Expression of matrix metalloproteinases (MMP-2 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in acute myelogenous leukaemia blasts: comparison with normal bone marrow cells

We compared the expression of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL-60, K-562 and KG-1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP-9 and/or MMP-2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP-1 and TIMP-2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP-1, the HL-60 cells also expressed TIMP-2. In contrast, normal steady-state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP-2 or MMP-9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP-9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP-1 and TIMP-2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP-2, the activated form of this enzyme was found in media conditioned by cells obtained from long-term cultures of normal and AML bone marrow adherent layers. Our finding of up-regulated production of gelatinases, TIMP-1 and TIMP-2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.     

Role of melatonin in regulating matrix metalloproteinase-9 via tissue inhibitors of metalloproteinase-1 during protection against endometriosis

Abstract:  Endometriosis is a gynecological disease of women and plausibly regulated by matrix metalloproteinases (MMPs). However, mechanisms of alterations in MMPs during endometriosis remain unclear. Human endometriotic tissues possessing varying degrees of severity were examined for expression of MMPs and tissue inhibitors of metalloproteinase (TIMP)-1. In addition, endometriosis was generated in mice and endometriotic tissues were tested for MMP-9 activity. Results show significant upregulation of secreted and synthesized proMMP-9 activity with duration and severity of endometriosis. Along with upregulation of activity, the expression of proMMP-9 was found increased while TIMP-1 expression followed an inverse trend. The effect of melatonin, a major secretory product of the pineal gland, on endometriosis was examined in preventive and therapeutic models in mice. The results show that melatonin arrested lipid peroxidation and protein oxidation and downregulated proMMP-9 activity and expression in a time and dose-dependent manner while protecting and regressing peritoneal endometriosis. Moreover, the attenuated activity and expression of proMMP-9 were associated with subsequent elevation in the expression of TIMP-1. Our study reveals for the first time the role of melatonin in arresting peritoneal endometriosis in mice and a novel marker, expression ratio of proMMP-9 versus TIMP-1, was identified for assessing severity and progression of endometriosis.     

Simultaneous up-regulation of matrix metalloproteinases 1, 2, 3, 7, 8, 9 and tissue inhibitors of metalloproteinases 1, 4 in serum of patients with chronic obstructive pulmonary disease

Background and objective: Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) have been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. However, the majority of studies have focused on single MMP, and there is limited information on parallel expression of MMP and their antagonists TIMP. We, therefore, investigated the serum profile of MMP 1–3, 7–9, 12 and 13, and TIMP 1–4 in COPD patients. Methods: Serum MMP 1–3, 7–9, 12 and 13, and TIMP 1–4 were measured in 74 COPD patients and 20 control subjects by multiple microsphere technology. Results: MMP 1–3 and MMP 7–9 were elevated in COPD patients compared with control subjects (P= 0.001–0.043). The increased concentrations of MMP 1, 8 and 9 paralleled GOLD stage (P= 0.002–0.007). TIMP 1 and 4 concentrations were elevated in COPD (P < 0.001). MMP 1, 8 and 9, and TIMP 1 and 4 serum levels in COPD non‐smokers were higher than in control non‐smokers (P= 0.002–0.025). MMP 12 and 13 levels were undetectable in our serum samples. Conclusions: This study provides further evidence for increased MMP 1, 7–9, and TIMP 1 serum levels in COPD, and demonstrates for the first time serum elevation of MMP 2 and 3, and TIMP 4. The finding that circulating TIMP 4 levels are increased in COPD and the observed relationship between serum levels of MMP 1, 8 and 9, and GOLD stage requires verification in an expanded patient cohort.     

Differential regulation of glomerular gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in obese Zucker rats

Summary The obese Zucker rat represents a model of obesity combined with insulin resistance and hyperlipidaemia, which over a period of several months develops spontaneous glomerulosclerosis. The present study addressed the question as to whether glomerular sclerosis was associated with alterations in the degradation of matrix components. In the early phase (up to 6 months) glomeruli from obese rats displayed increased total collagen content (+ 64 %) and decreased gelatinolytic activity (− 34 %) as compared to lean control animals. This decline in glomerular gelatinolytic activity was due to a reduction in gelatinase B [matrix metalloproteinase (MMP)-9]. Glomerular MMP-9 mRNA was reduced 4.6 ± 0.6-fold (n = 3; p < 0.05), MMP-9 protein was not detectable by Western blotting and MMP-9 activity was considerably suppressed in gelatin zymograms. MMP-2, in terms of mRNA expression and activity, was unchanged. Tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression, TIMP-1 protein (immunohistochemistry) and TIMP-1 activity (reverse zymography) were enhanced in glomeruli from obese rats, while TIMP-2 mRNA remained unchanged. Moreover, mRNA for the α₁ IV collagen chain was 2.1 ± 0.8-fold higher in glomeruli isolated from obese animals (n = 3; p < 0.05). These findings indicate that matrix expansion in glomeruli from obese Zucker rats is due to both enhanced synthesis of matrix components as well as reduced degradation by matrix metalloproteinases. Apparently the latter effect is based on a reduction in MMP-9 and up-regulation of its inhibitor TIMP-1. [Diabetologia (1997) 40: 1035–1043] Received: 16 January 1997 and in final revised form: 28 April, 1997     

COPD患者血浆金属蛋白激酶及组织抑制物含量的研究

目的研究慢性阻塞性肺病（Chronic obstructive pulmonary disease,COPD）患者血清基质金属蛋白酶（Matrix metal-loproteinase,MMP）-9和金属蛋白激酶组织抑制物（Tissue inhibitors of metalloproteinases,TIMPs）-1含量,以探讨它们与气道阻塞之间的关系。方法收集72例COPD,26例支气管哮喘（简称哮喘）以及66例对照患者的静脉血,利用ELISA方法测定上述血清TIMP-1和MMP-9含量,并对COPD进行呼吸功能测定,对TIMP-1、MMP-9含量与第一秒用力肺活量进行相关性分析。结果COPD患者血清TIMP-1含量（193.0±5.3μγ/L）显著高于对照组（119.9±6.5）μg/L和哮喘组（162.1±13.6）μg/L,并与COPD患者的第一秒用力肺活量呈负相关性,MMP-9/TIMP-1摩尔比值低于对照组,COPD急性加重期TIMP-1含量升高。结论血清TIMP-1含量在COPD气道阻塞和急性加重期升高。     

血清组织金属蛋白酶抑制因子-1在慢性病毒性肝炎肝纤维化非创伤性诊断中的应用

目的评价血清组织金属蛋白酶抑制因子诊断肝纤维化的应用价值.方法本研究测定了142例慢性病毒性肝炎病人的血清TIMP-1,并与血清生化指标、免疫学指标、影像学指标及肝活检组织学检查进行比较,以决定TIMP-1是否可作为慢性肝病肝纤维化诊断的一项有用的指标.结果TIMP-1与ALT、AST、GGT、ALT、SB、HA、APOAI呈正相关,与白蛋白呈负相关,与PGA、PGAA积分无相关.与肝炎病毒标志(HBsAg、抗-HBc、HBeAg、抗-HBe、HBVDNA、抗-HCV)无相关,与血脂(TG、TC、高密度脂蛋白,低密度脂蛋白)无相关,血清TIMP-1浓度与CD3、CD8、NK、IgG相关(P＜0.05).将TIMP-1与B超指标作相关性分析,发现与胆囊壁厚度,脾脏厚度和胆囊形态相关(P＜0.05);将TIMP-1与CT和MRI作相关性分析,发现与脾脏厚度相关(P＜0.01);TIMP-1与肝组织学指标之间的相关分析可以看出TIMP-1与肝内炎症和纤维化均显著相关.利用判别分析法计算,以血清TIMP-1判别肝内炎症程度、纤维化的有无和肝硬化有无的准确性分别为72.54%、40.85%和81.25%.结论血清TIMP-1水平反映了慢性肝病肝内炎症和纤维化的一些重要特征,在目前情况下是一种较好的肝纤维化无创伤性诊断和随访指标.     

Heterogeneity in the breakpoints of chromosome 19 among acute leukemia patients with the t(11;19)(q23;p13) translocation

Gene probes for insulin receptor (INSR) and c-ets-1 were hybridized to metaphase cells from three leukemic patients with the t(11;19)(q23;p13) translocation. Patients 1 and 2 were diagnosed as acute lymphocytic leukemia (ALL) (L2), and patient 3, as acute myelogenous leukemia (AML) (M4). The c-ets-1 gene was demonstrated to have translocated from chromosome 11 to the short arm of the rearranged chromosome 19 (19p+) in all three patients. On the other hand, the INSR gene translocated from chromosome 19 to the rearranged chromosome 11 (11q-) in the AML case, but remained on the rearranged chromosome 19 in the two ALL cases. Thus, the breakpoints of chromosome 19 are different among the patients studied, proximal to the INSR gene locus in the AML case and distal in the two ALL cases. Consequently, the c-ets-1 gene and the INSR gene remain separated in the AML case, whereas they become close to each other in the two ALL cases. Rearrangement of these two genes was studied in the two ALL patients, with no positive data being obtained. The results suggest that there may be heterogeneity in the breakpoints of chromosome 19 among the t(11;19)-associated acute leukemias.     

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets

Activated natural killer (A-NK) cells, a subset of CD56^dimCD3^– lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph⁺ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (±SD) of 83 ± 7% of CD56⁺CD3^– cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27 ± 33% CD56⁺CD3^– cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56⁺CD3^– cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl⁺ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl⁺ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P = 0.002 and P = 0.029, respectively) in the BCR/abl^– cultures than those in the six BCR/abl⁺ cultures. 5/6 BCR/abl^– cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56⁺CD3⁺ cells. Only 2/6 BCR/abl⁺ cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33⁺) were more frequently detected in the BCR/abl⁺ than BCR/abl^– A-NK cell cultures (P = 0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl⁺ cells from these cultures.     

Fibrosis in endstage human heart failure: Severe changes in collagen metabolism and MMP/TIMP profiles

Objectives

We studied fibrosis, collagen metabolism, MMPs/TIMPs and cytokine expression in various forms of human heart failure (HF) by quantitative immunofluorescent microscopy, Western blot, zymography, RT-PCR and in situ hybridization. In explanted human hearts with HF due to either dilated (DCM, n = 6) or ischemic (ICM-BZ-borderzone, ICM-RZ-remote zone, n = 7) or inflammatory (myocarditis, MYO, n = 6) cardiomyopathy and 8 controls MMP2, 8, 9, 19, and TIMP1, 2, 3, 4 as well as procollagens I and III (PINP, PIIINP), mature collagen III (IIINTP) and the cross-linked collagen I degradation product (ICTP) were measured.

Results

In comparison with controls, MMPs and TIMPs were significantly upregulated ranging (from highest to lowest) from ICM-BZ, DCM, ICM-RZ, MYO for all MMPs with the exception of MMP9 (highest in DCM), and for TIMPs from ICM-BZ, ICM-RZ, DCM and MYO. MMP2 and 9 were activated in all groups. The TIMP/MMP ratio was 1.3 for control, 1.9 in ICM-BZ (TIMP > MMP) and lowered to 1.0 in the other groups. Collagen I/collagen III ratio correlated significantly with the decrease in LVEDP. PINP was higher than ICTP in all groups. PIIINP elevation was present in DCM and ICM-RZ and IIINTP was up to 4-fold augmented in all groups. Fibrosin mRNA was upregulated in ICM-BZ, activin A in MYO but FGF1 and FGF2 remained unchanged. ANP mRNA was increased in all groups.

Conclusions

Although different degrees of severity of collagen metabolism, MMP/TIMP imbalance and cytokine expression in diverse forms of HF are present, the end product is collagen deposition. These findings suggest multiple mechanisms acting alone or in concert in fibrosis development in HF.     

Interphase FISH to detect PBX1/E2A fusion resulting from the der(19)t(1;19)(q23;p13.3) or t(1;19)(q23;p13.3) in paediatric patients with acute lymphoblastic leukaemia

Approximately 6% of paediatric patients with precursor B-cell acute lymphoblastic leukaemia (B-ALL) harbour a rearrangement involving the gene regions of PBX1 (1q23) and E2A (19p13.3) which is visualized cytogenetically either as a der(19)t(1;19)(q23;p13.3) or the less common balanced t(1;19)(q23;p13.3). Unfortunately, no commercial dual-colour, double fusion fluorescence in situ hybridization (D-FISH) strategies are available to detect this recurrent anomaly. Therefore, we have created a D-FISH assay to detect these translocations and monitor minimal residual disease. This probe set was created using four bacterial artificial chromosomes (BACs) corresponding to the PBX1 gene region at 1q23 and four BACs corresponding to the E2A gene region at 19p13.3. We analysed 30 negative bone marrow controls and 20 diagnostic and post-treatment specimens from 13 paediatric B-ALL patients with a cytogenetically defined 1;19 translocation. Once unblinded, the results demonstrated that our D-FISH method effectively identified all diagnostic samples as abnormal and identified disease in four post-treatment samples that were previously considered to be normal by conventional cytogenetic analysis. The development of this FISH strategy for the detection of der(19)t(1;19)(q23;p13.3) and t(1;19)(q23;p13.3) proved to be an effective technique, allowing both the detection of disease in diagnostic samples and in post-treatment samples.