Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Enhanced expression of vascular endothelial growth factor in metastatic melanoma

Tumour growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific cytokine. VEGF is angiogenic in vivo and it also acts as a vascular permeability factor. VEGF is overexpressed in many skin disorders characterized by angiogenesis and increased vascular permeability. We investigated VEGF expression in 22 primary cutaneous melanomas, 33 melanoma metastases and six naevocellular naevi using immunohistochemistry. VEGF accumulated on the vascular endothelia in the normal dermis, suggesting that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions. No VEGF was detected in the cells of naevocellular naevi or normal dermis. In contrast, 32% of the primary and 91% of the metastatic melanomas contained melanoma cells staining for VEGF. Expression of VEGF was more frequent in metastases than in primary melanomas (P <0.0001). Tumour-infiltrating inflammatory cells expressed VEGF in all melanomas. A high number of VEGF-expressing inflammatory cells was associated with high VEGF expression in melanoma cells (P = 0.003). Our results suggest that VEGF is up-regulated during the course of melanoma progression and dissemination and that tumour-infiltrating cells expressing VEGF may contribute to the progression of melanoma.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

目的 研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子(VEGF)及其受体( VEGFR)的影响。方法酶联免疫吸附法检测非细胞毒性浓度(5、10、20 ng/ml)青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果培养24、48 h后,无青蒿琥酯作用的SHI-1细胞培养上清液VEGF质量浓度分别为( 980.3±2.2)、(982.4±2.3) pg/ml,VEGFR-1表达率分别为(5.40±3.11)％和(4.45±2.85)％,VEGFR-2表达率分别为(13.90.± 2.26)％和（13.95±1.96）％。5、10、20 ng/ml青蒿琥酯作用24h后,SHI-1细胞培养上清液VEGF质量浓度分别为(234.6±1.8)、(114.9±1.6)、(108.8±1.5) pg/ml,作用48 h后分别为(62.3±1.7)、(60.9±1.6)、(32.7±1.7) pg/ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜ 0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义(均P＜ 0.05)。5、10、20 ng/ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为(4.30±2.21)％、(4.20±1.37)％和(3.90±1.86)％,作用48 h后分别为(3.80±2.87)％、(3.60±1.73)％和(3.00±1.82)％,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义(均P＞ 0.05);作用24h后,SHI-1细胞VEGFR-2阳性率分别为(4.40±1.15)％、(3.10±0.68)％和(1.10±0.72)％,作用48 h后分别为(3.00±1.68)％、(2.20±0.93)％和(0.60±0.92)％,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜ 0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞ 0.05）。结论SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

Regulation of the growth hormone-insulin-like growth factor I axis in developing and adult monkeys is affected by estradiol replacement and supplementation with insulin-like growth factor I

Developmental changes in the GH-insulin-like growth factor I (IGF-I) axis were evaluated in female rhesus monkeys to test the hypothesis that estradiol differentially regulates IGF-I secretion and molar ratios of IGF-I to IGF-binding protein-3 (IGFBP-3) from adolescence into adulthood and that estradiol can reestablish GH secretion in the face of enhanced IGF-I negative feedback inhibition of GH. Adult ovariectomized females were compared to ovariectomized adolescent females studied from 18-36 months of age, a period encompassing the juvenile phase through the expected age at first ovulation. A subgroup of adult (n = 5) and adolescent females (n = 5) was treated continuously with human IGF-I (110 micrograms/kg.day, s.c.) throughout the study period and were compared to age-matched, untreated adults (n = 5) and adolescent animals (n = 6). To further understand how IGF-I affects the GH-IGF-I axis, the acute response to IGF-I (100 micrograms/kg, s.c.) was assessed in adults and at two ages in developing females. Furthermore, all females were treated periodically with estradiol (4 micrograms/kg.day) to assess the effects on the parameters of the GH-IGF-I axis from adolescence into adulthood. Finally, the response to GHRH (1.0 microgram/kg, i.v.) was assessed in adult females and in adolescent females at 18 and 24 months during no estradiol and estradiol replacement. Serum IGF-I and IGFBP-3, in the absence of estradiol replacement, increased significantly throughout puberty before declining from late adolescence into adulthood. Supplementation with IGF-I resulted in a significant increase in both serum IGF-I and IGFBP-3 concentrations at all ages, although the effect was less in juvenile females. Nevertheless, the age-dependent increase and decline in IGF-I and IGFBP-3 were maintained in these supplemented animals. Estradiol replacement significantly increased both serum IGF-I and IGFBP-3 through adolescence, even in IGF-I-supplemented animals. However, with the transition from adolescence, estradiol suppressed serum IGF-I secretion, yet continued to increase IGFBP-3 in young adult and fully adult females. This change in proportionately less IGF-I compared with IGFBP-3 resulted in a significant age-dependent decrease in the molar ratio of IGF-I to IGFBP-3. Indeed, the molar ratio was highest during midadolescence, when both IGF-I and IGFBP-3 were at their zeniths. Serum IGFBP-1 was significantly higher in adolescent compared with adult females. However, estradiol replacement significantly elevated serum IGFBP-1 in adult, but not adolescent, females, abolishing the age differences observed under no estradiol conditions. Serum GH was significantly higher in adolescent compared with adult females; levels in juvenile animals were intermediate. Replacement with estradiol significantly elevated serum GH in adolescent and adult females, particularly in females supplemented with IGF-I. In contrast, estradiol had no effect on serum GH during the juvenile phase. Supplementation with IGF-I significantly dampened the response to GHRH in young and fully adult females, but not in juvenile animals. However, estradiol replacement restored the response to GHRH in these adult, IGF-I-supplemented females. These data indicate that in the absence of any ovarian influence, the decline in serum IGF-I and IGFBP-3 begins in postpubertal, young adult females and is not necessarily a consequence of old age. Furthermore, there is an age-dependent uncoupling of estradiol regulation of the GH-IGF-I axis, as estradiol stimulates GH and IGFBP-3 at all ages but increases serum IGF-I only during adolescent and decreases IGF-I in postpubertal, young adult females. Furthermore, IGF-I has a greater suppressive effect on GH secretion with advancing age, an effect reversed by estradiol replacement. These data suggest that the deficits in the GH-IGF-I axis observed in aged individuals may reflect a continuation of the regulatory changes that begin in young adult females.     

Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription

The growing amount of high quality molecular dynamics simulations generated using the latest methodological developments and force fields has led to a sharper understanding of the forces underlying the dynamics of biomolecular systems, as well as to stimulating insights into the structure and catalysis of nucleic acids. It is now clear that inclusion of long-range electrostatic interactions and of the aqueous and ionic environment is necessary for producing realistic and accurate simulations. Yet, many papers hint at a force field and protocol dependence of the results and thus contain the seeds for the future improvements that will be necessary for deepening our understanding of recognition phenomena and folding of nucleic acids.     

Preparation of N epsilon B28-monoazidobenzoyl insulin-like growth factor I and photoaffinity labeling of insulin-like growth factor I receptor

Recombinant human insulin-like growth factor I (hIGF-I) was reacted with azidobenzoyl hydroxysuccinimide to produce a mixture of photoactive hIGF-I derivatives. The mixture was purified by reversed-phase HPLC to yield three mono-substituted azidobenzoyl hIGF-Is. One of the derivatives was identified by amino acid sequencing as N epsilon B28-monoazidobenzoyl hIGF-I. This derivative was indistinguishable from native hIGF-I when bioassayed in Rat-1 fibroblasts. A 120-kDa band, the alpha subunit of the IGF-I receptor, was specifically labeled in Rat-1 plasma membranes by this photoprobe. The labeling of this band was reduced by hIGF-I at 1 nM and completely abolished by hIGF-I, but not insulin, at 100 nM, indicating the specificity of the photolabeling of the IGF-I receptor by this fully active IGF-I photoprobe.     

Retinoids downregulate vascular endothelial growth factor/vascular permeability factor production by normal human keratinocytes

PURPOSE: We describe the clinical presentation, angiographic findings, and clinical outcome in a group of patients with pseudoaneurysms treated by a new endovascular technique using Guglielmi electrolytically detachable platinum coils (GDCs). METHODS: We retrospectively reviewed the angiographic and clinical findings in a series of 11 patients with pseudoaneurysms occurring in a variety of locations: seven in the cavernous carotid artery, one in the petrous carotid artery, two in the anterior cerebral artery, and one in the cervical vertebral artery. RESULTS: All aneurysms were cured with GDC embolization. The only complication was a branch occlusion, which resolved with heparinization and produced no clinical sequelae. CONCLUSION: Pseudoaneurysms can be safely and effectively treated by embolization with GDCs. Consideration needs to be given to the anatomic location of the pseudoaneurysm and the acuity of onset. Treatment efficacy may by improved if there are bony confines around the aneurysm or if therapy takes place in the subacute period, when the wall of the pseudoaneurysm has matured and stabilized.     

Thalidomide inhibits corneal angiogenesis induced by vascular endothelial growth factor

BACKGROUND: Ocular diseases caused by neovascularization are among the leading causes of blindness. No specific pharmacological treatment is available. Among potential drugs, thalidomide deserves special interest since a wide body of clinical experience exists. However, its antiangiogenic effect is controversial. We therefore investigated the effect of thalidomide on corneal angiogenesis induced by vascular endothelial growth factor (VEGF), which has a special role among angiogenic growth factors. METHODS: Corneal neovascularization was induced in NZW rabbits by an intrastromal pellet loaded with 500 or 750 ng VEGF. Animals received two daily feedings of 200 mg/kg thalidomide. RESULTS: Significant inhibition of corneal angiogenesis (P < 0.0001) was caused by the teratogenic dose of thalidomide after the 5th day of treatment and persisted for more than 16 days. No obvious side effects were recorded. CONCLUSIONS: Thalidomide has a significant antiangiogenic effect against VEGF-induced neovasclar growth. Together with earlier findings this observation indicates that the drug inhibits two angiogenic pathways which are mediated through integrin adhesion molecules.     

Thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor

Thrombomodulin (TM) is a cell-surface receptor that plays a critical role in endothelial cell anticoagulant activity through its cofactor role in the thrombin-catalyzed activation of human protein C. In this study, we examined the effect of vascular endothelial growth factor (VEGF), a potent angiogenic factor, on surface anticoagulant activity and thrombomodulin expression. We show that thrombin-dependent activation of human protein C, measured on the endothelial cell surface, increased from 50 to 80% following exposure of cells to VEGF for 24 h. The effect was concentration dependent with the half-maximal stimulatory effect at approximately 100 pM. This increase in thrombin-dependent aPC generation correlated with a proportional and concentration-dependent increase in the level of cell-surface TM antigen. Both the total cellular TM antigen and the total cellular TM mRNA levels increased approximately 2.5-fold in VEGF-treated cells suggesting that most if not all of the regulation was at the message level. We further show that VEGF blocked IL-1 beta-induced suppression of both TM surface antigen and mRNA and was similarly capable of antagonizing the down-regulation of TM by TGF-beta and from cell activation by LPS. Our data suggest that VEGF regulation of TM may contribute to mechanisms that would maintain local hemostasis during angiogenesis and revascularization and could play a role in minimizing loss of vessel anticoagulant function during inflammatory processes.     

Human neutrophils secrete vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil-derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation.     

Regulation of insulin-like growth factor I (IGF-I) gene expression in brain of transgenic mice expressing an IGF-I-luciferase fusion gene

Insulin-like growth factor I (IGF-I) plays an important role in the development and function of the central nervous system (CNS). Little is known, however, about the factors and mechanisms involved in regulation of CNS IGF-I gene expression. To facilitate our goal to define mechanisms of IGF-I gene regulation in the CNS, we generated several lines of transgenic (Tg) mice that express firefly luciferase (LUC) under control of a 11.3-kb fragment from the 5' region of the rat IGF-I gene. Consistent with expression of the native IGF-I gene in murine brain, expression of the transgene predominated in neurons and astrocytes and used promoter 1, the major IGF-I promoter in the CNS and in most tissues. Transgene messenger RNA and protein expression rapidly increased after birth and peaked at postnatal (P) day 4 in all brain regions studied. LUC activities in all regions then gradually decreased to 0.5-4% of their peak values at P31, except for the olfactory bulb, which maintained about one third of its maximal activity. Compared with littermate controls, administration of dexamethasone decreased LUC activity and transgenic IGF-I messenger RNA abundance, whereas GH significantly increased the expression of the transgene. Addition of GH to cultured fetal brain cells from Tg mice for 12 h also increased LUC activity in a dose-dependent manner (77-388%). These results show that this IGF-I promoter transgene is expressed in a fashion similar to the endogenous IGF-I gene, and thus indicates that the transgene contains cis-elements essential for developmental, GH, and glucocorticoid regulation of IGF-I gene expression in the CNS. These Tg mice should serve as an useful model to study mechanisms of IGF-I gene regulation in the brain.     

Hepatic growth hormone receptor, insulin-like growth factor I, and insulin-like growth factor-binding protein messenger RNA expression in pediatric liver disease

BACKGROUND: Previous studies have shown "beat-to-beat" variation in systemic BP with high-frequency jet ventilation (HFJV). However, it is not clear if such changes are paralleled by changes in cardiac output. OBJECTIVE: To characterize the effect of HFJV near or equal to the heart rate (HR) on beat-to-beat cardiac output in an adult human subject with ARDS. DESIGN: Case study. SETTING: ICU, university teaching hospital. PATIENTS: One patient with end-stage liver disease complicated by sepsis, severe pancreatitis, ARDS, and multisystem organ failure. METHODS: The patient was intubated, sedated, paralyzed, and ventilated with controlled mechanical ventilation (CMV). Ventilatory mode was then switched to HFJV at fixed frequencies (f) near but not equal to the HR (f= 100, 110, and 120 beats/min; HR=108/min). HFJV was then synchronized to the ECG such that f and HR were equal. Continuous cardiac output (COc) was monitored during change of ventilator mode from CMV to fixed-rate HFJV to synchronized HFJV, then followed through progressive delays in jet triggering within the cardiac cycle during the synchronous HFJV mode. COc was monitored by arterial pulse-contour analysis, allowing assessment of beat-to-beat changes in cardiac output. MEASUREMENTS AND MAIN RESULTS: A cyclic variation in COc equal to the beat frequency difference between f and HR was observed (harmonic interaction) during fixed-rate HFJV. This COc oscillation was abolished during synchronous HFJV. COc was significantly greater during systolic synchronous HFJV as compared to diastolic synchronous HFJV or fixed-rate HFJV (10.1 to 9.0 [p<0.05] and to 8.6 [p<0.05] L/min, systolic synchronous to diastolic synchronous and to fixed-rate HFJV, respectively). CONCLUSIONS: This study demonstrates instantaneous variations in cardiac output in a human subject with fixed rates of HFJV near to the HR in humans. These variations are abolished by synchronous HFJV but cardiac output was dependent on the timing of the HFJV inspiration in relation to the cardiac cycle. COc is a potentially valuable method to monitor sudden changes in cardiac output and facilitate attempts to maximize cardiac output during synchronized HFJV.     

Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1. In vivo studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.     

Brain edema in meningiomas is associated with increased vascular endothelial growth factor expression

OBJECTIVE: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-specific cytokine, induces proliferation of endothelial cells and increases vascular permeability dramatically. All gliomas secrete significant amounts of VEGF, whereas meningiomas are variable in expression. Thus, we sought to determine whether the extent of VPF/VEGF expression in meningiomas correlated with differences in brain edema associated with these tumors. METHODS: Meningioma tissue samples from 37 patients (15 men, average age 65 +/- 13 yr; 22 women, average age 60 +/- 10 yr) who underwent surgery at or were referred to the University of Alabama Hospital were examined retrospectively for the extent of expression of immunoreactive VPF/VEGF. Additionally, peritumoral edema was assessed on a blinded basis radiographically from preoperative magnetic resonance imaging scans. Selected specimens were examined by in situ hybridization to document the source of VPF/VEGF. RESULTS: The predominant meningioma subclassifications were transitional (57%) or meningothelial (27%) subtypes. VPF/VEGF immunoreactivity ranged from 0 to 3.5, with a median value of 2 on a subjective 5-point scale; magnetic resonance imaging-assessed edema ranged in extent from 0 to 4 (subjective 5-point scale), with a median value of 2.5. The correlation of determination (R2) of magnetic resonance imaging-assessed tumor edema rating and VPF/VEGF staining intensity rating was 0.6087 (r = 0.78; P = 0.0001). In situ hybridization localized VPF/VEGF messenger ribonucleic acid in meningioma cells and not in normal parenchymal brain cells. CONCLUSION: These data suggest that meningioma-associated edema may be a result of the capacity of meningioma cells to produce VPF/VEGF locally, leading to increased tumor neovascularization and enhanced vascular permeability.