The effects of levonorgestrel implants on vascular endothelial growth factor expression in the endometrium.
(17/2914)
Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endometrium were studied in Norplant users and normal controls, using immunohistochemistry on formalin-fixed paraffin-embedded endometrial sections. The VEGF staining index was quantified using computerized image analysis. The VEGF staining index between stages of the menstrual cycle and between normal and Norplant endometria were compared. Norplant VEGF staining index was analysed for correlation with microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period up to 90 days prior to biopsy, and the length of time since the last bleeding/spotting episode. The results showed that immunoreactive VEGF was detected predominantly in endometrial glands but weakly expressed in the stroma throughout the menstrual cycle, and also in Norplant users. Large variation in the VEGF staining index between individuals was observed and no significant difference in the VEGF staining index was detected between stages of the menstrual cycle for the glands and stroma. The glandular and stromal VEGF staining indices were significantly higher in Norplant than in normal endometrium (P<1x10(-4)). No correlation was found between the Norplant VEGF staining index and endometrial microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period, and the length of time since the last bleeding/spotting episode. The VEGF staining index was higher in glands than stroma for both normal and Norplant endometrium. The results suggest a differential control of endometrial glandular versus stromal VEGF expression, and possible positive effects of levonorgestrel on VEGF expression. (+info)
Regulation of interleukin-8 expression by reduced oxygen pressure in human glioblastoma.
(18/2914)
Oxygen deprivation is an important biological feature of tumor growth. We previously showed that in glioma, anoxia increases expression of IL-8, a chemokine and angiogenic factor. Here, we analysed for the first time the biochemical mechanisms inducing the IL-8 gene upon anoxia in glioma cells, and showed that they differ from those inducing the VEGF gene. Both genes are induced in biologically and genetically heterogenous glioblastoma cell lines (LN-229, LN-Z308, U87MG, T98G), whereas, in gliosarcoma cells (D247MG), only the VEGF gene is induced. The kinetics of IL-8 and VEGF mRNA inductions differ in these cells and reoxygenation experiments showed that the induction is due to the anoxic stress per se. Furthermore, in LN-229 and LN-Z308 cell lines actinomycin D, DRB and nuclear run-on experiments showed that anoxia stimulates increased transcription of both genes. Electromobility shift assays show increased protein binding to the AP-1 site on the IL-8 promoter following anoxia treatment. Finally, in situ hybridization on glioblastoma sections shows that the in vivo expression patterns of IL-8 and VEGF genes overlap, but are not identical. Since intratumoral augmentation of IL-8 and VEGF secretion, following microenvironmental decreases in oxygen pressure, may promote angiogenesis, further definition of these pathways is essential to appropriately target them for antitumoral therapy. (+info)
In vivo significance of ICAM-1--dependent leukocyte adhesion in early corneal angiogenesis.
(19/2914)
PURPOSE: Numerous investigations have stressed the significance of leukocytes in early angiogenesis. Leukocytes invade the cornea, and the location of their extravasation corresponds to the site of vessel ingrowth. The interactions between leukocytes and vascular endothelium are mediated by various proteins, including adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). In this study, the role of ICAM-1 during early corneal angiogenesis was evaluated in vivo. METHODS: Corneal neovascularization was induced in New Zealand White rabbits by use of intrastromal pellets containing 750 ng vascular endothelial growth factor (VEGF). The fluorescent dye rhodamine 6G was used to stain leukocytes in vivo. Leukocyte adhesion and vessel growth were quantified in vivo by high-resolution fluorescence angiography. To inhibit ICAM-1 interactions a microemulsion containing anti-ICAM-1 antibody was applied topically. RESULTS: Limbal vessels showed increased leukocyte adhesion 24 hours after pellet implantation: The number of rolling and sticking leukocytes was significantly increased compared with the number in control animals (P < 0.01). Treatment with anti-ICAM-1 antibody resulted in reduced leukocyte sticking and increased leukocyte rolling. The area covered by new blood vessels was significantly diminished in eyes treated with anti-ICAM-1 (P < 0.05). CONCLUSIONS: The results support the hypothesis that ICAM-1-mediated leukocyte adhesion is a key event in early angiogenesis. This model may serve for investigation of the significance of adhesion molecules by in vivo observation and quantification. (+info)
Role for nitric oxide in the hyperpermeability and hemodynamic changes induced by intravenous VEGF.
(20/2914)
PURPOSE: To explore the effects of brief intravenous (IV) infusion of vascular endothelial growth factor (VEGF) on vascular albumin permeability, blood flow, and vascular conductance (blood flow normalized to arterial blood pressure) in ocular tissues and brain and to assess the role of nitric oxide in mediating these changes. METHODS: A quantitative, double-tracer, radiolabeled albumin permeation method was combined with radiolabeled microspheres for assessment of changes in vascular permeability and blood flow, respectively, induced in ocular tissues by IV infusion of recombinant human VEGF165 for 20 minutes (80-450 picomoles/kg body weight). An inhibitor of nitric oxide synthase (NOS), NG-monomethyl-L-arginine (L-NMMA; 50 micromoles/kg body weight infused simultaneously with VEGF), was used to explore the role of nitric oxide in mediating the vascular changes induced by VEGF. RESULTS: Infusion of VEGF165 in thiopental-anesthetized rats dose-dependently increased 125I albumin permeation in the retina, anterior uvea, and choroid/sclera and in brain, aorta, lung, kidney, small intestine, and peripheral nerve. Mean arterial blood pressure, cardiac output, and stroke volume were decreased only at the highest dose of VEGF, whereas heart rate remained unchanged. Blood flow was increased in the anterior uvea, and vascular conductance was increased in retina, anterior uvea, choroid/sclera, and brain at the highest dose of VEGF. The NOS inhibitor, L-NMMA, blocked VEGF-induced vascular hyperpermeability in all ocular and nonocular tissues, prevented the increase in vascular conductance in all ocular tissues, and blocked the decrease in mean arterial blood pressure, cardiac output, and stroke volume. Infusion of L-NMMA alone decreased vascular conductance in choroid/sclera and kidney, slightly increased mean arterial blood pressure, and in general, did not affect 125I-albumin permeation. (L-NMMA slightly decreased albumin permeation in the retina and increased it in the brain.) CONCLUSIONS: Intravenous infusion of VEGF can acutely impair endothelial cell barrier functional integrity and relax resistance arterioles in ocular tissues and brain through a mechanism involving activation of NOS. (+info)
Human Muller cells express VEGF183, a novel spliced variant of vascular endothelial growth factor.
(21/2914)
PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor expressed as multiple RNA transcripts due to alternative splicing. During an investigation of the expression of VEGF mRNA in human Muller cells cultured under hypoxic conditions, a cDNA species was isolated whose size was incompatible with known VEGF transcripts. This study was performed to determine the nucleotide sequence of the candidate VEGF species and examine the effects of hypoxia on its expression. METHODS: Cultured human Muller cells were exposed to normoxic (20% O2, 5% CO2, 75% N2) or hypoxic (2% O2, 5% CO2, 93% N2) conditions at 37 degrees C for 4 hours and processed for reverse transcription-polymerase chain reaction (RT-PCR), molecular cloning, Southern hybridization, nucleotide sequencing, semiquantitative RT-PCR, and ribonuclease protection assay. RESULTS: The nucleotide sequence of the novel VEGF species isolated from human Muller cells had a short exon 6-encoded sequence without 18-bp nucleotides immediately upstream of the exon 7-encoded sequence in VEGF189. The 18-bp deletion (corresponding to the six amino acids Tyr-Lys-Ser-Trp-Ser-Val) was compatible with a polypeptide containing 183 amino acids (VEGF183). Although VEGF183 mRNA was found in all tissues studied, its expression seemed to be higher than that of VEGF 189 in the brain and spleen; lower in the kidney, retina, skeletal muscle, and liver; and at similar level in the heart. Exposure to hypoxic conditions for 4 hours promoted increased levels of VEGF mRNA including that of VEGF183. CONCLUSIONS: The expression of the novel isoform VEGF 183 in human Muller cells, its variable tissue expression, and its modulation by hypoxia may provide another pathway for VEGF induction of angiogenesis in the retina. (+info)
VEGF deprivation-induced apoptosis is a component of programmed capillary regression.
(22/2914)
The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis. (+info)
Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.
(23/2914)
Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease. (+info)
Chemotactic, mitogenic, and angiogenic actions of UTP on vascular endothelial cells.
(24/2914)
Endothelial cells express receptors for ATP and UTP, and both UTP and ATP elicit endothelial release of vasoactive compounds such as prostacyclin and nitric oxide; however, the distinction between purine and pyrimidine nucleotide signaling is not known. We hypothesized that UTP plays a more important role in endothelial mitogenesis and chemotaxis than does ATP and that UTP is angiogenic. In cultured endothelial cells from guinea pig cardiac vasculature (CEC), both UTP and vascular endothelial growth factor (VEGF) were significant mitogenic and chemotactic factors; in contrast, ATP demonstrated no significant chemotaxis in CEC. In chick chorioallantoic membranes (CAM), UTP and VEGF treatments produced statistically significant increases in CAM vascularity compared with controls. These findings are the first evidence of chemotactic or angiogenic effects of pyrimidines; they suggest a role for pyrimidine nucleotides that is distinct from those assumed by purine nucleotides and provide for the possibility that UTP serves as an extracellular signal for processes such as endothelial repair and angiogenesis. (+info)