膜1型基质金属蛋白酶在骨代谢作用中的研究进展

基质金属蛋白酶是一类锌依赖性内肽酶,可降解大多数细胞外基质.膜1型基质金属蛋白酶(membrane type-1 matrix metalloproteinase,MT1-MMP或MMP14)属于基质金属蛋白酶家族,是唯一一种能够直接促进细胞向3 D胶原蛋白基质入侵的胶原酶.骨细胞胞外基质在骨形成和骨吸收过程中发挥重要...     

Novel protection strategy for pulmonary transplantation

BACKGROUND: Ischemia-reperfusion injury continues to represent a significant challenge to successful lung transplantation. Traditional pulmonary ischemic protection is performed using hypothermic hyperkalemic depolarizing solutions to reduce the metabolic demands of the ischemic organ. Measures to further reduce the effects of ischemic injury have focused on the reperfusion period. We tested the hypothesis that novel physiologic hyperpolarizing solutions-using ATP-dependent potassium channel (K(ATP)) openers-given at the induction of ischemia, will reduce cellular injury and provide superior graft function even after prolonged periods of ischemia. METHODS: An isolated blood-perfused ventilated rabbit lung model was used to study lung injury. Airway, left atrial, and pulmonary artery pressures were measured continuously during the 2-h reperfusion period. Oxygenation, as a surrogate of graft function, was measured using intermittent blood gas analysis of paired left atrial and pulmonary artery blood samples. Graft function was measured by oxygen challenge technique (F(i)O(2) = 1.0). Wet-to-dry ratio was measured at the conclusion of the 2-h reperfusion period. Control (Group I) lungs were perfused with modified Euro-Collins solution (depolarizing) and reperfused immediately (no ischemia). Traditional protection lungs were perfused with modified Euro-Collins flush solution and stored for 4 h (Group II) or 18 h (Group III) at 4 degrees C before reperfusion. Novel protection (Group IV) lungs were protected with a hyperpolarizing solution containing 100 nM Aprikalim, a specific K(ATP) channel opener, added to the modified Euro-Collins flush solution and underwent 18 h of ischemic storage at 4 degrees C before reperfusion. RESULTS: Profound graft failure was measured after 18 h of ischemic storage with traditional protection strategies (Group III). Graft function was preserved by protection with hyperpolarizing solutions even for prolonged ischemic periods (Group IV). Wet-to-dry weight ratio, airway, left atrial, and pulmonary artery pressures were not significantly different between the groups. CONCLUSIONS: We have created a model of predictable lung injury. Membrane hyperpolarization with a K(ATP) channel opener (PCO) provides superior prolonged protection from ischemia-reperfusion injury in an in vitro model of pulmonary transplantation.     

Anticancer effects of zoledronic acid against human osteosarcoma cells.

Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N = 9). Exposure to ZOL at low micromolar concentrations induced a dose- and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy.     

Titanium particles up-regulate the activity of matrix metalloproteinase-2 in human synovial cells

Purpose

Wear debris particle-induced osteolysis and subsequent aseptic loosening is one of the major causes of failure of total joint replacement. The purpose of this study was to investigate the effect of titanium implant material and inflammatory cytokines on human synovial cells and the development to osteolysis and aseptic loosening.

Methods

This study investigated the effect of titanium implant material on the ECM-degraded MMP-2 in human synovial cells and analyzed the contribution of synovial cells in osteolysis and aseptic loosening.

Results

When human synovial cells are exposed to titanium materials, MMP-2 activity is induced by 1.72 ± 0.14-fold with Ti disc and 3.95 ± 0.10-fold with Ti particles, compared with that of the controls, respectively. Inflammatory cytokines TNFα and IL-1β are also shown to induce MMP-2 activity by 3.65 ± 0.28-fold and 6.76 ± 0.28-fold, respectively. A combination of Ti particles and cytokines induces MMP-2 activities to a higher level (10.54 ± 0.45-fold). Inhibitors of various signal pathways involved in MMP-2 reverse Ti particle-induced MMP-2 activities.

Conclusions

Synovial cells surrounding the bone–prosthesis interface may contribute to production of MMP-2, and NFκB inhibitors may be explored as potential therapeutics to alleviate wear debris-induced osteolysis and aseptic loosening.     

Experimental hindlimb ischemia leads to neutrophil-mediated increases in gastrocnemius MMP-2 and -9 activity: a potential mechanism for ischemia induced MMP activation

INTRODUCTION: Matrix metalloproteinases (MMP)-2 and -9 are Type 4 collagenases instrumental in basement membrane degradation, a process necessary for angiogenesis to occur. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of both PMN-derived serine protease and membrane type 1 (MT1)-MMP, are able to activate pro-MMP-2 following hindlimb ischemia. We hypothesized that neutrophil depletion (ND) of animals prior to hindlimb ischemia (HI) would abrogate the activation of pro-MMP-2 and decrease the level of MMP-9 MATERIALS AND METHODS: 12 FVB/N Tie2/LacZ-182 SATO female mice were randomly divided into four blinded groups; HI + PBS, HI + anti-PMN antibody (GR-1), HI + isotype matched control antibody (IgG(2b,K)), and no HI + PBS. PMN depletion was achieved prior to the time of ischemia and maintained until sacrifice. HI was achieved by unilateral femoral artery ligation. Three days postligation the animals were sacrificed and the gastrocnemius muscle from each hindlimb was harvested. MMP-2 and -9 (gelatin zymography) and MT1-MMP (Western blot) expression and activation were quantified by densitometry and NIH Image Analysis software. MMP values were expressed as a ratio of ischemic-to-nonischemic hindlimbs and compared between groups. Statistical significance was determined with analysis of variance (ANOVA) RESULTS: Zymograms revealed a greater than 10-fold increase in active MMP-9 and greater than 4-fold increase in active MMP-2 from HI + PBS compared to no HI + PBS (P < 0.05). HI + anti-PMN antibody demonstrated reduction of both active MMP-2 and -9 levels to that of the nonischemic group. Pro-MMP-2 was constitutively expressed in all four groups with no significant differences between any group (P = NS). There was no difference between the HI + isotype-matched antibody group and the HI + PBS group throughout the experiments (P = NS). ND did not affect MT1-MMP activation or expression CONCLUSIONS: Limb ischemia causes activation of MMP-2 and -9, which is eliminated by ND. ND animals undergoing hindlimb ischemia exhibit identical levels of active MMP-2 and -9 as animals that did not have hindlimb ischemia. Neutrophils may be an important activator of MMP-2 and the suppliers of MMP-9 in the ischemic hindlimb and may be essential for tissue remodeling, basement membrane degradation, and angiogenesis in ischemic limbs     

Serial analysis of matrix metalloproteinase-2 in dialysate of rat sclerosing peritonitis models

Background. Sclerosing peritonitis (SP) is a serious complication of continuous ambulatory peritoneal dialysis (CAPD). In order to carry out CAPD safely, it is important to analyze the development of SP. Methods. We prepared animal models of SP by the intraperitoneal administration of chlorhexidine gluconate (CHX) or talc, using male Sprague-Dawley rats. The peritoneal equilibration test and histological examinations were performed in the model rats, and dialysate drained from them was analyzed by gelatin zymography. Results. In the two types of SP animal models, matrix metalloproteinase-2 (MMP-2) level in dialysate correlated with the changes of inflammation, thickness of peritoneum, D/D0 glucose level (glucose level of drained dialysate obtained 90 min after the injection of 2.5% glucose containing peritoneal dialysis fluid divided by that obtained immediately after the injection), and net ultrafiltration. Conclusions. From these results, MMP-2 level in drained dialysate was considered to change with the development and progression of SP in rat models. Thus, MMP-2 has potential as a diagnostic marker for SP. Received: November 9, 2000 / Accepted: February 13, 2001     

Inhibition of mTOR signaling by oleanolic acid contributes to its anti‐tumor activity in osteosarcoma cells

Oleanolic acid (OA), a pentacyclic triterpenoid exhibits potent anti‐tumor activity against many tumor cell lines. But the mechanisms through which OA inhibits osteosarcoma cells are not known. The mammalian target of rapamycin (mTOR) serves as a central regulator of cell growth, proliferation, survival, and metabolism by integrating intracellular and extracellular signals. In this study, we examined effects of OA on proliferation, cell cycle progression, apoptosis in osteosarcoma cells, and involvement of mTOR signaling in this process. OA inhibited cell proliferation and colony formation, induced G1 arrest in osteosarcoma MG63 and Saos‐2 cells dose and time dependently. The protein level of cyclin D1, which plays critical role in G1 to S phase transition and servers as a downstream target of mTOR complex 1 (mTORC1) was down‐regulated by OA. Phosphorylation of p70 ribosomal S6 kinase 1 (p70 S6K1) (T389) and S6 (S235/236), mediators of mTORC1 signaling in controlling protein translation and cell growth, was also inhibited by OA. Furthermore, OA inhibited phosphorylation of Akt, a pro‐survival factor and substrate for mTORC2. Inactivation of Akt correlated with pro‐apoptotic role of OA in osteosarcoma cells, as manifested by an increase in annexin V‐FITC binding, cleavage of poly (ADP‐ribose) polymerase (PARP) and activation of caspases 3. Our results suggest that OA is a promising agent for treatment of osteosarcoma and mTOR signaling may contribute to its anti‐tumor effects on osteosarcoma cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:846–852     

血管生成拟态和基质金属蛋白酶-2在前列腺癌中的表达及意义

目的 研究血管生成拟态( vasculogenic mimicry,VM)在人前列腺癌组织中的分布及与预后的关系,探讨基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)在前列腺癌VM形成中的作用. 方法 选取天津医科大学第二医院1995年1月至2008年12月收治的前列腺癌患者96例.年龄59 ～ 72岁,平均(66.7±11.0)岁.术前血清PSA为15.6 ～ 76.7 μg/L,平均(34.6±1.7)μg/L.术前经MRI检查及穿刺活检均诊断为局灶性前列腺癌.收集前列腺根治性切除术后的组织标本,采用血小板-内皮细胞黏附分子-1(platelet-endothelial cell adhesion molecule-1,PECAM-1/CD31)免疫组化和过碘酸雪夫氏( periodic acid-sciff,PAS)组织化学双重染色后,光镜下观察VM结构,比较VM表达阳性组和阴性组的预后情况,分析VM表达与预后的关系.免疫组化法检测MMP-2在前列腺癌组织中的表达,分析MMP-2蛋白表达与VM形成的关系和作用. 结果 96例中24例存在VM结构,VM阳性组平均Gleason评分8.0±0.3,PSA( 37.7±2.3) μg/L,均高于VM阴性组Gleason评分6.2±0.3,PSA(19.5±2.1) μg/L,差异有统计学意义(P＜0.05).VM阴性组无生化复发中位生存期96个月,高于VM阳性组无生化复发中位生存期39个月,差异有统计学意义(P＜0.05).MMP-2表达和VM的形成呈明显正相关(rs =0.60,P＜0.01). 结论 前列腺癌组织中VM形成与组织恶性程度相关,肿瘤细胞分泌MMP-2可能促进了VM的形成,VM阳性和MMP-2高表达与前列腺癌预后不良有关.     

Pericellular matrix formation alters the efficiency of intracellular uptake of oligonucleotides in osteosarcoma cells

One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-kappaB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-kappaB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-kappaB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-kappaB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.     

siRNA特异性抑制骨肉瘤细胞株HER-2基因的表达

[目的]观察RNA干扰技术对骨肉瘤细胞株（OS-732细胞）HER-2（表皮生长因子受体-2）表达的抑制效应。[方法]体外化学合成HER-2序列特异性双链RNA（siRNA），转染入OS-732骨肉瘤细胞株中，分别用RT-PCR、Western Blot和免疫细胞化学法检测转染前后HER-2基因的mRNA和蛋白的表达情况，用MTT（四甲基偶氮唑盐比色法）检测细胞的增殖和化疗敏感性，观察细胞生物学特性的改变。[结果]HER-2 siRNA对OS-732细胞HER-2 mRNA表达明显抑制，作用约持续10d；干涉第9d，干涉组细胞蛋白表达减弱；细胞对顺铂的敏感性提高约10倍。[结论]体外合成的siRNA能有效抑制骨肉瘤OS-732细胞中HER-2基因的表达，抑制细胞的增殖，并提高了细胞对顺铂的敏感性。     

Clinical and biological significance of PIM1 kinase in osteosarcoma

Osteosarcoma is the most prevalent histological form of primary malignant bone tumor. The majority of osteosarcoma patients have limited alternative therapeutic options and metastatic patients generally have a poor prognosis. Proto‐oncogene serine/threonine‐protein kinase PIM1 is associated with growth and survival of many kinds of tumor cells. However, the role of PIM1 in osteosarcoma remains largely unknown. In this study, we investigated the functional and therapeutic relevance of PIM1 as a putative target in osteosarcoma. We found PIM1 was highly expressed in various osteosarcoma cell lines and in tumor tissues from osteosarcoma patients. Tissue microarray and immunohistochemistry analysis showed that the overall and disease‐free survival rate of patients with high levels of PIM1 protein expression were significantly shorter than patients with low levels. High levels of PIM1 were also associated with present metastasis and can be considered as an independent prognostic factor in osteosarcoma patients. Knockdown of PIM1 expression by synthetic siRNA or shRNA greatly inhibited cell growth, migration, and invasion. Moreover, these changes accompanied with down‐regulation of anti‐apoptotic protein Bcl‐2. The similar results were obtained in osteosarcoma cells treated with PIM1 specific inhibitor (SMI‐4a). These results suggest that PIM1 kinase is critical for the growth and metastasis of osteosarcoma cells and can be a potential therapeutic target for osteosarcoma treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1185–1194, 2016.     

Urine matrix metalloproteinases and their extracellular inducer EMMPRIN in children with chronic kidney disease

Abstract

Background: Transforming growth factor (TGF)beta1 and matrix metalloproteinases (MMPs) play an essential role in CKD-related tissue remodeling. However, there are no data on urine MMPs and their extracellular inducer EMMPRIN in CKD patients. The aim of study was to assess the concentrations of MMP-2, MMP-7, MMP-9, EMMPRIN and TGFbeta1 in serum and urine of CKD children and to analyze the potential relations between those parameters. Methods: Forty-one pre-dialysis CKD children and 23 age-matched controls were enrolled in the study. The concentrations of analyzed parameters were assessed by ELISA. Results: Serum and urine values of MMP-2, MMP-7, MMP-9, EMMPRIN and TGFbeta1 were significantly elevated in CKD patients versus controls. The MMP-2 and MMP-9 levels in urine correlated significantly with the corresponding values in serum, whereas MMP-7, EMMPRIN and TGFbeta1 urine concentrations did not. There were also significant correlations between urine values of all parameters. Conclusions: The increased urine levels of MMPs, EMMPRIN and TGFbeta1 indicate enhanced proteolysis and renal tissue remodeling. In the case of MMP-7, EMMPRIN and TGFbeta1 those disturbances seem independent of enhanced serum activity of the corresponding enzymes. The urine MMP-7 and EMMPRIN concentrations may serve as new independent indices of tissue remodeling and renal interstitial fibrosis in children with CKD.     

ROCK1 as a potential therapeutic target in osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. Patients with localized disease are routinely treated with surgery and chemotherapy. Unfortunately, many of these patients eventually relapse even after high‐dose pre‐ and postoperative chemotherapy. Upon recurrence of the tumor locally or distantly, they have limited treatment options that are usually unsuccessful. Our prior studies screening lentiviral shRNA libraries, searching for kinases involved in osteosarcoma cell growth and proliferation have identified the Rho‐associated coiled‐coil containing protein kinase 1 (ROCK1) as a possible hit. We show in this study that ROCK1 is highly expressed in various tumor cell lines and tumor tissues from osteosarcoma patients. ROCK1 knockdown by synthetic siRNA decreases cell proliferation, viability and induces apoptosis in osteosarcoma cell lines KHOS and U‐2OS. Finally, we established the relationship between expression levels of ROCK1 and clinical prognosis in osteosarcoma patients by using immunohistochemistry. There were significant differences in overall survival between cohorts of patients with ROCK1 levels categorized as high‐staining, moderate‐staining, and low‐staining. High levels of ROCK1 were associated with poor outcomes in clinical osteosarcoma. These findings suggest that knockdown of ROCK1 inhibits proliferation and induces apoptosis in osteosarcoma cell lines. ROCK1 may be a promising therapeutic target for the treatment of osteosarcoma patients. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1259–1266, 2011