MMP-8, MMP-9 and Neutrophil Elastase in Peripheral Blood and Exhaled Breath Condensate in COPD

Chronic obstructive pulmonary disease (COPD) is characterised by progressive and irreversible airflow limitation associated with chronic inflammation involving cytokines and metalloproteinases (MMPs). MMP-8, MMP-9 and neutrophil elastase (NE) are known to be implicated in COPD but the factors influencing activation and suppression remain unclear. This study aimed to compare MMP-8, MMP-9 and NE in the peripheral blood of COPD patients and controls and to likewise assess exhaled breath condensate (EBC) for these MMPs. Peripheral blood micro(mi)RNA139-5p levels, which may regulate MMPs in COPD, were also measured. Blood and EBC were collected from COPD patients (stable and during exacerbations) and healthy controls. Expression of mRNA for MMP-8, MMP-9, NE and miRNA-139-5p expression in peripheral blood mononuclear cells (PBMCs) was measured using qRT-PCR. MMP-8, MMP-9 and NE protein in plasma as well as MMP-8 and MMP-9 protein in EBC were analysed by enzyme-linked immunoassays. PBMCs from COPD patients showed greater expression of mRNA for MMP-8 (p = 0.0004), MMP-9 (p = 0.0023) and NE (p = 0.0019). PBMC expression of mRNA for NE was significantly higher in COPD exacerbations compared to stable cases (p < 0.05). Expression of mRNA for MMP-9 and NE correlated negatively with spirometry in patients (p < 0.05). Plasma from COPD patients showed greater levels of protein for MMP-8 (p = 0.003), MMP-9 (p = 0.046) and NE (p = 0.018). MMP-8 protein levels were lower in the EBC of COPD patients (p < 0.0001). In PBMCs, enhanced expression of mRNA for MMP-9 and NE is associated with COPD and may correlate with disease severity and exacerbations.     

Recurrence or metastasis of HCC:predictors, early detection and experimental antiangiogenic therapy

AIM To investigate the predictors for recurrence or metastasis of HCC, and to evaluate the effect of antiangiogenic therapy on the growth of transplantable human HCC in nude mice. METHODS RT-PCR was used to measure the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in 56 pairs of nontumorous liver and tumor samples. Sixty blood samples from human HCC were examined by nested RT-PCR to find out AFP mRNA. Recombinant human endostatin and polyclonal antibody against VEGF were administered to treat human HCC transplanted in nude mice. RESULTS Thirty of 56 HCC samples showed stronger expression of MMP-9 in tumorous tissues than in nontumorous tissues. Fifteen of the 26 patients with relative expression level of MMP-9 more than 0.34 developed tumor recurrence or metastasis, whereas only 7 of 30 patients with relative expression level less than 0.34 developed tumor recurrence (P＜0.05).There was no significant difference in the relative expression level of VEGF between patients with postoperative recurrence or metastasis and those without recurrence. AFP mRNA was detectable in 53.3% of patients with HCC. The sensitivity and specificity of AFP mRNA as a marker to detect hematogenous dissemination of HCC cells was 81.8% and 84.4%, respectively. Recombinant human endostatin and polyclonal antibody against VEGF inhibited the growth of transplantable HCC in nude mice by 52.2% and 45.7%, respectively.CONCLUSION MMP-9 expression in HCC correlates with the postoperative recurrence or metastasis of HCC. Patients with high level of MMP-9 expression in HCC are susceptible to metastasis. AFP mRNA could serve as an indicator of hematogenous spreading of HCC cells in circulation and a predictor of recurrence or metastasis of HCC. Antiangiogenesis may be an adjuvant therapy for HCC.     

Expression of vascular endothelial growth factor in surgical specimens of hepatocellular carcinoma

Purpose: Vascular endothelial growth factor (VEGF) has been reported to play an important role in angiogenesis in hepatocellular carcinoma (HCC). However, there is great variation in reports on the distribution of VEGF expression, especially in non-carcinoma liver cells. Furthermore, some reports have mentioned that endothelial cells were positive for VEGF antibody but have not evaluated its significance. In this study, we focused our attention to these problems and try to solve them. We also analyzed the factors influencing VEGF expression and evaluated the prognostic potential of VEGF protein in HCC. Methods: We examined the VEGF expression in specimens surgically removed from 46 HCC patients and 3 patients with liver cancer metastatic from the colon, and in 4 specimens of liver tissue with benign disease, by immunohistochemical methods. Results/conclusions: VEGF was expressed in HCC cells and hepatocytes and on vascular endothelial cells. Our finding that about seven times more endothelial cells were positive for VEGF antibody in carcinoma areas than in non-carcinoma areas (P < 0.001) suggests that VEGF is a very important angiogenesis factor for HCC growth. VEGF expression in HCC cells and non-carcinoma liver cells and on endothelial cells did not closely correlate with the disease recurrence rate (P > 0.05), suggesting that VEGF expression may not be useful as an individual factor for estimating the prognosis of HCC. A statistical analysis of the relationships between VEGF expression and clinicopathological variables revealed the following: preoperative transcatheter arterial embolization enhanced VEGF expression in both HCC cells and non-carcinoma liver cells. The histological grade of HCC and the level of alanine aminotransferase was related to VEGF expression in non-carcinoma liver cells and on endothelial cells in HCC areas. Tumor size and the histological status of the accompanying chronic hepatitis also influenced the VEGF expression on endothelial cells. Our findings concerning not only HCC but also the surrounding liver and endothelial cells may provide useful information for further research on the role of VEGF expression in HCC patients. Received: 29 July 1999 / Accepted: 11 October 1999     

肺鳞癌、腺癌中Survivin表达及其与VEGF和P53表达的相关性研究

目的探讨肺鳞癌、腺癌组织中survivin表达、临床意义及其与VEGF、P53表达的相关性。方法采用免疫组织化学（Envision二步法）检测survivin、VEGF和P53在116例肺鳞癌、腺癌组织及15例肺良性病变组织中的表达情况。结果 survivin、VEGF、P53的阳性率分别为62.1%（72/116）、72.4%（84/116）、55.2%（64/116）,显著高于肺良性病变组织的survivin表达与肺鳞癌、腺癌的低分化（P〈0.05）、淋巴结转移呈正相关（P〈0.05）,与患者预后负相关（P〈0.05）;VEGF和p53的表达与肺癌组织的分化程度、TNM分期及淋巴结转移均有关（P〈0.05）;并且Survivin表达与VEGF、P53表达呈正相关（P〈0.05）。结论 Survivin的表达与肺鳞癌、腺癌的低分化、淋巴结转移正相关;过度表达提示预后不良;Survivin有望成为肺癌诊断和基因治疗的新靶点;survivin、VEGF和P53三者的协同表达可能是促进肺癌恶性进展的重要因素。     

Expression of breast cancer anti‐estrogen resistance 1 in relation to vascular endothelial growth factor,p53, and prognosis in esophageal squamous cell cancer

The purpose of this study was to clarify the role of breast cancer anti‐estrogen resistance 1 (BCAR1) expression in relation to vascular endothelial growth factor (VEGF), p53, and proliferation in esophageal squamous cell cancer (ESCC). Expression of BCAR1, VEGF, p53, and the ki‐67 proliferative index were examined by tissue microarray and immunohistochemistry in 106 specimens with ESCC and matched adjacent normal tissues. Among them, 40 cases were simultaneously examined by Western blot. Both Western blot and immunohistochemistry showed that BCAR1 expression was substantially higher in ESCC than in adjacent normal tissues (P < 0.001). BCAR1 expression was significantly connected with degree of tumor differentiation, with poorly differentiated tumors showing higher BCAR1 expression (P < 0.001). BCAR1 expression was significantly and positively correlated with VEGF and p53 expression levels (r= 0.541, P < 0.001; r= 0.374; P < 0.001) but not proliferative index (r= 0.44; P= 0.066). Additionally, a significant relationship was also observed between VEGF and p53 (r= 0.321; P= 0.001). Kaplan–Meier survival analysis revealed that patients with high BCAR1 expression had significantly shorter survival times than those with low BCAR1 expression levels (median survival 40 months vs. 27 months, P= 0.09). Multivariate analysis also revealed that levels of BCAR1 expression (hazard ratio 2.250, P= 0.015) was a significant and independent prognostic indicator. High expression of BCAR1 is associated with elevated VEGF and p53 expression levels, as well as poor prognosis in ESCC. Therefore, BCAR1 may be a potential candidate for predicting prognosis and a new therapy target for ESCC.     

Relationship between survivin expression and recurrence, and prognosis in hepatocellular carcinoma

AIM： To study the expression of the inhibitor of apoptosis protein survivin in hepatocellular carcinoma （HCC）, and its correlation with clinicopathological factors, cell proliferation, recurrence and prognosis after hepatectomy. METHODS： Immunohistochemical staining of survivin and Ki-67 was performed by the standard streptavidin- peroxidase technique on paraffin sections of 55 cases of HCC. RESULTS： The positive rate of survivin in HCC was 52.7% （29/55）. Significant correlation was found between survivin expression with portal vein thrombi and intrahepatic matastasistic nodes （P 〈 0.05）. The recurrent rate in survivin-positive HCC was significantly higher than that in survivin-negative HCC after hepatectomy, the 1- and 3-year survival rate in patients with survivin-positive tumors was significantly lower than that in patients with survivin-negative tumors （58.62 and 10.34% vs 76.92 and 30.77%, P 〈 0.05, log-rank test）. The proliferation index （Ki-67） in survivin-positive HCC （33.83% ± 18.90%） was significantly higher than that in survivin-negative HCC （19.60% ± 19.35%） （P 〈 0.05）. CONCLUSION： Survivin may play an important role in progression of HCC by promoting cell proliferation, and may be positively correlated with high risk of disease recurrence and poor prognosis in HCC. Its expression may serve as a prognostic factor for patients with HCC after hepatectomy.     

Construction and clinical significance of a predictive system for prognosis of hepatocellular carcinoma

AIM: The aims of this study were to explore individualized treatment method for hepatocellular carcinoma (HCC) patients whose maximum tumor size was less than 5 cm to improve prognosis and survival quality. METHODS: Thirty cases of primary HCC patients undergoing tumor resection were retrospectively analyzed (resection group). All the tumors were proved as primary HCC with pathologic examination. The patients were divided into two groups according to follow-up results: group A, with tumor recurrence within 1 year after resection; group B, without tumor recurrence within 1 year. Immunohist-ochemical stainings were performed using 11 kinds of monoclonal antibodies (AFP, c-erbB2, c-met, c-myc, HBsAg, HCV, Ki-67, MMP-2, nm23-H1, P53, and VEGF), and expressing intensities were quantitatively analyzed. Regression equation using factors affecting prognosis of HCC was constructed with binary logistic method. HCC patients undergoing percutaneous microwave coagulation therapy (PMCT) were also retrospectively analyzed (PMCT group). Immunohistochemical stainings of tumor biopsy samples were performed with molecules related to HCC prognosis, staining intensities were quantitatively analyzed, coincidence rate of prediction was calculated. RESULTS: In resection group, the expressing intensities of c-myc, Ki-67, MMP-2 and VEGF in cancer tissue in group A were significantly higher than those in group B (t = 2.97, P= 0.01; t = 2.42, P= 0.03<0.05; t = 2.57, P= 0.02<0.05; t = 3.43, P = 0.004<0.01, respectively); the expressing intensities of 11 kinds of detected molecules in para-cancer tissue in groups A and B were not significantly different (P>0.05). The regression equation predicting prognosis of HCC is as follows: P(1) = 1/[1+e-(3.663-0.412mycc-2.187kl-67c-0.397vegfc)]. It demonstrates that prognosis of HCC in resection group was related with c-myc, Ki-67 and VEGF expressing intensity in cancer tissue. In PMCT group, the expressing intensities of c-myc, Ki-67 and VEGF in cancer tissue in group A were significantly higher than those in group B (t = 4.57, P= 0.000<0.01; t = 2.08, P= 0.04<0.05; t = 2.38, P= 0.02<0.05, respectively); the expressing intensities of c-myc, Ki-67 and VEGF in para-cancer tissue in groups A and B were not significantly different (P>0.05). The coincidence rate of patients undergoing PMCT in group A was 88.00% (22/25), in group B 68.75% (11/16), the total coincidence rate was 80.49% (33/41). CONCLUSION: The regression equation is accurate and feasible and could be used for predicting prognosis of HCC, it helps to select treatment method (resection or PMCT) for HCC patients to realize individualized treatment to improve prognosis.     

Endocan Expression Correlated with Poor Survival in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in China. We aimed to first present the expression of endocan in HCC tissue and its correlation with the clinicopathological features and overall survival of patients with HCC after curative hepatectomy. Immunohistochemical detection of endocan, CD34, and vascular endothelial growth factor (VEGF) were performed on samples from 100 patients with HCC. Endocan protein was expressed in endothelium of HCC tissue in all specimens, but was not expressed in endothelium of pericarcinomatous liver tissue and normal liver tissue. Microvessel density (MVD) denoted by endocan (endocan-MVD) in HCC was correlated with microscopic venous invasion and VEGF expression (P < 0.05). Survival analysis showed that overall survival of patients was inversely associated with endocan-MVD (P < 0.01). Multivariate analysis showed that endocan-MVD was an independent prognostic marker for overall survival of HCC (P < 0.01). In conclusion, endocan-MVD was a significant factor to predict the prognosis of HCC patients after curative hepatectomy.     

c-Myc和基质金属蛋白酶2等4种分子表达对肝癌手术切除预后的影响

目的 探讨c-Myc、Ki-67、基质金属蛋白酶(MMP)-2、血管内皮生长因子(VEGF)表达对肝癌手术切除预后的影响。 方法 回顾分析外科手术切除的原发性肝细胞癌资料,最大径≤5 cm,均为单发结节,切除前未行肝动脉化疗栓塞。术后跟踪随访,记录有无复发及复发时间,分为两组,术后1年内复发组15例(A组);术后2年后复发或未复发组15例(B组)。蜡块重新切片,行c-Myc、Ki-67、MMP2、VEGF免疫组化染色定量分析,比较其在两组病例癌组织及癌旁组织表达强度差异。 结果 c-Myc、Ki-67、MMP-2、VEGF在A组病例癌组织的表达强度高于B组(P<0.05),其在A、B两组病例癌旁组织的表达强度差异无显著性。 结论 癌组织c-Myc、Ki-67、MMP-2、VEGF表达与肝癌手术切除后复发有关。     

Influence of hepatitis B virus reactivation on the recurrence of HBV‐related hepatocellular carcinoma after curative resection in patients with low viral load

It is unclear whether the reactivation of hepatitis B virus (HBV) influences the prognosis of hepatocellular carcinoma (HCC) after resection in patients with chronic hepatitis B. The aim of this study was to identify the influence of HBV reactivation on the recurrence of hepatitis B‐related HCC after curative resection in patients with low viral load (HBV DNA <2000 IU/mL). We retrospectively analysed a total of 130 patients who underwent curative resection for HBV‐related early stage HCC (single nodule; <5 cm/two or three nodules; <3 cm) with pre‐operative HBV DNA levels <2000 IU/mL with serial HBV DNA tests. The predictive factors including HBV reactivation for the recurrence of HBV‐related HCC after curative resection were investigated. Fifty‐three patients (41%) had HBV reactivation after resection among 130 patients. HBV reactivation was observed in 22 of 53 patients with undetectable baseline HBV DNA and in 31 of 77 patients with detectable baseline HBV DNA. Cumulative recurrence rates after resection at 1, 2 and 3 years were 17.0%, 23.3% and 31.4%, respectively. The multivariable analysis demonstrated that the risk factors for the recurrence were the presence of microvascular invasion (hazard ratio (HR) 2.62, P = 0.003), multinodularity (HR 4.61, P = 0.005), HBV reactivation after resection (HR 2.03, P = 0.032) and HBeAg positivity (HR 2.06, P = 0.044). HBV reactivation after curative resection is associated with the recurrence of HBV‐related HCC in patients with low viral load.     

Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma

AM: To investigate expression and significance of inhibitor of apoptosis protein survivin in hepatocellular carcinoma (HCC). METHODS: The expression of survivin and vascular endothelial growth factor (VEGF) was investigated in 38 cases of HCC tissues and 38 liver cirrhosis tissues by immunohistochemistry and Western blot. The relationship between the expression of survivin and clinicopathological factors of HCC was analyzed. RESULTS: Survivin protein was detected in 23 (60.5%) of 38 HCCs and 3 (7.9%) of 38 liver cirrhosis tissues. In 23 cases of HCC which expressed survivin, the expression of VEGF was positive in 18 cases and slight positive or negative in 5 cases. While in 15 cases of HCC which did not express survivin, 12 cases did not express or slightly expressed, and 3 cases expressed VEGF. In liver cirrhosis tissues, the expression of VEGF was as follows: 24 cases were negative, 10 cases were weak positive and 4 cases were strong positive. The expression of survivin was coincident with the expression of VEGF in HCC (P<0.01). The expression of survivin in HCC had no relationship with the patients' age, gender, tumor size and differentiation level of HCC, while it was related to the metastasis of HCC. The protein quantitative analysis by Western blot also showed that overexpression of survivin in HCC was closely correlated to the expression of VEGF (P<0.01). Furthermore, stronger expression of survivin and VEGF was also found in patients with metastasis rather than in those with no metastasis (P<0.01). CONCLUSION: Survivin plays a pivotal role in the metastasis of HCC, and it has some correlation with tumorigenesis. The expression of survivin in the primary lesion is very useful as an indicator for metastasis and prognosis of HCC. It could become a new target of gene therapy of HCC.     

The expression of CXCR4 and its relationship with matrix metalloproteinase‐9/vascular endothelial growth factor in esophageal squamous cell cancer

Esophageal cancer (EC) is a highly aggressive neoplasm with poor prognosis. The main reason for this disappointing outcome is the strong behavior of esophageal cancer cell's invasion and metastasis. CXC chemokine receptor 4 (CXCR4) was found to be expressed in many tumors and significantly correlated with invasion, angiogenesis, metastasis, and prognosis. In the present study, we investigated the expressions of CXCR4, matrix metalloproteinase‐9 (MMP‐9), and vascular endothelial growth factor (VEGF) in esophageal squamous cell cancer (ESCC) and analyzed the relationship among the three proteins. Sections of paraffin‐embedded tissues were obtained from 127 patients with ESCC undergoing esophagectomy at Zhongshan Hospital, Fudan University in 2005. The CXCR4, MMP‐9, and VEGF expressions in EC tissues were evaluated according to the immunohistochemical staining area and intensity. The correlations between patients' prognosis and covariates were analyzed by Kaplan–Meier method (univariate analysis) and Cox regression (multivariate analysis). The overall expression rate of CXCR4, MMP‐9, and VEGF was 88.2%, 93.7%, and 79.5%, respectively. CXCR4 expression was significantly associated with tumor grade, tumor size, tumor depth, regional lymph node metastasis, and tumor, node, metastasis (TNM) stage (P < 0.05). MMP‐9 expression was significantly associated with age and tumor grade (P < 0.05). VEGF expression was significantly associated with tumor grade, tumor depth, and TNM stage (P < 0.05). CXCR4 expression was positively correlated with MMP‐9 expression (P < 0.01, r= 0.365) and VEGF expression (P < 0.01, r= 0.380). However, there was no significant correlation between MMP‐9 and VEGF expression (P > 0.05). In univariate analysis, CXCR4 expression, tumor size, tumor depth, lymph node metastasis, and TNM stage were correlated with patients' prognosis (P < 0.05); in multivariate analysis, tumor size and lymph node metastasis were the independent factors of poor prognosis. CXCR4 was highly expressed in ESCC and correlated with MMP‐9, VEGF, clinicopathological features and prognosis. We speculated CXCR4 play an important role during the progression of this disease and there might be some regulatory mechanism existing between CXCR4 and MMP‐9/VEGF in ESCC.     

肝癌组织中survivin、VEGF的表达及意义

目的探讨survivin与血管内皮生长因子（VEGF）在肝细胞肝癌（HCC）组织中的表达及其临床意义。方法采用免疫组化法检测50例HCC组织和20例正常肝组织中survivin、VEGF蛋白的表达,并分析其与HCC临床病理参数的关系。结果50例HCC组织中survivin、VEGF蛋白的阳性表达率分别为64.0%、68.0%,在正常肝组织中的表达率分别为0、10%,两者相比,P均〈0.05;survivin、VEGF蛋白表达与HCC临床分期及淋巴结转移相关（P〈0.05）;HCC中survivin、VEGF蛋白表达呈正相关（r=0.886,P〈0.05）。结论survivin和VEGF蛋白在HCC中表达率较高,为肝癌的分子靶向治疗提供了新的靶点;survivin和VEGF在HCC中的表达关系密切,对肝癌的发展可能有协同作用;测定HCC中survivin、VEGF蛋白的表达,对判断患者预后及指导治疗有较大帮助。     

A local imbalance between MMP and TIMP may have an implication on the severity and course of appendicitis

Background Matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs) have been demonstrated to be involved in inflammatory conditions in the intestine. The purpose of this study was to investigate whether the alterations of the MMP/TIMP balance might reflect the course of the inflammatory process in acute appendicitis and if the expression and localisation of MMPs and TIMP is variable in the various clinical manifestations of appendicitis. Materials and methods The study comprises 40 patients (26 men and 14 women) having emergency appendectomy and a control group constituting of 10 patients (5 men and 5 women) having a hemicolectomy for other reasons. MMP and TIMP expressions were assessed and compared in tissue specimens from phlegmonous (n = 15), gangrenous (n = 7), perforated appendicitis (n = 11) and controls with noninflamed appendices (n = 10) by means of enzyme-linked immunosorbent assay technique. Localisation of the enzymes was performed by immunohistochemistry. Results MMP-1 was significantly higher in gangrenous and perforated appendicitis compared with phlegmonous appendicitis and controls (p < 0.05) while MMP-2 was significantly lower in gangrenous appendicitis compared with phlegmonous appendicitis and controls. MMP-2 was also lower in perforated appendicitis when compared with controls (p < 0.01). Elevated expression of MMP-9 was demonstrated in all groups of appendicitis compared with the controls (p < 0.001). Conclusions MMP-9 is the most abundantly expressed MMP of those investigated in inflamed appendix. We postulate that a local imbalance between MMP-9 and TIMP-1 may trigger a perforation. These results suggest that MMPs might be useful as biomarkers of appendices prone to perforation.     

Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma

AIM: To determine the expressions of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9) in hepatocellular carcinoma (HCC) and to investigate the relationship between iNOS and MMP-9 expression and their effects on angiogenesis and progression of HCC. METHODS: In this study, we examined iNOS, MMP-9, and CD34 expression in specimens surgically removed from 32 HCC patients and 7 normal liver tissues by immunohistochemical staining. Meanwhile, microvessel density (MVD) was determined as a marker of angiogenesis by counting CD34-positive cells. RESULTS: The positive rates of iNOS and MMP-9 expression were 71.88% (23/32) and 78.13% (25/32) in HCC. MMP-9 expression was significantly correlated with tumor size, capsule status, TNM stage, and risk of HCC recurrence (P= 0.032, P= 0.033, P= 0.007, and P= 0.001, respectively). There was also a significant relationship between iNOS expression and capsule status and risk of HCC recurrence (P= 0.049 and P= 0.004, respectively), but no correlation between iNOS expression and tumor size and TNM stage. There was a positive association between MVD and TNM stage and risk of HCC recurrence (P= 0.037 and P= 0.000, respectively). The count of MVD was significantly different in different iNOS and MMP-9 immunoreactivity groups (F= 17.713 and 17.097, P= 0.000 and P= 0.000, respectively). The examination of Spearman's rank correlation coefficient showed that there was a significant positive correlation between MVD and iNOS, MMP-9 immunoreactivity (r= 0.754 and 0.751, P= 0.000 and A=0.000, respectively). There was also a significant association between MMP-9 and iNOS expression in HCC (P= 0.010). CONCLUSION: Nitric oxide (NO) produced by iNOS could modulate MMP-9 production and therefore contribute to tumor cell angiogenesis and invasion and metastasis in HCC. The strong expression of iNOS and MMP-9 in HCC may be helpful in evaluating the recurrence of HCC, predicting poor prognosis. For patients with strong expression of MMP-9 and iNOS, the optimal treatment scheme needs to be selected.     

Luteinizing hormone acts directly at granulosa cells to stimulate periovulatory processes: modulation of luteinizing hormone effects by prostaglandins

The midcycle surge of luteinizing hormone (LH) triggers events within the primate periovulatory follicle that culminate in follicle rupture and luteinization of the follicle wall; these events include the shift from primarily estrogen to primarily progesterone production, vascularization of the granulosa cell layer, and expression of matrix metalloproteinases and their inhibitors (MMPs and TIMPs) thought to be necessary for follicle rupture. However, it is unknown if LH acts directly at granulosa cells to regulate these important periovulatory processes. The ovulatory LH surge also stimulates the production of prostaglandins (PGs) by the follicle just before follicle rupture, suggesting that LH may have both PG-dependent and PG-independent actions. To address these questions, gonadotropins were administered to adult female rhesus monkeys to stimulate the development of multiple, large preovulatory follicles. Granulosa cells were aspirated and maintained in vitro for up to 48 h in serum-free, chemically defined medium. Granulosa cells were cultured with LH alone or in combination with PGs to determine if these hormones act directly at granulosa cells to induce the production of factors implicated in periovulatory processes. LH treatment increased media progesterone (p<0.05) and vascular endothelial growth factor (VEGF; p<0.05) levels as well as stimulating expression of mRNAs for MMP-1 (p=0.05), MMP-9 (p<0.05), and TIMP-1 (p<0.05), similar to the effects of an ovulatory dose of gonadotropin in vivo. PGE2 alone elevated media progesterone levels but decreased LH stimulation of MMP-1 mRNA (p<0.05). PGF2α reduced LH-stimulated TIMP-1 mRNA (p<0.05) levels. These studies suggest a direct action of LH on granulosa cells to stimulate the processes involved in tissue remodeling and neovascularization, i.e., MMPs/TIMPs and angiogenic factors, as well as steroidogenesis. LH-stimulated PGs may have a regulatory role to modulate some effects of the LH surge, such as MMP/TIMP expression.     

Survivin gene expression in endometriosis

Survivin is a novel inhibitor of apoptosis and is expressed during fetal development and in cancer tissues, but its expression has not been reported in normal adult tissues or benign diseases. We investigated survivin gene and protein expression in a tumor-like benign disease, endometriosis, and correlated them with apoptosis and invasive phenotype of endometriotic tissues. Gene expression levels of survivin, matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1 (MT1)-MMP in 63 pigmented or nonpigmented endometriotic tissues surgically obtained from 35 women with endometriosis were compared with those in normal eutopic endometrium obtained from 12 women without endometriosis. Survivin, MMP-2, MMP-9, and MT1-MMP mRNA expression levels in clinically aggressive pigmented lesions were significantly higher than those in normal eutopic endometrium, and survivin gene expression in pigmented lesions was also higher than that in nonpigmented lesions (P < 0.05). There was a close correlation between survivin and MMP-2, MMP-9, or MT1-MMP gene expression levels in 63 endometriotic tissues examined (P < 0.01). Apoptotic cells detected by the dUTP nick-end labeling were rare in 11 ovarian endometriotic tissues, which showed positive immunohistochemical expression for survivin and MMPs. Our findings suggest that up-regulation of survivin and MMPs may cooperatively contribute to survival and invasion of endometriosis.     

Angiopoietin 2 displays a vascular endothelial growth factor dependent synergistic effect in hepatocellular carcinoma development in mice

BACKGROUND: Orchestration of two major classes of angiogenic factors-namely, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2)-has been shown to play a pivotal role in tumour angiogenesis, including hepatocellular carcinoma (HCC). However, few studies have focused on the direct interaction of these factors on in vivo tumour development and angiogenesis. AIM: To examine the interaction between both factors in murine HCC. METHODS: We examined the combination effect of VEGF and Ang-2 overexpression by means of a combination of a retroviral tetracycline (tet) regulated gene manipulating system in vivo, by providing tet in the drinking water, and a conventional plasmid gene expression system. RESULTS: Neither Ang-2 nor VEGF overexpression induced proliferation of HCC cells in vitro. In vivo, although overexpression of Ang-2 did not increase tumour development, simultaneous expression of Ang-2 and VEGF synergistically augmented tumour growth and angiogenesis in murine HCC. Ang-2 plus VEGF induced tumour development was markedly attenuated by treatment with neutralising monoclonal antibodies against VEGF receptors. Ang-2 plus VEGF overexpression significantly increased the activities of matrix metalloproteinase (MMP)-2 and MMP-9 in the tumour. Suppression of intratumoral VEGF almost completely abolished this augmentation of MMPs. CONCLUSIONS: These results suggest that Ang-2 synergistically augments VEGF mediated HCC development and angiogenesis. This proangiogenic activity was exerted only in the presence of VEGF, at least partly mediated via induction of MMP-2 and MMP-9 in the tumour.     

Angiogenesis factor pattern differs in acute lymphoblastic leukemia and chronic lymphocytic leukemia

Angiogenesis is an important event in the survival and progression of solid tumors. The angiogenic status and the exact role of the angiogenic cytokines in lymphoid leukemia has not been fully elucidated.

We have investigated the profile of the systemic components of angiogenic regulation in B-lineage acute lymphoblastic leukemia (B-ALL) and B-chronic lymphocytic leukemia (B-CLL), namely vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), endostatin and matrix metalloproteinase-9 (MMP-9) using enzyme-linked immunosorbent assay (ELISA).

In B-ALL patients, sVEGF, and MMP-9 were significantly lower than control levels at diagnosis (p < 0.001) and increased to near control levels in remission (p > 0.05). Both serum TNF-α and endostatin levels showed no significant difference at diagnosis (p > 0.05) and in remission (p > 0.05) compared to control levels. VEGF, TNF-α, MMP-9 and endostatin levels were not significantly correlated with peripheral white cell count or bone marrow blast cell count, but were positively correlated with platelet count.

In B-CLL patients, serum VEGF, MMP-9 and TNF-α were significantly higher (p < 0.001 = 0.009, 0.007, respectively) and decreased to near control levels in remission (p > 0.05 for all). Serum endostatin levels showed no significant difference at diagnosis and in remission compared to control levels (p > 0.05). A significant positive correlation between VEGF, TNF-α, MMP-9 and peripheral white cell counts, bone marrow lymphocytic count and platelets count were found.

In conclusion, our data suggest that the driving forces of angiogenic factors (VEGF, TNF-α and MMP-9) in adult B-ALL appears different from that in B-CLL patients.     

Effect of c-myc, Ki-67, MMP-2 and VEGF expression on prognosis of hepatocellular carcinoma patients undergoing tumor resection

AIM: To explore the effect of c-myc, Ki-67, MMP-2 and VEGF expression on prognosis of hepatocellular carcinoma (HCC) patients undergoing tumor resection. METHODS: Primary HCC patients underwent tumor resection were retrospectively analysed. The maximum size of the tumor was less than 5 cm, there was only one nodule in each patient. No chemoembolization was performed before resection. They were followed up after resection, and the time of recurrence was recorded. They were divided into 2 groups: group A (15 cases): tumor recurrence within 1 year after tumor resection, and group B (15 cases): with or without tumor recurrence 2 years after tumor resection. Pathological slices were made with tumor wax-sample. Immunohistochemistry staining was performed with c-myc, Ki-67, MMP-2 and VEGF monoclonal antibodies. Staining intensity was quantitatively analysed with a pathological diagram-writing analyzing system. The expressing intensity differences of stained molecules in cancer tissue and para-cancer were analysed. RESULTS: c-myc, Ki-67, MMP-2 and VEGF expressing intensities in cancer tissue in group A were higher than those in group B (P values were 0.010, 0.030, 0.022 and 0.004, respectively), but they were not significantly different in para-cancer tissue in groups A and B (P values were 0.334, 0.343, 0.334 and 0.334, respectively). CONCLUSION: The expression of c-myc, Ki-67, MMP-2 and VEGF in cancer tissue is related to the recurrence of HCC after tumor resection.