Hemoglobin induces the expression and secretion of vascular endothelial growth factor from human malignant cells

Vascular endothelial growth factor (VEGF) is an angiogenic hormone that increases the growth of many malignant tumors. Tissue factor (TF), the initiator of blood coagulation, is implicated in VEGF regulation. We recently reported that hemoglobin (Hb) upregulates TF on malignant cells. Therefore, to explore the role of Hb in angiogenesis, we examined its effect on VEGF production in A375 melanoma and J82 bladder carcinoma (TF+) and KG1 myeloid leukemia (TF-) cells. Hb (0.50 mg/ml) induced VEGF expression and secretion in TF+ malignant cells. VEGF secretion was inhibited by cycloheximide (85%) and the specific inhibitors of protein tyrosine kinase, genistein (71+/-0.74 and 55+/-4.90%) and mitogen-activated protein (MAP)-kinase, PD098059 (82+/-2.0 and 59+/-6.7%) in A375 and J82 cells respectively. In contrast, Hb (2.0 mg/ml) did not increase VEGF in KG1 cells. Hb-induced VEGF was purified from the culture medium of J82 cells using immunoaffinity chromatography and two isoforms (46 and 30 kd) identified. We conclude that Hb-induced synthesis of VEGF in TF-bearing malignant cells is mediated by protein tyrosine kinase and by MAP-kinase pathways.     

Hemoglobin enhances tissue factor expression on human malignant cells.

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.     

Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cell-induced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (Abciximab) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab')(2) (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. Abciximab significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by Abciximab (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab')(2) reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.     

Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cellinduced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (AbciximabTM) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab') 2 (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. AbciximabTM significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by AbciximabTM (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab') 2 reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.     

Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.     

Reductions of circulating matrix metalloproteinase 2 and vascular endothelial growth factor levels after treatment with pegvisomant in subjects with acromegaly

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in activation of the matrix metalloproteinase (MMP) system; the latter is implicated in atherosclerosis and cardiovascular disease. Patients with acromegaly have reduced life expectancy primarily due to cardiac disease. AIM: This study assessed plasma MMPs and VEGF levels in patients with active acromegaly (IGF-I > 130% upper limit of normal), and on treatment with pegvisomant. SUBJECTS AND METHODS: Twenty patients [nine female, mean age 56.1 +/- 13.8 yr (mean +/- sd)] were studied at baseline and on pegvisomant therapy and compared with data from 25 healthy volunteers (12 female; 56.6 +/- 14.2 yr). Plasma MMP-2, MMP-9, and VEGF levels were measured. RESULTS: Serum IGF-I fell from a baseline (mean +/- sd) level of 620.1 +/- 209.3 ng/ml to 237.5 +/- 118.5 ng/ml on pegvisomant (doses 10-60 mg; P < 0.001). MMP-2 levels at baseline were significantly higher in patients compared with healthy controls (380.7 +/- 204.8 vs. 207.4 +/- 62.6 ng/ml; P < 0.001), but with treatment a significant reduction in MMP-2 [380.7 +/- 204.8 vs. 203.0 +/- 77.4 ng/ml; P < 0.001] and VEGF (283.4 +/- 233.6 vs. 229.1 +/- 157.4 pg/ml; P = 0.008) was noted. There was no significant difference in MMP-9 levels between patients and controls at baseline (797.5 +/- 142.1 vs. 788.3 +/- 218.0 ng/ml; P = 0.87) or between baseline and posttreatment levels (797.5 +/- 142.1 vs. 780.0 +/- 214 ng/ml; P = 0.76). CONCLUSIONS: Our novel data demonstrate that treatment of acromegaly with pegvisomant leads to reductions in MMP-2 and VEGF concentrations. Further studies are required to determine the significance of these findings with relation to cardiac disease.     

骨桥蛋白促进人肝癌细胞株SMMC-7721恶性表型的实验研究

目的研究骨桥蛋白(OPN)对低侵袭性人肝癌细胞株SMMC-7721恶性表型的影响。方法pcDNA 3,1(-)/OPN重组质粒转染SMMC-7721细胞,以空质粒转染作对照,用RT- PCR反应、Western blot检测OPN表达水平,用ELISA检测细胞培养上清液OPN、MMP-2、-9、尿激酶纤溶酶原活化因子(uPA)水平,并用体外功能试验观察转染前后恶性表型的变化。结果重组质粒转染SMMC-7721后OPN表达明显升高,细胞培养上清液OPN为(3.02±0.12)ng/ml,对照组为(1.43±0.07)ng/ml,MMP-2重组质粒转染组为(43.04±3.06)ng/ml,对照组为(22.15±4.34)ng/ml、uPA重组质粒转染组水平明显高于空质粒转染组,分别为(4.78±0.70)ng/ml和(1.61±0.34)ng/ml,两组差异均有统计学意义,t值分别为19.89、6.81和7.03,P值均<0.01。MMP-9水平分别为(7.82±2.25)ng/ml和(7.70±1.92)ng/ml,两组差异无统计学意义。体外功能试验提示SMMC-7721转染OPN重组质粒后细胞黏附、运动和侵袭能力明显增强,细胞黏附率为75.33%±10.59%,对照组为57.34%±2.52%,t=2.86,P<0.05。运动试验透膜细胞数分别为(14.3±2.5)个和(6.3±1.5)个,t=4.70,P<0.05。侵袭试验透膜细胞数分别为(8.2±1.5)个和(4.1±1.3)个,t=4.11,P<0.05。而细胞增殖能力无明显改变。结论OPN可能是通过增加MMP-2、uPA分泌促进人肝癌细胞株SMMC-7721的恶性表型。     

Preoperative plasma concentrations of vascular endothelial growth factor and matrix metalloproteinase 9 are associated with stage progression in papillary thyroid cancer

OBJECTIVE: Vascular endothelial growth factor (VEGF), a potent angiogenic peptide, and matrix metalloproteinase 9 (MMP-9), a proteolytic enzyme, are found to be abundantly expressed in several types of cancer and correlate with tumour progression. In this study, we investigated the relationship between preoperative plasma VEGF, MMP-9 levels and disease stages in papillary thyroid cancer. DESIGN: Plasma samples were consecutively collected from 30 patients with papillary thyroid cancer preoperatively (seven males and 23 females with a mean age of 45 +/- 16 years) and control plasmas obtained from 30 patients with benign goitre (seven males and 23 females with a mean age of 44 +/- 18 years) and 23 healthy persons (four males and 19 females with a mean age 44 +/- 15 years). Plasma VEGF and MMP-9 concentrations were determined by enzyme-linked immunosorbent assay. Cancer progression was staged by the TNM classification of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC). RESULTS: In thyroid cancer patients, the plasma VEGF and MMP-9 values were higher than those in controls (51.8 +/- 11.5 ng/l vs. 27.0 +/- 0.8 ng/l; 72.3 +/- 23.3 micro g/l vs. 22.1 +/- 3.0 micro g/l, respectively; P < 0.05), and those in benign goitre (51.8 +/- 11.5 ng/l vs. 39.7 +/- 8.8 ng/l; 72.3 +/- 23.3 micro g/l vs. 23.0 +/- 2.2 micro g/l, respectively; P < 0.05). But, there was no difference of plasma VEGF and MMP-9 between patients with goitre and normal subjects. A comparison of VEGF and MMP-9 levels in patients at each cancer stage and in patients with benign nodule found that plasma VEGF and MMP-9 values in TNM stages III and IV, but not stage I or II, were significantly elevated (P < 0.05). When cancer patients were grouped according to clinopathological features, the plasma VEGF and MMP-9 concentrations were both significantly elevated in patients with large tumour size (P < 0.01), lymph node involvement (P < 0.01), extrathyroidal invasion (P < 0.05), distant metastasis (P < 0.05) or advanced stages (P < 0.01). CONCLUSION: Our study demonstrated that circulating VEGF and MMP-9 levels correlated with progression of papillary thyroid cancer, suggesting plasma VEGF and MMP-9 may serve as preoperative adjuvant markers for assessing disease activity in papillary thyroid cancer. However, they should not be used as a diagnostic tool for differentiating malignant from benign thyroid disorders.     

Expression of tissue factor and vascular endothelial growth factor is associated with angiogenesis in colorectal cancer

We examined the expression of tissue factor (TF) and vascular endothelial growth factor (VEGF) and the microvessel density (MVD) in 100 patients with colorectal cancer, and we investigated the relationship of the expression of TF or VEGF with angiogenesis. TF antigen was positive in 57.0% of all specimens. Incidence of TF expression was 41.2%, 45.5%, 52.6%, 84.6%, and 81.3% in tumors from patients in clinical stages I, II, IIIA, IIIB, and IV, respectively. TF expression was correlated with the Dukes' classification (P = 0.01) and the clinical stage of colorectal cancer (P = 0.02). VEGF antigen was positive in 64.0% of all specimens. Incidence of VEGF expression was 41.2%, 57.6%, 73.7%, 84.6%, and 75.0% in tumors from patients in clinical stages I, II, IIIA, IIIB, and IV, respectively. VEGF expression was correlated with the Dukes' classification (P = 0.01) but showed a weak association with the clinical stage (P = 0.08). MVD was significantly associated with the depth of invasion (P = 0.01), lymph node metastasis (P = 0.001), and liver metastasis (P = 0.02). The mean values of MVD were 7.5 +/- 2.8, 10.1 +/- 5.7, 14.6 +/- 5.8, 13.5 +/- 3.9, and 15.9 +/- 4.2 in tumors from patients in clinical stages I, II, IIIA, IIIB, and IV, respectively. A close relationship between VEGF and MVD (P < 0.001) and a significant correlation between TF expression and MVD were observed (P = 0.02). TF-positive carcinomas presented high MVD and VEGF expression (P < 0.001) more frequently than did TF-negative tumors. These results suggest that involvement of TF in the process of metastasis and progression of colorectal cancer may depend on increased angiogenesis.     

肝细胞癌患者血清ECM1、MMP-9和VEGF水平的变化及其意义

目的检测肝细胞癌患者血清ECM1水平,分析其与MMP-9和VEGF水平间的关系。方法应用酶联免疫吸附法测定40例肝癌患者血清ECM1、MMP-9和VEGF水平,分析其与肿瘤临床与病理特征间的关系。结果 40例肝癌患者血清ECM1、MMP-9和VEGF平均水平分别为150.01±5.85pg/ml、467±30.81μg/l和751.29±51.08pg/ml;三者水平均与肿瘤包膜、TNM分级、发生淋巴结转移和肿瘤分期有关(P0.005),而ECM1和VEGF还与血管侵犯有关(P0.005);ECM1与MMP-9和VEGF水平呈显著正相关(r=0.438,P=0.005r;=0.427,P=0.006)。结论 ECM1可能通过与MMP-9和VEGF相互作用而促进了肝癌血管生成和侵袭转移。     

慢性阻塞性肺疾病患者肺组织中基质金属蛋白酶抑制剂-1及基质金属蛋白酶-9与细胞黏附因子表达的关系

目的 研究COPD患者肺组织中基质金属蛋白酶抑制剂-1(TIMP-1)、基质金属蛋白酶-9(MMP-9)、细胞黏附因子-1(ICAM-1)蛋白和mRNA的分布和表达,探讨其与气流阻塞的关系及吸烟对其影响.方法 取39例因肺癌行肺叶切除的癌旁肺组织标本,其中不吸烟不伴COPD组(A组)9例、吸烟不伴COPD组(B组)11例、吸烟伴COPD组(C组)19例.用免疫组化和逆转录聚合酶链反应方法检测TIMP-1、MMP-9、ICAM-1的蛋白和mRNA表达,并进行相关性分析.结果 MMP-9表达于肺泡上皮细胞、支气管上皮细胞、血管平滑肌细胞、肺泡巨噬细胞、间质细胞,C组MMP-9免疫组化阳性细胞数(54.0±15.0)明显高于A组和B组(1.2±0.7和1.4±0.8);TIMP-1蛋白表达的主要部位为肺泡巨噬细胞、肺泡上皮细胞、毛细血管内皮细胞、血管平滑肌细胞,C组弱表达,A组和B组无表达;ICAM-1主要表达于肺泡上皮细胞,C组ICAM-1免疫组化阳性细胞数(52.1±13.4)明显高于A组和B组(2.1±1.1和4.5±2.4).C组MMP-9、TIMP-1、ICAM-1的mRNA平均吸光度值(0.71±0.16、0.47±0.10、0.62±0.15)明显高于A组(0.17±0.05、0.20±0.06、0.37±0.11)和B组(0.20±0.08、0.26±0.08、0.44±0.12).C组肺组织TIMP-1、MMP-9与ICAM-1的mRNA表达水平呈直线正相关,MMP-9与ICAM-1蛋白表达水平呈显著正相关.MMP-9和ICAM-1的mRNA及蛋白表达水平、TIMP-1的mRNA表达水平与FEV1占预计值%、FEV1/FVC占预计值%均呈显著负相关.结论 TIMP-1、MMP-9和ICAM-1在促进炎性细胞迁移进入细胞外基底膜及气道上皮细胞,导致肺组织破坏和重塑,引起及加重COPD患者的气流阻塞中起着重要作用.     

Matrix metalloproteinases and their inhibitors in malignant and autoreactive pericardial effusion

Matrix metalloproteinases (MMPs) are proteolytic enzymes essentially involved in tissue remodeling and tumor invasion, and their activity is counterbalanced by endogenous antagonists, the tissue inhibitors of matrix proteinases (TIMPs). Recent reports have suggested a potential role of MMPs in the evolution of pericardial effusion (PE). In this study, we determined the levels of MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 in 19 patients who had malignant PE that was confirmed by histology or cytology and 30 patients who had nonmalignant, autoreactive PE compared with pericardial fluid of 19 patients who had preserved left ventricular function and who underwent aortocoronary bypass surgery for control. Samples were assayed by zymography, immunoblotting, and quantitative enzyme-linked immunosorbent assay. We found significantly higher MMP-2 levels in malignant PE than in pericardial fluid (2,906 +/- 348 vs 1,493 +/- 114 ng/ml, p = 0.0005) or autoreactive PE (2,079 +/- 269 ng/ml, p = 0.01). No significant differences in MMP-9 levels were found between malignant PE and autoreactive PE (83 +/- 28.6 vs 106 +/- 30.4 ng/ml, p = 0.22), whereas MMP-9 was below the detection limit in pericardial fluid. No differences in TIMP-1 levels were found across the different study groups, whereas compared with pericardial fluid, TIMP-2 levels were significantly lower in autoreactive PE (113 +/- 18.9 vs 187 +/- 12.2 ng/ml, p = 0.002). In addition, there was a trend to lower TIMP-2 levels in malignant PE (137 +/- 27.1 ng/ml, p = 0.07). The present findings indicate that proteolytic enzymes and their inhibitors are involved in the pathogenesis of PE, with an expression pattern that depends on etiology. The involvement of MMP-2 in the pathogenesis of malignant PE may indicate a potential role of MMP inhibitors in the control of malignant PE.     

Effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM To study the effects of hypoxia,hyperoxia on theregulation of expression and activity of matrixmetalloproteinase-2 (MMP-2) in hepatic stellate cells(HSC).METHODS The expressions of MMP-2,tissue inhibitor ofmatrix metalloproteinass-2 (TIMP-2) and membrane typematrix matalloproteinass-1 (MT1-MMP) in cultured rat HSCwere detected by immunocytochemistry (ICC) and in situhybridization (ISH).The contents of MMP-2 and TIMP-2 inculture supernatant were detected with ELISA and theactivity of MMP-2 in supernatant was revealed byzymography.RESULTS In the situation of hypoxia for 12h,theexpression of MMP-2 protein was enhanced (hypoxiagroup positive indexes:5.7±2.0,n=10;control:3.2±1.0,n=7;P<0.05),while TIMP-2 protein was decreasedin HSC (hypoxla group positive indexes:2.5±0.7,n=10;control:3.6±1.0,n=7;P<0.05),and the activity(total A) of MMP-2 in suparnatant declined obviously(hypoxla group:7.334±1.922,n=9;control:17.277±7.424,n=11;P<0.01).Compared the varied duration ofhypoxia,the changes of expressions Including mRNA andprotein level as well as activity of MMP-2 were mostnotable in 6 h group.The highest value (Ahypoxla~-Acontrol) ofthe protein and the most intense signal of mRNA were inthe period of hypoxia for 6 h,along with the lowestactivity of MMP-2.In the situation of hyparoxia for 12 h,the contents (A_(450)) of MMP-2 and TIMP-2 in supernatantwere both higher than those in the control,especially theTIMP-2 (hyperoxla group:0.0499±0.0144,n=16;control:0.0219±0.0098,n=14;P<0.01),and so wasthe activity of MMP-2 (hyperoxia group:5.252±0.771,n=14;control:4.304±1.083,n=12;P<0.05),and the expression of MT1-MMP was increased.CONCLUSION HSC is sensitive to the oxygen,hypoxiaenhances the expression of MMP-2 and the effect is moremarked at the early stage;hyperoxia mainly raises theactivity of MMP-2.     

Endurance training reduces circulating inflammatory markers in persons at risk of coronary events: impact on plaque stabilization?

Inflammatory pathways are involved in destabilization of atherosclerotic plaques. We assessed the hypothesis that endurance training decreases circulating concentrations of inflammatory markers in persons with coronary artery disease (CAD) and cardiovascular risk factors (CVRFs). Thirty-two subjects with CAD and/or CVRFs joined a 12-week supervised endurance training. We found a significant decrease of the chemokines interleukin (IL)-8 (pre: 3.9+/-0.6, change: -1.2+/-0.4 pg/ml, -21%, p=0.002) and monocyte chemoattractant protein-1 (pre: 213+/-9, change: -20.4+/-8.2 pg/ml, -5%, p=0.03). Diabetes mellitus (DM) significantly influenced changes of IL-8 (p=0.002). IL-8 substantially dropped by 39% in diabetics. Moreover, matrix metalloproteinase-9 (MMP-9) highly significantly decreased in response to training (pre: 750+/-98, change: -278+/-77 ng/ml, -18%, p=0.005). Exercise-induced changes of MMP-9 were influenced by concomitant use of statins (p=0.038). We observed a particularly strong MMP-9 reduction of 44% in patients treated with statins. Acute phase reactants IL-6 (pre: 1.7+/-0.3, change: +0.25+/-0.7 pg/ml, +4%, p=0.58) and high sensitivity C-reactive protein (pre: 2.1+/-0.5, change: -0.25+/-0.4 mg/l, -9%, p=0.54) did not change in response to training. In conclusion, endurance training decreased circulating chemokines and MMP-9, which may in part explain its beneficial effect on coronary risk. Patients with DM or treated with statins because of hypercholesterolemia may particularly take advantage.     

Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor

Tissue factor (TF), a transmembrane receptor for coagulation factor VII/VIIa, is aberrantly expressed in human cancers. We demonstrated a significant correlation between TF and vascular endothelial growth factor (VEGF) production in 13 human malignant melanoma cell lines (r(2) = 0.869, P < 0.0001). Two of these cell lines, RPMI-7951, a high TF and VEGF producer, and WM-115, a low TF and VEGF producer, were grown s.c. in severe combined immunodeficient mice. The high-producer cell line generated solid tumors characterized by intense vascularity, whereas the low producer generated relatively avascular tumors, as determined by immunohistologic staining of tumor vascular endothelial cells with anti-von Willebrand factor antibody. To investigate the structure-function relationship of TF and VEGF, a low-producer melanoma cell line (HT144) was transfected with a TF cDNA containing the full-length sequence, a cytoplasmic deletion mutant lacking the coding sequence for the distal three serine residues (potential substrates for protein kinase C), or an extracellular domain mutant, which has markedly diminished function for activation of factor X. Cells transfected with the full-length sequence produced increased levels of both TF and VEGF. Transfectants with the full-length sequence and the extracellular domain mutant produced approximately equal levels of VEGF mRNA. However, cells transfected with the cytoplasmic deletion mutant construct produced increased levels of TF, but little or no VEGF. Thus, the cytoplasmic tail of TF plays a role in the regulation of VEGF expression in some tumor cells.     

Serial analyses of serum vitamin D-binding protein in preterm infants from birth to postconceptual maturity

Serial measurements of vitamin D-binding protein (DBP) were made in 28 infants of 29.7 +/- 2 weeks gestation and 1162 +/- 344 g birthweight. Mean (+/- SD) values (micrograms per ml) were: cord, 375 +/- 97; 1 week, 396 +/- 82; 3 weeks, 382 +/- 67; 6 weeks, 393 +/- 78; 9 weeks, 372 +/- 120; and 12 weeks, 490 +/- 130. At 12 weeks of age (40-42 weeks postconceptual age), DBP, 25-hydroxyvitamin D (25OHD), calcium, phosphorus, copper, and albumin were all significantly increased (by paired t test). At this point, but not earlier, DBP was correlated with 25OHD (r = 0.69; P less than 0.01) and phosphorus (r = 0.49; P less than 0.05), and 25OHD was correlated with phosphorus (r = 0.48; P less than 0.05) and calcium (r = 0.44; P less than 0.05). This may represent a concomitant maturation of the liver's protein synthetic abilities and the absorption and/or 25-hydroxylation in the liver of the parent vitamin D.     

基质金属蛋白酶-9、血管内皮生长因子和增殖细胞核抗原在胃腺癌表达的相关性及临床意义

目的 研究胃腺癌中基质金属蛋白酶-9(MMP-9)及其相关因子血管内皮生长因子(VEGF)和增殖细胞核抗原(PCNA)蛋白的表达,并分析其临床意义.方法 利用组织芯片技术,通过免疫组化法检测45例胃腺癌组织及其相应的45例癌旁组织和10例正常胃黏膜组织中MMP-9、VEGF和PCNA蛋白的表达,并分析它们的相关性及其与胃腺癌的分化程度和发生发展的关系.结果 本研究中MMP-9、VEGF和PCNA分别在胃腺癌、癌旁组织、正常胃黏膜中检出的阳性率为:MMP-9,82.2%(37/45)、64.4%(29/45)、30.0%(3/10),差异有统计学意义(P=0.019);VEGF,73.3%(33/45)、62.2%(28/45)、30.0%(3/10),差异有统计学意义(P=0.029);PCNA,84.4%(38/45)、71.1%(32/45)、10.0%(1/10),差异有统计学意义(P=0.001).MMP-9、VEGF和PCNA分别在高、中、低分化胃腺癌组织中的阳性率为:MMP-9,70.0%(7/10)、80.0%(8/10)、88.0%(22/25),差异有统计学意义(P=0.015);VEGF,50.0%(5/10)、60.0%(6/10)、88.0%(22/25),差异有统计学意义(P=0.000);PCNA,60.0%(6/10)、90.0%(9/10)、92.0%(23/25),差异有统计学意义(P=0.004).等级相关分析表明MMP-9、VEGF和PCNA呈两两正相关(P<0.05).结论 组织芯片技术是胃腺癌中各种蛋白表达的一种强有力的分析工具.MMP-9、VEGF和PCNA蛋白参与胃腺癌的发生发展过程,可以成为临床上评价预后的指标.     

血管内皮生长因子及其受体在肺气肿患者肺组织中的表达

目的探讨血管内皮生长因子(VEGF)及其受体2(VEGF受体2/KDR)在肺气肿患者肺组织中的表达及其与肺气肿的相关性。方法取35例行肺叶切除术患者[A组(吸烟伴肺气肿组)16例,B组(不吸烟肺功能正常组)14例,C组(吸烟但肺功能正常组)5例]的外周肺组织标本,ELISA法检测肺组织匀浆中VEGF的含量,免疫组化法检测KDR蛋白表达,RT PCR检测VEGF和KDRmRNA水平,TUNEL法检测肺泡隔细胞的凋亡。结果A组患者肺组织VEGF、KDR表达均低于B组(P<0.01),肺泡隔细胞凋亡率高于B组(P<0.01)。C组与B组相比,VEGF及KDR表达差异无统计学意义(P>0.05)。结论VEGF及KDR水平减少与肺泡隔细胞凋亡的增加可能与肺气肿的发生相关。     

Expression and correlation of CD44v6, vascular endothelial growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-9 in Krukenberg tumor

AIM: To explore the expression and correlation of CD44v6, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and matrix metalloproteinase (MMP)-9 in Krukenberg and primary epithelial ovarian carcinoma. METHODS: The expressions of CD44v6, VEGF, MMP-2 and MMP-9 were detected by immunohistochemical method in 20 cases of normal ovarian tissues, 38 cases of Krukenberg tumor and 45 cases of primary epithelial ovarian carcinoma. RESULTS: The expression of CD44v6 (primary epithelial ovarian carcinoma tissue vs normal ovarian tissue: x2 = 4.516, P= 0.034; Krukenberg tumor tissue vs normal ovarian tissue: x2 = 19.537, P= 0.001) and VEGF (primary epithelial ovarian carcinoma tissue vs normal ovarian tissue: P = 0.026; Krukenberg tumor tissue vs normal ovarian tissue: x2 = 22.895, P= 0.001) was significantly higher in primary epithelial ovarian carcinoma tissue and Krukenberg tumor tissue than in normal ovarian tissue. The positive expression rate of MMP-2 and MMP-9 was 0% in the normal ovarian tissue. The positive expression rate of CD44v6 (x2 = 10.398, P= 0.001), VEGF (x2 = 13.149, P= 0.001), MMP-2 (x2= 33.668, P= 0.001) and MMP-9 (x2 = 38.839, P = 0.001) was remarkably higher in Krukenberg tumor than in primary epithelial ovarian carcinoma. The correlation of CD44v6, VEGF, MMP-2, and MMP-9 was observed in primary epithelial ovarian carcinoma and Krukenberg tumor. CONCLUSION: CD44v6, VEGF, MMP-2, and MMP-9 are involved in ovarian carcinoma, gastric cancer and Krukenberg tumor. Detection of CD44v6, VEGF, MMP-2 and MMP-9 may contribute to the diagnosis of ovarian carcinoma, gastric cancer, and Krukenberg tumor.     

Variability of vascular endothelial growth factor in normal human breast tissue in vivo during the menstrual cycle

Exposure to sex steroids increases the risk of breast cancer, but the mechanisms are poorly understood. Angiogenesis is crucial in tumor development and progression. Very little is known about the regulation of angiogenesis in the normal breast. Vascular endothelial growth factor (VEGF) has a key stimulatory role in angiogenesis. Interferon-inducible protein 10 (IP-10) is a potent inhibitor of angiogenesis in vivo. These factors function in autocrine/paracrine pathways; therefore, direct measurements in the target tissue are needed. I measured VEGF and IP-10 in normal human breast tissue in situ in healthy women, using microdialysis, in the follicular and luteal phase of the menstrual cycle. In breast tissue, VEGF levels increased in the luteal phase, compared with the follicular phase (17.8 +/- 4 pg/ml to 34 +/- 9 pg/ml, P < 0.05). Plasma VEGF did not show a cyclic variation (10.6 +/- 2.8 pg/ml vs. 14.6 +/- 3.5 pg/liter, P = 0.3). IP-10 levels did not vary during the menstrual cycle either in breast tissue (65 +/- 17 pg/ml vs. 75 +/- 21 pg/ml, P = 0.6) or in plasma (64 +/- 7 pg/ml vs. 81 +/- 10 pg/ml, P = 0.06). The data suggests that, in the luteal phase, VEGF and IP-10, in the normal human breast, exhibit a proangiogenic profile. This may be one mechanism by which sex steroids contribute to breast cancer development.