factor (VEGF) but did suppress hypoxia-induced VEGF. HIF1α induced submesothelial thickening and angiogenesis in peritoneal tissue. The fibrogenic effects of HIF1α were Smad3 dependent. In summary, submesothelial hypoxia may be an important secondary factor, which augments TGFβ-induced peritoneal injury. The hypoxic response is mediated partly through HIF1α and the mTOR inhibitor rapamycin blocks the hypoxic-induced angiogenic effects but does not affect the direct TGFβ-mediated fibrosis and angiogenesis.     

NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.