缺氧诱导人肾间质成纤维细胞血管内皮生长因子的表达

目的 阐明缺氧对人肾间质成纤维细胞血管内皮生长因子(VEGF)表达的影响。方法 原代培养获得人肾间质成纤维细胞,传至第3代用于本实验。细胞经缺氧处理24 h或正常培养后以半定量RT-PCR法检测VEGF mRNA表达量,以流式细胞术检测细胞表面VEGF蛋白含量,以双抗夹心ELISA法检测培养上清VEGF蛋白含量。结果 (1)缺氧能显著增加人肾间质成纤维细胞VEGF mRNA表达(0.34±0.04 vs 0.10±0.01,n=4,P<0.01)。(2)缺氧后细胞表面VEGF蛋白含量较正常对照显著增加(平均荧光强度:1.053±0.055 vs 0.763±0.057,n=4,P<0.01)。(3)缺氧培养细胞上清VEGF含量显著高于正常对照组[(2.009±0.144)mg/L vs(1.647±0.175)mg/L,n=4,P<0.05]。结论 缺氧能诱导人肾间质成纤维细胞表达VEGF,这在缺氧引起的肾脏病理改变中可能起到一定的作用。     

Elevated interleukin-6 expression in keloid fibroblasts

Keloids are characterized by a net accumulation of collagen. To date, the role of growth factors and various cytokines in the pathogenesis of these lesions has not been fully characterized. Interleukin-6 (IL-6) is an important immunoregulatory cytokine that has been implicated in a number of fibrotic autoimmune diseases such as scleroderma, interstitial nephritis, and pulmonary interstitial fibrosis. However, the role of IL-6 in the development of keloids has yet to be defined. This study demonstrates increased expression of the IL-6 gene in fibroblasts isolated from patients with keloids when compared with control fibroblasts using the ribonuclease protection assay. Subsequent detection of increased levels of IL-6 secretion by keloid fibroblasts is also demonstrated under unstimulated and stimulated conditions using serum and interferon gamma (IFN-gamma) (unstimulated: 0.3694 + 0.2499 pg/cell vs 0.0662 + 0.0786 pg/cell, P = 0.0137; serum: 1.066 + 0.513 pg/cell vs 0.233 + 0.231 pg/cell, P = 0.0027; serum and IFN-gamma: 1.286 + 0.395 pg/cell vs 0.244 + 0.199 pg/cell, P < 0.0001). These results suggest that IL-6 may play a significant role in the pathogenesis of keloids.     

The interleukin-6 and vascular endothelial growth factor in hematopoietic stem cell transplantation

We studied the correlation between changes in the serum levels of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) with complications such as acute graft versus host disease (aGVHD), veno-occlusive disease (VOD) or occurrence of infection after hematopoietic stem cell transplantation (HSCT). Serum VEGF and IL-6 levels were sequentially measured by enzyme-linked immunosorbant assay (ELISA) in 35 patients who had undergone HSCT. Serum levels of IL-6 in patients with aGVHD were increased in comparison with patients without aGVHD, but the difference was not statistically significant. Serum levels of VEGF were only increased in patients with aGVHD during the early days after transplantation. No significantly altered levels of IL-6 and VEGF were observed in patients with VOD or sepsis. These results demonstrate that rising levels of VEGF and IL-6 may be good and specific biomarkers for transplant aGVHD.     

Increased vascular endothelial growth factor production in fibroblasts isolated from strictures in patients with Crohn's disease

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that is implicated in early wound healing and fibrosis. Fibroblasts may initiate stricture formation in Crohn's disease through overexpression of VEGF. The aim of this study was to examine VEGF expression and regulation in fibroblasts isolated from patients with Crohn's disease. METHODS: Fibroblasts were isolated by a primary explant technique from serosal biopsies of non-strictured and strictured segments of bowel from eight patients undergoing resection for Crohn's disease, and normal colon from six patients undergoing resection for benign and malignant colorectal disease. Fibroblasts were cultured with transforming growth factor (TGF) beta and corticosteroids. After 24 h the culture supernatant was collected for VEGF assay by enzyme-linked immunosorbent assay. RESULTS: VEGF production was significantly higher in fibroblasts isolated from strictures (mean(s.e.m.) 1980(260) pg/ml) than from non-strictured segments (1116(165) pg/ml) in patients with Crohn's disease or control fibroblasts (898(93) pg/ml). TGF-beta increased VEGF production in normal and non-strictured Crohn's fibroblasts. Corticosteroids suppressed unstimulated VEGF production in all groups. CONCLUSION: Enhanced serosal fibroblast VEGF production might play a role in initiating stricture formation in Crohn's disease. VEGF production in serosal fibroblasts is sensitive to stimulation with TGF-beta. Corticosteroids may reduce stricturing through suppression of VEGF.     

Neutralizing vascular endothelial growth factor activity inhibits thyroid cancer growth in vivo

BACKGROUND: Without angiogenesis, tumor growth is limited to a few millimeters, the limit of diffusion. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen and a major regulator of angiogenesis. METHODS: To investigate the relationship between VEGF and thyroid tumor angiogenesis, we xenografted human dermal matrix inoculated with FTC-133 cells into nude mice or directly injected FTC-133 cells subcutaneously. To block the function of VEGF, the neutralizing anti-VEGF monoclonal antibody A.4.6.1 (mAb A.4.6.1) was injected intraperitoneally twice weekly. As control, an antibody of the same isotype (Ab 5B6) or phosphate buffer saline solution (PBS) was used. To evaluate the dermal matrix as a model for angiogenesis studies, recombinant human VEGF was inoculated into the dermal matrix pocket and xenografted into mice. RESULTS: In the dermal matrix angiogenesis model, the number of blood vessels paralleled the concentration of recombinant human VEGF and was highest at 100 ng/mL. Mice that were treated with the mAb A4.6.1 developed fewer blood vessels (mean, 6.6 per HPF) than control mice (18 per HPF in Ab 5B6 and 22 per HPF in PBS; P <.01). Tumors from mice that were treated with mAb A.4.6.1 were much smaller (mean +/- SD, 0.09 +/- 0.02 gm) at 5 weeks, compared with the tumors treated with Ab 5B6 (5.38 +/- 1.15 gm) or PBS (4.0 +/- 0.72 gm; P <.001). CONCLUSIONS: VEGF is produced by the follicular thyroid cancer cell line and stimulates angiogenesis and growth of thyroid cancer. This stimulation can be blocked by mAb A.4.6.1.     

Vascular endothelial growth factor inhibits mitogen-induced vascular smooth muscle cell proliferation

INTRODUCTION: Delivery of vascular endothelial growth factor (VEGF) protein or gene transfer has been shown to accelerate re-endothelialization and attenuate neointimal hyperplasia in various arterial injury models, including balloon injury, stent implantation, and vein grafts. In addition to stimulating re-endothelialization, we hypothesize that VEGF has further vascular protective functions to prevent neointimal hyperplasia by directly inhibiting mitogen-induced proliferation of vascular smooth muscle cells (VSMCs) via the mitogen-activated protein kinase pathway. MATERIALS AND METHODS: Human aortic VSMCs were seeded and serum starved for 24 h. The cells were then stimulated with a mitogen, recombinant human platelet derived growth factor at 20 ng/mL together with 0, 10, 20, 30, 40, 50 ng/mL recombinant human VEGF. A proliferation assay was used to quantitate bromodeoxyuridine uptake into newly synthesized DNA. Western immunoassay was used to quantify extracellular signal-regulated kinase (ERK) 2 protein and phosphorylation of retinoblastoma and ERK 1/2 protein. RESULTS: VEGF inhibited bromodeoxyuridine incorporation into mitogen-induced VSMC in a dose-dependent manner, reaching statistical significance at concentrations of 30 (P < 0.05), 40 (P < 0.05), and 50 ng/mL (P < 0.01). Densitometry of western immunoblots revealed an inhibition of phosphorylation of retinoblastoma at VEGF concentrations of 40 and 50 ng/mL and ERK 1/2 phosphorylation at concentrations of 30, 40 and 50 ng/mL. CONCLUSION: In addition to stimulating re-endothelialization, VEGF appears to have a vascular protective function by directly inhibiting VSMC proliferation. This effect occurs in the absence of endothelial cells and via the mitogen-activated protein kinase pathway. VEGF may serve as an important modulator of mitogen-induced VSMC proliferation after vascular injury.     

Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation

OBJECTIVE: The goal of this work was to test the functional role of vascular endothelial growth factor (VEGF) in promoting the vigorous granulation tissue formation, wound fluid accumulation, and angiogenic responses characteristic of this wound model. BACKGROUND: Formation of vessel-rich granulation tissue is central to wound repair and is thought to be regulated by locally liberated angiogenic factors. Despite the clinical importance of granulation tissue formation in the early stage of wound healing, surprisingly little is known about the molecular identity of signals leading to granulation tissue invasion of a wound space. Methods. A ventral hernia, surgically created in the abdominal wall of 15 swine, was repaired using silicone sheeting and skin closure. An osmotic minipump, inserted in a remote subcutaneous pocket, delivered saline (n = 5), an irrelevant control antibody (n = 5), or neutralizing anti-VEGF antibody (n = 5) into the wound environment. Serial ultrasonography on Days 2, 4, 7, 9, 11, and 14 was used to determine the dimensions of the subcutaneous granulation tissue and wound fluid compartment. VEGF and transforming growth factor beta1 (TGF-beta1) levels in serial wound fluid samples were quantitated by ELISA. On Day 14, animals were sacrificed and the abdominal wall was harvested for histologic, biochemical, and molecular analyses. RESULTS: In animals receiving saline or an irrelevant antibody, a nearly linear 4-fold increase in granulation tissue thickness and 7-fold increase in wound fluid volume were measured over the 14-day study interval. In contrast, in animals receiving anti-VEGF neutralizing antibody, Day 14 granulation tissue thickness and wound fluid volume measurements were essentially unchanged from Day 2 values. Moreover, in the anti-VEGF animals, ultrasonography was unable to resolve the "angiogenic zone" typical of both controls, and correspondingly, wound vessel count and vascular surface area estimates derived from image analysis of histological sections were 3-fold lower in the anti-VEGF animals compared with the saline and antibody controls. Finally, VEGF levels in wound fluid detectable by ELISA analysis were strikingly (10-fold) reduced in anti-VEGF animals on Postsurgery Days 7-14. In contrast, TGF-beta1 levels were unaffected by the anti-VEGF treatment. CONCLUSION: Functional VEGF is a key mediator in wound angiogenesis, fluid accumulation, and granulation tissue formation.     

Alveolar and plasma concentrations of interleukin-8 and vascular endothelial growth factor following oesophagectomy

The acute respiratory distress syndrome occurs in approximately 10% of all patients undergoing elective oesophagectomy. Local increases in lung pro-inflammatory cytokines have been previously detected in high-risk patients before the development of the acute respiratory distress syndrome. We hypothesised that similar changes would occur following oesophagectomy. Two groups of patients were studied. In the collapsed lung group (n = 11), interelukin-8 and vascular endothelial growth factor were measured in bronchoalveolar lavage samples obtained from the intra-operative collapsed lung after operation. In the ventilated lung group (n = 10), bronchoalveolar lavage was performed after operation from the ventilated lung and cytokines measured. Cytokines were also measured in peripheral blood samples before and after operation. Bronchoalveolar lavage cytokine levels in both lungs were of an order of magnitude greater than in peripheral blood. Pulmonary pro-inflammatory cytokine release occurs following oesophageal surgery and may indicate subclinical lung injury.     

Resveratrol inhibits fibrogenesis and induces apoptosis in keloid fibroblasts

Keloids are benign dermal fibrotic tumors arising during the wound healing process. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Resveratrol, a dietary compound, has anticancer properties and, from recent studies, it has been suggested that resveratrol may have an antifibrogenic effect on organs such as the liver and kidney. Based on this idea, we investigated its effect on the regulation of extracellular matrix expression, proliferation, and apoptosis of keloid fibroblasts. Type I collagen, α‐smooth muscle actin, and heat shock protein 47 expression decreased in resveratrol‐treated keloid fibroblasts in a dose‐dependent manner. In addition, resveratrol diminished transforming growth factor‐β1 production by keloid fibroblasts. We also demonstrated that it suppressed their proliferation and induced apoptosis of the fibroblasts. Conversely, resveratrol did not decrease type I collagen, α‐smooth muscle actin, and heat shock protein 47 mRNA expression in normal skin fibroblasts and barely suppressed cell proliferation. Our data indicate that resveratrol may have an antifibrogenic effect on keloid fibroblasts without any adversely effects on normal skin fibroblasts, suggesting the potential application of resveratrol for the treatment of keloids.     

Elevated vascular endothelial growth factor production in islets improves islet graft vascularization

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, of which only approximately 30% become stably engrafted. Rapid and adequate revascularization of transplanted islets is important for islet survival and function. Delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in islet cell death and early graft failure. To improve islet revascularization, we delivered human vascular endothelial growth factor (VEGF) cDNA to murine islets, followed by transplantation under the renal capsule in diabetic mice. Diabetic animals receiving a marginal mass of 300 islets that were pretransduced with a VEGF vector exhibited near normoglycemia. In contrast, diabetic mice receiving an equivalent number of islets that were transduced with a control vector remained hyperglycemic. Immunohistochemistry with anti-insulin and anti-CD31 antibodies revealed a relatively higher insulin content and greater degree of microvasculature in the VEGF vector-transduced islet grafts, which correlated with significantly improved blood glucose profiles and enhanced insulin secretion in response to glucose challenge in this group of diabetic recipient mice. These results demonstrate that VEGF production in islets stimulates graft angiogenesis and enhances islet revascularization. This mechanism might be explored as a novel strategy to accelerate islet revascularization and improve long-term survival of functional islet mass posttransplantation.     

血管内皮生长因子基因在原代培养大鼠肝细胞的转染表达

目的　研究血管内皮生长因子基因 (VEGF12 1)在原代培养大鼠肝细胞中转染表达。方法　构建 pIRES EYFP /VEGF12 1质粒 ,逆转录 聚合酶链反应 (RT PCR)、WesternBlot和荧光显微镜观察VEGF基因在原代培养大鼠肝细胞中的转染表达。结果　pIRES EYFP/VEGF12 1质粒成功构建并转染原代培养大鼠肝细胞 ,RT PCR观察到 2 5 8bp的VEGF特异性条带 ,WesternBlot检测到相对分子质量 42× 10 3 的特异性条带 ,荧光显微镜观察到转染阳性肝细胞发出黄绿色荧光。结论　pIRES EYFP/VEGF12 1质粒在原代培养大鼠肝细胞转染并表达 ,为VEGF基因修饰肝细胞移植和基因治疗奠定基础。     

血管内皮生长因子在原发性肝细胞癌中的表达及其作用

目的：探讨血管内皮生长因子（VEGF）的表达与原发性肝细胞癌（PHCC）侵袭和转移的关系。方法：采用免疫组织化学链酶卵白素－辣根过氧化物酶（LSAB）法对30例PHCC和20例癌旁组织手术切除后的石蜡标本组织中的VEGF表达及微血管密度（MVD）进行检测.结果 VEGF表达,MVD在PHCC组织中比癌旁组织明显增高,在PHCC中,有转移者及包膜不完整者VEGF表达,MVD明显高于无转移者及包膜完整者,VEGF表达,MVD在大肿瘤（直径＞5厘米）与小肿瘤（直径小于等于5厘米）两者间无显著性差异,VEGF的表达与MVD相关,结论：PHCC组织中的VEGF表达,MVD与肿瘤的侵袭和转移行为密切相关,可作为判定PHCC发生,转移及预后的指标,为监测和治疗提供了新思路。     

Interaction between stromal fibroblasts and colorectal cancer cells in the expression of vascular endothelial growth factor

BACKGROUND: Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been implicated in metastasis of colorectal cancer (CRC). The present study aimed to clarify whether cancer-stromal interaction induces the production of VEGF. MATERIALS AND METHODS: Human colonic fibroblasts (CCD-18Co) and CRC (SW480, SW620) cells were analyzed in this study. The cell cycle of colonic fibroblasts during co-culture was analyzed by flow cytometry. VEGF and TGF-beta1 released into the conditioned media in co-culture models were measured. Northern blot with human specific VEGF probe was performed to identify the expression of VEGF in this model. RESULTS: Co-culture of colonic fibroblasts with CRC cells increased the viability of fibroblasts during co-culture. Cell cycle analysis revealed that most of the fibroblasts co-cultured with CRC cells were arrested at G1 phase and few cells were in sub-G1 phase that indicates apoptosis. Although VEGF protein was detected in the culture media of all of the monocultures, co-cultivation of CRC with fibroblasts resulted in synergistic increase of VEGF production compared with monocultures. However TGF-beta1 protein was not detected in any conditioned medium. VEGF mRNA was detected in both CRC and fibroblasts. Under co-culture condition, an abundance of VEGF mRNA expression was noted in fibroblasts relative to CRC cells. CONCLUSIONS: The present study suggests that CRC manipulates the host stroma to suppress apoptosis and up-regulate VEGF production.     

血清血管内皮生长因子与肿瘤

肿瘤血管形成在实体肿瘤生长及转移过程中起重要作用。肿瘤血管形成是生长刺激因子 (如ＶＥＧＦ ,ｂＦＧＦ)与抑制因子 (ａｎｇｉｏｓｔａｔｉｎ ,ｅｎｄｏｓｔａｔｉｎ)相互作用的结果。测定肿瘤组织血管密度、ＶＥＧＦ、ｂＦＧＦ表达、血清或尿液中ＶＥＧＦ、ｂＦＧＦ浓度 ,有望成为判定肿瘤病期进展、转移及预后新的敏感指标[1] 。实体肿瘤生长在直径 >1ｍｍ时必须有ＶＥＧＦ等参与下的血管形成并网络化。目前 ,文献报道肿瘤组织ＶＥＧＦ、血管计数是肿瘤病期进展及预后的标志。血清ＶＥＧＦ(Ｓ ＶＥＧＦ)来源于肿瘤细胞。有资料报道Ｓ …