Increased expression of angiogenic factors in human colonic angiodysplasia

OBJECTIVE: Angiodysplasia of the colon is a distinct vascular abnormality characterized by focal accumulation of ectatic vessels in the mucosa and submucosa. To investigate whether angiogenesis contributes to the pathogenesis of human colonic angiodysplasia, we examined the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and its endothelial cell receptors flt-1 and KDR. METHODS: Immunohistochemistry was performed in sections of specimens obtained from 18 patients with colonic angiodysplasia and from eight patients with colon cancer and its adjacent, histologically normal margins of resection. We used affinity-purified rabbit polyclonal antibodies and a streptoavidin-biotin peroxidase method. RESULTS: We detected strong immunoreactivity for vascular endothelial growth factor, homogeneously distributed in the endothelial lining of blood vessels of all sizes in 16 (89%) specimens of colonic angiodysplasia and in seven (88%) patients with colon cancer. In contrast, very limited immunoreactivity was found in normal colon. Vascular staining for flt-1 was observed in eight (44%) and one (12.5%) of the colonic angiodysplasia or colon cancer specimens, respectively, but not in normal colon. Vascular immunoreactivity for basic fibroblast growth factor was observed in seven (39%) specimens from patients with colonic angiodysplasia, whereas either very limited or no immunostaining was found in sections from specimens of patients with colon cancer and its normal margins. CONCLUSIONS: In human colonic angiodysplasia, increased expression of angiogenic factors is likely to play a pathogenic role.     

Accelerated orthotopic hepatocellular carcinomas growth is linked to increased expression of pro‐angiogenic and prometastatic factors in murine liver fibrosis

Background: Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy. We questioned whether a pre‐existing fibrosis affects tumour development of implanted syngenic hepatoma cells. To further investigate a selected panel of factors involved in tumour growth, tumour organ samples were characterized for gene expression of vascular endothelial growth factor (VEGF)‐A/‐C, VEGF receptors Flt1, Flk‐1, Flt‐4 and for VEGF‐A protein levels. Results: The presented data show that tumour sizes were 3.7‐fold increased and fibrotic livers had numerous satellites. Increased tumour sizes were associated with elevated intratumoral VEGF‐A protein amounts and intratumoral increased VEGF receptor gene expression levels in tumour tissue from fibrotic livers as compared with non‐fibrotic livers. Additionally, intratumoral gene expression levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 were elevated in fibrotic mice. Conclusion: Our results indicate that liver fibrosis stimulates tumour development of implanted syngenic hepatoma cells. Accelerated tumour growth was going along with elevated intratumoral VEGF‐A and VEGF‐A receptor status, which most probably mediated pro‐angiogenic and prometastatic effects in this model. Furthermore, advanced tumour spread was associated with increased MMP‐2/‐9 expression. These data suggest that the intratumoral VEGF‐A proteins levels and VEGF receptor status contribute to accelerated hepatocellular carcinoma development in fibrotic mice and that elevated MMP‐2, MMP‐9 and VEGF‐C levels could promote tumour metastasis in this model.     

Conformational requirements of suramin to target angiogenic growth factors

The conformational requirements of suramin for its binding to basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) were examined by molecular modeling and docking simulations using the conformational features of suramin determined by the present proton nuclear magnetic resonance (1H-NMR) studies and the crystal structures of growth factors reported previously. The assignment of resonances of suramin to individual protons was accomplished by the combined analysis of the coupling constants, two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY). The NOESY data obtained for suramin were utilized in a conformational search algorithm with constraints to generate a family of conformers which were further refined by restrained energy minimization. A family of nine conformers generated by restrained modeling falls primarily into one of two categories, either the conformer's two naphthyl rings are far apart, approximately 28-30 A, from one another or the conformer's two naphthyl rings are relatively close, approximately 16-18 A. The NMR data provide evidence for the presence of more than one conformer in solution. The modeling and docking simulation studies suggest that suramin binds efficiently to bFGF and PDGF by an induced-fit mechanism, wherein suramin complements bFGF or PDGF by adjusting its conformational freedom around the two pairs of single bonds that link the middle phenyl rings to the secondary amide backbone. The interaction of suramin with bFGF or PDGF primarily involves ion-pair, hydrophobic and hydrogen bonding interactions, in addition to van der Waals' contacts. The results indicate that suramin not only sterically blocks the receptor binding loop of the growth factors, but also competes for the binding sites of agonists such as heparin. The results suggest that suramin's propensity to bind to several polypeptides of varying size and structure is due to its conformational flexibility. Collectively, the data emphasize that conformationally constrained suramin analogs that selectively and competitively target angiogenic growth factors could be designed to reduce non-specific binding and, accordingly, toxicity.     

Correlation of postoperative survival and angiogenic growth factors in pancreatic carcinoma

BACKGROUND/AIMS: VEGF (vascular endothelial growth factor) and EGF (epidermal growth factor) are promoters of angiogenesis. It was the aim of this study to investigate a possible coexpression of both growth factors in tumor samples of pancreatic cancer patients in relation to survival after resection of the tumor. METHODOLOGY: We investigated the expression of VEGF165 and EGF in tumor specimen from 19 patients that underwent pancreaticoduodenectomy. Growth factor expression was determined using immunohistochemical methods. RESULTS: Coexpression of VEGF165 and EGF was observed in tumor samples of 9 (47%) patients. VEGF165 and EGF expression in the same tumor correlates significantly (P < 0.05, Fisher-test). UICC stage III pancreatic carcinoma patients with VEGF165 negative tumor cells had a significantly better outcome after surgery compared to UICC stage III patients with VEGF165-positive tumor cells (median survival time 19 months vs. 9 months respectively; P < 0.05, Wilcoxon-test). CONCLUSIONS: Antiangiogenic therapy after surgery for pancreatic cancer may be beneficial, especially for UICC III patients.     

扶正化瘀方对非酒精性肝纤维化小鼠肝组织血管生成基因的调节作用及其对肝纤维化的影响*

目的 探讨血管生成基因在非酒精性脂肪性肝纤维化形成中的作用,以阐明扶正化瘀方治疗非酒精性脂肪性肝纤维化的作用机制。方法 选用7~8w龄健康雄性C57BL/6J小鼠,给予高脂、蛋氨酸-胆碱缺乏（MCD）饮食喂养8w,建立非酒精性脂肪性肝纤维化模型。采用扶正化瘀方进行干预,以蛋氨酸-胆碱充足饮食设立对照组。以HE和Masson染色观察小鼠肝组织脂肪变、炎症和纤维化程度;采用RT-PCR、免疫组化和Western blot法检测α-平滑肌肌动蛋白（α-SMA）、缺氧诱导因子-1α（HIF-1α）及其下游因子血管内皮生成因子（VEGF)和诱导型一氧化氮合酶（iNOs）mRNA及其蛋白水平。结果 模型组小鼠肝细胞出现大泡性为主的脂肪变性,小叶内可见点灶状肝细胞坏死及炎细胞浸润、窦周纤维化,汇管区纤维组织增生,纤维间隔形成;应用扶正化瘀方干预组动物肝损伤、炎症及纤维化程度显著改善,肝组织α-SMA、HIF-1α、VEGF和iNOs mRNA水平亦较模型组显著减低,依次为（3.83±0.53） 对 （8.33±2.32）、（3.02±0.55）对（4.50±0.78）、（3.23±0.94）对（7.40±1.54）和（3.60±0.96）对 （9.94±1.60）,（P<0.01）;上述蛋白表达水平与基因水平呈一致变化趋势,扶正化瘀方干预组与模型组α-SMA、HIF-1α、VEGF和iNOs表达水平依次为（0.53±0.04） 对 （1.08±0.20）、（0.44±0.02）对（0.55±0.08）、（0.54±0.07） 对 （1.08±0.03）、和（1.61±0.21）对（3.30±0.88）,（P<0.05）。结论 扶正化瘀方可通过抑制肝星状细胞活化,下调HIF-1α及其下游促血管生成因子VEGF和iNOs表达,阻止或减缓非酒精性脂肪性肝纤维化的进展。     

Epithelial microchimerism: consistent finding in human liver transplants

BACKGROUND: Eleven liver biopsies from six male patients who received a liver transplant (LT) from female donors were examined in order to determine whether male host-derived hepatic cells were present in female grafts that exhibited minimal or important inflammatory damage. METHODS: Immunohistochemistry for epithelial cell type differentiation (anticytokeratin monoclonal antibody) and fluorescence in situ hybridization for XY chromosomes identification were performed on each slide. RESULTS: Host-derived hepatic cells were found in all except one transplant, with a frequency ranging from 2.3 to 25 per thousand of the total hepatocytes in the biopsy specimen. They were usually found as isolated cells scattered throughout the hepatic lobule; in one patient they were grouped into little clusters. Host-derived hepatic cells persisted throughout the histological follow up (up to 535 days after LT). Polyploidy for XY chromosome was observed. CONCLUSION: Hepatocytes derived from extra-hepatic stem cells are frequently found in small numbers in human liver grafts and persist over time.     

Mesoderm induction and blood island formation by angiogenic growth factors and embryonic inducing factors

Summary Factors which induce mesoderm, including endothelium lined cavities and primitive blood cells in omnipotent amphibian ectoderm, have been isolated from different sources. Recently it was shown that angiogenic factors, which belong to the protein families of the heparin binding growth factors (acidic and basic fibroblast growth factor) and the transforming growth factors (TGF- 1 and - 2), also induce mesodermal tissues in amphibian ectoderm. In Triturus ectoderm, capillary like endothelial networks are induced preferentially by the transforming growth factors. The relationship between growth factors and inducing fators is discussed.Supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie     

Brain-derived neurotrophic factor increases expression of MnSOD in human circulating angiogenic cells

Existing evidence suggests that brain-derived neurotrophic factor (BDNF) promotes survival and proliferation of endothelial cells, stimulates mobilization of hematopoietic progenitors, and induces angiogenesis in ischemic tissues. However, the mechanisms underlying vascular protective function of BDNF are poorly understood. We hypothesized that BDNF increases antioxidant capacity of circulating angiogenic cells. Human mononuclear cells were isolated from peripheral blood of 30 healthy male volunteers (48±2 years old), and cultured in endothelial growth medium-2 for 4-5 days. The attached cells (so called early endothelial progenitor cells [early EPCs], or circulating angiogenic cells) expressed BDNF receptors, tropomyosin-related kinase B and p75 neurotrophin receptor. Treatment of early EPCs with recombinant human BDNF for 24 h significantly increased manganese superoxide dismutase (MnSOD) expression, but had no effect on expression of other antioxidant enzymes including copper zinc SOD (CuZnSOD), catalase, and glutathione peroxidase-1. BDNF stimulated phosphorylation of IκB kinase (IKK)α/β and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK); however it did not activate p38, Erk, or AKT. Treatment with nuclear factor κB inhibitor, PDTC, or JNK inhibitor, SP600125, attenuated BDNF-augmented MnSOD protein expression. BDNF treatment inhibited apoptosis induced by a superoxide anion generator LY83583, and serum starvation-induced cell detachment. These findings suggest that BDNF protects EPCs by increasing expression of MnSOD thereby enhancing their antioxidant capacity.     

Altered expression of angiogenic factors in the VEGF-Ets-1 cascades in inflammatory bowel disease

Background The VEGF-Ets-1 cascades play important roles in angiogenesis by converting endothelial cells to an angiogenic phenotype. The aim of this study was to clarify whether the VEGF-Ets-1 cascades are involved in the pathogenesis of inflammatory bowel disease (IBD).Methods Colonic specimens were taken from 42 patients with ulcerative colitis (UC), 37 with Crohns disease (CD), 8 with non-IBD colitis, and 21 normal controls. (1) Expression of vascular endothelial growth factor (VEGF), VEGF receptors (Flt-1, KDR), and Ets-1 proteins in colonic mucosa was immunohistochemically examined using specific antibodies. (2) Expression of Ets-1 protein or VEGF, Flt-1, KDR, and Ets-1 mRNA in colonic mucosa was measured by Western blot or RT-PCR.Results (1) The number of VEGF-containing cells was significantly increased in active UC (P < 0.05). The numbers of positive blood vessels (mean ± SE /mm²) to Flt-1, KDR, and Ets-1 antibodies were significantly increased in active UC (Flt-1: 4.0 ± 0.84; KDR: 2.4 ± 0.37; Ets-1: 5.5 ± 0.77) compared to active CD (Flt-1: 0.6 ± 0.30; KDR: 0.77 ± 0.28; Ets-1: 2.0 ± 0.56) (P < 0.01), non-IBD colitis (Flt-1: 1.0 ± 0.45; KDR: 1.83 ± 0.54; Ets-1: 3.0 ± 1.0), and controls (Flt-1: 0.88 ± 0.40; KDR: 0.60 ± 0.22; Ets-1: 1.67 ± 0.47) (P < 0.01). The numbers of positive cells to these antibodies were also increased in active UC. (2) Expression of Ets-1 protein and Flt-1, KDR, and Ets-1 mRNA was increased in active UC.Conclusions Angiogenic factors in the VEGF-Ets-1 cascades were upregulated in UC, but they were relatively downregulated in CD. These alterations might be involved in the pathogenesis of both diseases.     

Myocardial gene expression of angiogenic factors in human chronic ischemic myocardium: influence of acute ischemia/cardioplegia and reperfusion

OBJECTIVE: Angiogenic therapies in animals have demonstrated the development of new blood vessels within ischemic myocardium. However, results from clinical protein and gene angiogenic trials have been less impressive. The present study aimed to investigate the expression of angiogenic genes in human chronic ischemic myocardium and the influence of acute ischemia/cardioplegia and reperfusion on their expression. METHODS: Myocardial biopsies were taken from chronic ischemic and nonischemic myocardium in 15 patients with stable angina pectoris during coronary bypass surgery. Tissue samples were evaluated by oligonucleotide microarray and quantitative real-time PCR for the expression of angiogenic factors. RESULTS: There was identical baseline expression of VEGF-A and VEGF-C mRNA in chronic ischemic myocardium compared with nonischemic myocardium. Reperfusion increased the gene expression of VEGF-A and VEGF-C mRNA both in nonischemic and ischemic myocardium. VEGF-A protein was detected mainly in the extracellular matrix around the cardiomyocytes in ischemic myocardium. CONCLUSION: These data suggest that the nonconclusive VEGF gene therapy trials chronic coronary artery disease was not due to a preexisting upregulation of VEGF in chronic ischemic myocardium. There might be room for further therapeutic angiogenesis in chronic ischemic myocardium.