Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.     

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation

Survival and proliferation of many types of vascular endothelial cells are influenced by fibroblast growth factor (FGF)1. Removal of FGF from the medium of human umbilical vein endothelial cells (HUVEC) in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide, an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in culture. The process of active death of vascular endothelial cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels.     

Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells

Apoptosis in vascular endothelial cells is suppressed by fibroblast growth factor (FGF)1. In order to investigate the signal transduction system that regulates endothelial apoptosis, we studied the effects of several mitogenic factors. Apoptosis occurred in human vascular endothelial cells under serum-free conditions, and FGF inhibited apoptosis without a requirement of any cooperative factors, as distinct from the mitogenic response. Other mitogenic agents, such as epidermal growth factor, transferrin, transforming growth factor beta, and interleukin 1 etc., with the exception of dexamethasone, had no such inhibitory effects. The effect of FGF was mimicked by a phorbol ester and was prevented by an inhibitor of protein kinase C. The results suggest that the FGF and protein kinase C are important in endothelial apoptosis.     

Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity: Impact of vascular endothelial growth factor

Endothelial cells play a crucial role in the pathogenesis of many diseases and are highly sensitive to low gravity conditions. Using a three-dimensional random positioning machine (clinostat) we investigated effects of simulated weightlessness on the human EA.hy926 cell line (4, 12, 24, 48 and 72 h) and addressed the impact of exposure to VEGF (10 ng/ml). Simulated microgravity resulted in an increase in extracellular matrix proteins (ECMP) and altered cytoskeletal components such as microtubules (alpha-tubulin) and intermediate filaments (cytokeratin). Within the initial 4 h, both simulated microgravity and VEGF, alone, enhanced the expression of ECMP (collagen type I, fibronectin, osteopontin, laminin) and flk-1 protein. Synergistic effects between microgravity and VEGF were not seen. After 12 h, microgravity further enhanced all proteins mentioned above. Moreover, clinorotated endothelial cells showed morphological and biochemical signs of apoptosis after 4 h, which were further increased after 72 h. VEGF significantly attenuated apoptosis as demonstrated by DAPI staining, TUNEL flow cytometry and electron microscopy. Caspase-3, Bax, Fas, and 85-kDa apoptosis-related cleavage fragments were clearly reduced by VEGF. After 72 h, most surviving endothelial cells had assembled to three-dimensional tubular structures. Simulated weightlessness induced apoptosis and increased the amount of ECMP. VEGF develops a cell-protective influence on endothelial cells exposed to simulated microgravity.     

Anti-tumor effects of vascular endothelial growth factor/vascular endothelial growth factor receptor binding domain-modified chimeric antigen receptor T cells

Background aimsThe vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling pathway plays an important role in angiogenesis and lymphangiogenesis, which are closely related to tumor cell growth, survival, tissue infiltration and metastasis. Blocking/interfering with the interaction between VEGF and VEGFR to inhibit angiogenesis/lymphangiogenesis has become an important means of tumor therapy.MethodsHere the authors designed a novel chimeric antigen receptor (CAR) lentiviral vector expressing the VEGF-C domain targeting both VEGFR-2 and VEGFR-3 (VEGFR-2/3 CAR) and then transduced CD3-positive T cells with VEGFR-2/3 CAR lentivirus.ResultsAfter co-culturing with target cells, VEGFR-2/3 CAR T cells showed potent cytotoxicity against both VEGFR-2- and VEGFR-3-positive breast cancer cells, with increased simultaneous secretion of interferon gamma, tumor necrosis factor alpha and interleukin-2 cytokines. Moreover, CAR T cells were able to destroy the tubular structures formed by human umbilical vein endothelial cells and significantly inhibit the growth, infiltration and metastasis of orthotopic mammary xenograft tumors in a female BALB/c nude mice model.ConclusionsThe authors’ results indicate that VEGFR-2/3 CAR T cells targeting both VEGFR-2 and VEGFR-3 have significant anti-tumor activity, which expands the application of conventional CAR T-cell therapy.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.     

Signal transduction by vascular endothelial growth factor receptors

VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.     

Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.     

Choline phospholipid metabolites of human vascular endothelial cells altered by cyclooxygenase inhibition,growth factor depletion,and paracrine factors secreted by cancer cells

Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO), reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs). We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.     

Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina

Mice or humans with photoreceptor degenerations experience permeability and dropout of retinal capillaries. Loss of photoreceptors results in decreased oxygen usage and thinning of the retina with increased oxygen delivery to the inner retina. To investigate the possibility that increased tissue oxygen plays a role in the vascular damage, we exposed adult mice to hyperoxia, which also increases oxygen in the retina. After 1, 2, or 3 weeks of hyperoxia, there was a statistically significant decrease in retinal vascular density that was not reversible, and endothelial cell apoptosis was demonstrated by TUNEL staining. Mice exposed to hyperoxia and mice with photoreceptor degeneration both showed decreased expression of VEGF in the retina. After complete or near-complete degeneration of photoreceptors, there was increased expression of VEGF in RPE cells, which may explain the association of photoreceptor degeneration and neovascularization in or around the RPE. Increased expression of VEGF in photoreceptors of transgenic mice failed to prevent hyperoxia-induced retinal capillary dropout. These data suggest that increased oxygen in the retina, either by increased inspired oxygen or by photoreceptor degeneration, results in endothelial cell death and dropout of capillaries. Decreased expression of VEGF may be a contributing factor, but the situation may be more complicated for mature retinal vessels than it is for immature vessels, because VEGF replacement does not rescue mature retinal vessels, suggesting that other factors may also be involved.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Eicosanoid regulation of vascular endothelial growth factor expression and angiogenesis in microvessel endothelial cells

12(R)-Hydroxy-5,8,14-eicosatrienoic acid (HETrE) is a potent inflammatory and angiogenic eicosanoid in ocular and dermal tissues. Previous studies suggested that 12(R)-HETrE activates microvessel endothelial cells via a high affinity binding site; however, the cellular mechanisms underlying 12(R)-HETrE angiogenic activity are unexplored. Because the synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its interactions with vascular endothelial growth factor (VEGF) in rabbit limbal microvessel endothelial cells. Addition of 12(R)-HETrE (0.1 nm) to the cells increased VEGF mRNA levels with maximum 5-fold increase at 45 min. The increase in VEGF mRNA was followed by an increase in immunoreactive VEGF protein. 12(R)-HETrE (0.1 nm) rapidly activated the extracellular signal-regulated kinases (ERKs) ERK1 and ERK2. Moreover, preincubation of cells with PD98059, a selective inhibitor of MEK-1, inhibited 12(R)-HETrE-induced VEGF mRNA. Addition of VEGF antibody to cells grown in Matrigel-coated culture plates inhibited 12(R)-HETrE-induced capillary tube-like formation, suggesting that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation of ERK1/2 and that VEGF mediates, at least in part, the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea after hypoxic injury, their interaction may be an important determinant in the development of neovascularized tissues.     

Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin

Follistatin, a hormone which acts to suppress the release of follicle-stimulating hormone (FSH) by pituitary-derived gonadotrophs, has previously been identified only in the liquor folliculi of ovarian follicles. By microsequencing of fractions derived from conditioned medium, we show here that bovine pituitary-derived folliculo stellate cells are also capable of producing and secreting this hormone. These results suggest that folliculo stellate cells may serve as a source of follistatin within the pituitary itself and that the regulation of FSH release from the pituitary could therefore involve a paracrine mechanism.     

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.     

Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner.

Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival.     

Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells

Fibulin-5 (FBLN-5) is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. It is also a gene target of TGF-beta in fibroblasts and endothelial cells that regulates cell proliferation and motility in a context-specific manner. Whereas FBLN-5 expression is low in adult vasculature, its expression is high in developing and injured vasculature, implicating FBLN-5 in regulating angiogenesis and endothelial cell function. We show here that TGF-beta stimulates FBLN-5 expression in endothelial cells, and that this response was inhibited by coadministration of the proangiogenic factor, VEGF. FBLN-5 expression was downregulated significantly during endothelial cell tubulogenesis, implying that FBLN-5 expression antagonizes angiogenesis. Accordingly, FBLN-5 overexpression in or recombinant FBLN-5 treatment of endothelial cells abrogated their ability to undergo angiogenic sprouting, doing so by inhibiting endothelial cell proliferation and invasion through Matrigel matrices. Moreover, FBLN-5 antagonized VEGF signaling in endothelial cells, as well as enhanced their expression of the antiangiogenic factor, thrombospondin-1. Finally, the ability of FBLN-5 to antagonize angiogenic processes was determined to be independent of its integrin-binding RGD motif. Collectively, our findings establish FBLN-5 as a novel antagonist of angiogenesis and endothelial cell activities, and offer new insights into why tumorigenesis downregulates FBLN-5 expression.