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Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay

AMERICAN JOURNAL OF PATHOLOGY 2003年第5期162卷 1431-1439页

作者： Guedez, L Rivera, AM Salloum, R Miller, ML Diegmueller, JJ Bungay, PM Stetler-Stevenson, WG NCI Pathol Lab Ctr Canc Res NIH Bethesda MD 20892 USA NCI Extracellular Matrix Sect Ctr Canc Res Bethesda MD 20892 USA NCI Vasc Biol Fac Ctr Canc Res Bethesda MD 20892 USA NIH Div Bioengn & Phys Sci Off Res Serv Bethesda MD 20892 USA

One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 mul of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and greater than or equal to50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAN allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC50 = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAN efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 mumol/L) and demonstrates a partial inhibition of angiogenesis (similar to40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAN as an assay to compare potencies of angiogenic f

关键词：生物学鉴定法毛细血管通透性/生理学内皮生长因子/药理学内皮血管/病理学明胶酶类/代谢胞间信号肽类和蛋白质类/药理学淋巴因子/药理学新生血管化病理性/病理学新生血管化病理性/病理生理学重组蛋白质类/药理学可重复性结果凝血酶敏感蛋白1/生理学血管内皮生长因子A 血管内皮生长因子类动物人类小鼠

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Mechanical stresses keep endothelial cells healthy: Beneficial effects of a physiological level of cyclic stretch on endothelial barrier function

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AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY 2003年第4期285卷 L782-L784页

作者： Fujiwara, K Univ Rochester Med Ctr Cardiovasc Res Ctr Rochester NY 14642 USA

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Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2) - A reassessment using novel receptor-specific vascular endothelial growth factor mutants

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第5期276卷 3222-3230页

作者： Gille, H Kowalski, J Li, B LeCouter, J Moffat, B Zioncheck, TF Pelletier, N Ferrara, N Genentech Inc Dept Mol Oncol San Francisco CA 94080 USA Genentech Inc Dept Prot Engn San Francisco CA 94080 USA Genentech Inc Dept Metab & Pharmacokinet San Francisco CA 94080 USA Genentech Inc Dept Prot Chem San Francisco CA 94080 USA

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability, VEGF mutants that bind to either KDR or Fit-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase C gamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF, We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.

关键词： 1-磷脂酰肌醇3-激酶/代谢毛细血管通透性/生理学细胞运动/生理学细胞培养的内皮生长因子/代谢内皮血管/细胞学内皮血管/生理学酶激活同工酶类/代谢淋巴因子/代谢丝裂原激活蛋白激酶类/代谢突变新生血管化生理性/生理学磷脂酶Cγ 原癌基因蛋白质类/生理学受体蛋白质酪氨酸激酶类/生理学受体生长因子/生理学受体血管内皮生长因子信号传导/生理学血管内皮生长因子A 血管内皮生长因子受体1 血管内皮生长因子类人类

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Serine/threonine phosphatase 2B regulates protein kinase C-α activity and endothelial barrier function

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AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY 2001年第3期281卷 L544-L545页

作者： Mehta, D Univ Illinois Coll Med Dept Pharmacol Chicago IL 60612 USA

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Pathogenesis of classical swine fever:: Renal haemorrhages and erythrodiapedesis

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JOURNAL OF COMPARATIVE PATHOLOGY 2000年第1期123卷 47-54页

作者： Gómez-Villamandos, JC Ruiz-Villamor, E Bautista, MJ Quezada, M Sánchez, CP Salguero, FJ Sierra, MA Univ Cardoba Fac Vet Dept Pathol Anat Cordoba 14014 Spain Univ Concepcion Dept Patol & Med Prevent Chillan Chile INIA Ctr Invest Sanidad Anim Madrid Spain

Thirty pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (hog cholera) virus to establish the chronological occurrence of lesions in the kidney and to determine the mechanism responsible for renal haemorrhages. The study included the use of histopathological, ultrastructural, immunohistochemical (detection of viral antigen gp55, MAC387, lambda chains, CD3 and Clq) and morphometrical techniques (vascular area). Renal interstitial oedema and haemorrhages were detected from 7 da's post-inoculation (dpi), associated with a slight interstitial mononuclear infiltrate and evidence of viral infection in macrophages and fibroblasts, and in a small proportion of lymphocytes. Viral infection was not detected in capillary endothelial cells. An intense mononuclear infiltrate, with B cells, T cells and small numbers of macrophages, was detected from 10 dpi. In the final phase of the experiment (14 dpi, slight proliferation and degranulation of mast cells were observed. Increased expression of the Clq component of complement was also detected. A significant increase in vascular area was observed from 7 dpi. These results suggest that haemorrhages observed in the kidneys of pigs inoculated with the Quillota strain resulted from erythrodiapedesis and increased vascular permeability, probably aggravated by mast cell degranulation in the final stage of the experiment. The results suggested that mast cell degranulation was linked to activation of the complement system. (C) 2000 Harcourt Publishers Ltd.

关键词：抗原病毒/分析生物学标记/分析毛细血管/代谢毛细血管/超微结构毛细血管/病毒学毛细血管脆性/生理学毛细血管通透性/生理学细胞脱颗粒/生理学细胞运动/生理学猪瘟/病因学猪瘟/免疫学猪瘟/病理学猪瘟病毒/免疫学胞质小泡/生理学胞质小泡/超微结构水肿/病因学水肿/病理学内皮血管/代谢内皮血管/超微结构内皮血管/病毒学红细胞/细胞学红细胞/生理学出血/病因学出血/病理学肾疾病/病因学肾疾病/病理学肥大细胞/生理学肥大细胞/超微结构猪动物女(雌)性男(雄)性

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Effects of temperature on the wall strength and compliance of frog mesenteric microvessels

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JOURNAL OF PHYSIOLOGY-LONDON 2000年第3期526卷 613-622页

作者： Neal, CR Michel, CC Univ London Imperial Coll Sci Technol & Med Div Biomed Sci London SW7 2AZ England

1. In single perfused mesenteric microvessels of pithed frogs, we assessed wall strength from the critical pressure, P-B, which has to be applied within the vessel in order to induce openings in the walls through which fluid and cells can extravasate. P-B was determined in capillaries and venules of tissues at 12-20 degrees C and after cooling to 0-5 degrees C. 2. The P-B (mean +/- S.E.M.) in 22 vessels between 12 and 20 degrees C was 52.64 +/- 3.88 cmH(2)O. In 19 microvessels cooled to less than 5 degrees C, P-B was 92.0 +/- 7.40 cmH(2)O which was significantly higher than at room temperature (P < 0.001). 3. The compliance of the vessel wall was estimated using both the red cell method and the oil meniscus technique. There was no measurable effect of temperature on wall compliance. The compliance of vessels from which the cells had teen removed by previous perfusion with detergent solutions tvas very similar to that of intact Vessels between 12 and 20 degrees C and between 0 and 5 degrees C. 4. The negligible effects of temperature upon compliance suggest that microvessel walls have to be distended to a greater extent in cold tissue before P-B is reached. This, together with their rapid closure, is consistent with the hypothesis that pressure-induced openings in microvascular walls are dependent on an active response of the endothelium rather than being the result of stress failure of the basement membrane.

关键词：基膜/生理学血流速度/药物作用血压/生理学毛细血管/生理学毛细血管通透性/生理学顺应性内皮血管/药物作用内皮血管/生理学内皮血管/超微结构红细胞/细胞学肠系膜/血液供给肠系膜/细胞学肠系膜/超微结构微循环/药物作用微循环/生理学微循环/超微结构显微镜检查电子辛苯昔醇/投药和剂量温度血管阻力/药物作用血管阻力/生理学小静脉/生理学动物

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Lesion characterization using vessel permeability map to new blood pool contrast agent calculated from dynamic magnetic resonance images

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JOURNAL OF DIGITAL IMAGING 2000年第2期13卷 193-195页

作者： Kim, S Kim, JH Moon, WK Min, BG Seoul Natl Univ Hosp Clin Res Inst Seoul 110744 South Korea Seoul Natl Univ Hosp Dept Diagnost Radiol Seoul 110744 South Korea Seoul Natl Univ Coll Engn Dept Biomed Engn Seoul South Korea

Use of contrast enhancement in conjunction with magnetic resonance (MR) imaging provides a means to evaluate tissue function, as well as morphology. Moreover, physiologic properties derived from kinetic analysis of dynamic contrast-enhanced data can improve the specificity of MR examinations. In this study, quantitative analysis of microvascular characteristics based on dynamic MR imaging were performed both for malignant and benign lesions using two types of contrast agents (CAs). A new MR macromolecular contrast medium (MMCM), 24 gadolinium-tetraazacyclododecanetetraacetic acid (DOTA)-dendrimer, was found to have a greater ability to distinguish benign from malignant lesions. When a blood pool agent was used, permeability differences in the two types of lesions were the most significant findings among all parameters considered. Copyright (C) 2000 by W.B. Saunders Company.

关键词：脓肿/病理生理学毛细血管通透性/生理学造影剂钆DTPA 杂环化合物图像增强磁共振成像肿瘤实验性/血液供给有机金属化合物敏感性与特异性睾丸肿瘤/血液供给肿瘤细胞培养的动物人类男(雄)性兔

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Neural immunoregulation: emerging roles for nerves in immune homeostasis and disease

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IMMUNOLOGY TODAY 2000年第6期21卷 281-289页

作者： Downing, JEG Miyan, JA Univ London Imperial Coll Sci Technol & Med Sch Med Dept Neuroendocrinol London W6 8RF England Univ Manchester Inst Sci & Technol Dept Biomol Sci Manchester M60 1QD Lancs England

In this review, James Downing and Jaleel Miyan outline emerging evidence for neural mechanisms that contribute to specific categories of host defence. Involvement of direct innervation in the adaptive control of immunological responses complements an established view of neuroendocrine-immune modulation. The challenge remains to understand the integrative and homeostatic functions of 'hardwiring' of peripheral immune effector sites, its bearing on disorder and potential for therapeutic modification.

关键词：传入通路/生理学毛细血管通透性/生理学细胞运动肠神经系统/生理学糖皮质激素类/生理学内环境稳定下丘脑-垂体系统/生理学免疫系统/生理学肠黏膜/免疫学肠黏膜/神经支配杀伤细胞天然/免疫学肥大细胞/免疫学模型免疫学模型神经学神经纤维/生理学神经免疫调节/生理学神经元传入/生理学神经分泌系统/生理学垂体肾上腺系统/生理学脾/细胞学脾/免疫学应激/免疫学应激/病理生理学交感神经纤维节后/生理学交感神经系统/生理学迷走神经/生理学动物大鼠

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Acute effects of bradykinin on cerebral microvascular permeability in the anaesthetized rat

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JOURNAL OF PHYSIOLOGY-LONDON 2000年第1期528卷 177-187页

作者： Sarker, MH Hu, DE Fraser, PA Kings Coll London Ctr Cardiovasc Biol & Med Div Physiol London SE1 1UL England

1. The permeability response to acutely applied bradykinin and [des-Arg(9)]-bradykinin on single cerebral venular capillaries has been investigated using the low molecular mass fluorescent dyes Lucifer Yellow and Sulforhodamine B with the single vessel occlusion technique. 2. When bradykinin was applied repeatedly for up to 2 h, the permeability increase was small and reversible for concentrations that ranged from 5 nM to 50 muM. 3. The logEC(50) of the permeability response to bradykinin was -5.3 +/- 0.15 (logM;mean +/- S.E.M.). This was reduced to -6.37 +/- 0.24 with the angiotensin-converting enzyme inhibitor captopril, to -6.33 +/- 0.19 with the neutral endopeptidase inhibitor phosphoramidon and to -7.3 +/- 0.20 with captopril and phosphoramidon combined. 4. The permeability response to bradykinin was blocked by the bradykinin B-2 receptor antagonist HOE 140, by inhibition of the Ca2+-independent phospholipase A(2), by the scavenging of free radicals, or by inhibition of both cyclo-oxygenase and lipoxygenase in combination. Block of Ca2+ entry channels with SWP 96365 had no effect on the response. 5. Application of [des-Arg(9)] bradykinin also increased permeability over the concentration range 5 nM to 50 muM, with a logEC(50) of -5.6 +/- 0.37. This response was not affected by free radical scavenging, but was completely blocked by the histamine H-2 receptor blocker cimetidine, 6. These results imply that the acute permeability response to bradykinin is mediated via the release of arachidonic acid, which is acted on by cyclo-oxygenase and lipoxygenase resulting in the formation of free radicals, and that the response to [des-Arg(9)]-bradykinin is mediated via histamine.

关键词：麻醉血管紧张素转换酶抑制药/药理学花生四烯酸/代谢缓激肽/类似物和衍生物缓激肽/代谢缓激肽/药理学毛细血管/药物作用毛细血管/代谢毛细血管通透性/药物作用毛细血管通透性/生理学脑血管循环/药物作用脑血管循环/生理学剂量效应关系药物自由基清除剂/药理学自由基/拮抗剂和抑制剂自由基/代谢大鼠 Wistar 第二信使系统/药物作用第二信使系统/生理学动物女(雌)性男(雄)性大鼠

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Anti-inflammatory effects of mutant forms of secretory leukocyte protease inhibitor

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AMERICAN JOURNAL OF PATHOLOGY 2000年第3期156卷 1033-1039页

作者： Mulligan, MS Lentsch, AB Huber-Lang, M Guo, RF Sarma, V Wright, CD Ulich, TR Ward, PA Univ Michigan Sch Med Dept Pathol Ann Arbor MI 48109 USA Univ Michigan Sch Med Dept Surg Ann Arbor MI 48109 USA Univ Louisville Sch Med Dept Surg Louisville KY 40292 USA Amgen Inc Thousand Oaks CA 91320 USA

The secretory leukocyte protease inhibitor (SLPI) is found in a variety of secreted fluids in mammals and is a known inhibitor of serine proteases. Wild-type (WT) SLPI has recently been shown to block nuclear factor kappa B (NF-kappa B) activation in rat lungs and to interfere with the ensuing inflammatory response and recruitment of neutrophils after an intrapulmonary deposition of IgG immune complexes. In this study, WT SLPI and SLPI mutants with various degrees of protease-inhibitory capacity (for trypsin, chymotrypsin, and elastase) were evaluated for their ability to suppress the lung-vascular leak, neutrophil accumulation, and NF-kappa B activation in the lung inflammatory model The SLPI mutant with Gly(72) (replacing Leu(72)) lost its ability to block in vivo activation of NF-kappa B, as well as its ability to suppress the lung vascular leak and neutrophil recruitment. The Phe(72) and Gly(20) mutants were as effective as the WT SLPI in suppressing NF-kappa B activation and neutrophil recruitment. The Lys(72) mutant had the most suppressive effects of the lung vascular leak and for neutrophil recruitment into the lung. The in vivo suppressive effects of SLPI mutants on lung vascular permeability, neutrophil recruitment, and NF-kappa B activation appear to be most closely related to their trypsin-inhibiting activity. These data suggest that the suppressive effects of SLPI on the intrapulmonary activation of NF-kappa B and neutrophil recruitment into the lung may be linked to their antiprotease activity, directed, perhaps, at the intracellular proteases.

关键词：急性病肺泡炎外源性变应性/代谢肺泡炎外源性变应性/病理生理学氨基酸取代消炎药抗原抗体复合物/免疫学毛细血管通透性/生理学疾病模型动物免疫球蛋白G/免疫学肺/血液供给肺/代谢肺/病理学诱变定点 NF-κB/代谢中性白细胞浸润/生理学蛋白酶抑制蛋白质类分泌蛋白质类/遗传学蛋白质类/生理学大鼠 Long-Evans 分泌型白细胞蛋白酶抑制因子丝氨酸蛋白酶抑制剂/遗传学丝氨酸蛋白酶抑制剂/生理学无特异性病原体有机物动物男(雄)性大鼠

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