Immunohistochemical changes in vulnerable rat brain regions after reversible global brain ischaemia

Human global ischaemia was simulated in adult rats by inducing 20 min brain ischaemia and 60 min post-ischaemic recirculation. Immunohistochemical expression of MMP-9, TIMP-3, Bax and Bcl-2, and DNA fragmentation (with the TUNEL reaction) were investigated. The morphological data showed different neuronal responses in the hippocampus compared with the cerebral and cerebellar cortices. MMP-9 immunoreactivity was different in the hippocampus, particularly in dentate gyrus and the CA1 region, compared with these cortices. Negative TIMP-3 staining in ischaemic hippocampal neurons may indicate a loss of its inhibitory activity on MMP-9 that could enhance cell death. Bcl-2 down regulation, Bax positivity and TUNEL+ type II cells in the dentate gyrus granular layer could be responsible for induction of apoptotic death in CA1 hippocampal pyramidal cells via loss of fibre input. Results suggest differential behaviours of neural cells after 60 min reperfusion.     

TIMP-1-GPI in combination with hyperthermic treatment of melanoma increases sensitivity to FAS-mediated apoptosis

Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8°C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes. Elfriede Noessner, Peter J. Nelson are equal contributors.     

基质金属蛋白酶及其抑制剂在乳腺癌中的表达

目的:研究基质金属蛋白酶2(Matrix Metalloproteinase-2,MMP-2),基质金属蛋白酶7(MMP-7),基质金属蛋白酶9(MMP-9),膜型基质金属蛋白酶(Membrane Type-1 Matrix Metalloproteinase,MT1-MMP),金属蛋白酶组织抑制剂1(Tissue Inhibitor of Metalloproteinase,TIMP-1),金属蛋白酶组织抑制剂2(TIMP-2)在乳腺癌组织中mRNA的表达,及与临床病理变量之间的关联。方法:采用150例乳腺癌患者的组织样本。使用半定量逆转录-聚合酶链反应(RT-PCR)法来测定肿瘤组织和正常乳腺组织中MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2的mRNA表达。结果:MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2在乳腺癌中的mRNA表达显著高于正常组织。结论:MMP-2,MMP-7,MMP-9,和MTI-MMP的表达增加和临床病理参数之间的关联,可以用来预测乳腺癌的侵害行为。     

The role of the apoptosis and the genes regulating apoptosis in the early differentiation of human embryo

Apoptosis (programmed cell death) is an important process participating in the formation of organs and tissues during embryogenesis. Our aim of the work is studying the role of the apoptosis during the human embryonic differentiation. We tend to give acquired findings into the correlation with expression of proteins Bcl-2 and Bax (products of genes regulating apoptosis). Detection of the apoptosis was carried out on 25 routinely processed human embryos by means of TUNEL technique. The level of expression of Bcl-2 and Bax was determined using standard three-step immunohistochemical procedure. Results were achieved by the comparison of apoptoic index and the level expression of Bcl-2 and Bax was semiquantitatively evaluated. The low value of apoptotic index was mostly accompanied by the high expression of Bcl-2 and the Bax expression was not proportionally related to the value of apoptic index.     

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.     

Collagenase expression and activity is modulated by the interaction of collagen types, hypoxia, and nutrition in human lung cells

Hypoxia not only controls organogenesis, embryogenesis, and wound repair, but also triggers tumor progression and metastasis. Matrix metalloproteinases (MMP), especially gelatinases (MMP-2, MMP-9) regulate the composition and stability of the extracellular matrix (ECM), which affects cell proliferation, migration, and differentiation. This study investigated the effect of hypoxia alone and in combination with ECM compounds and nutrition on MMP-2 and MMP-9 expression, activity, and synthesis in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMC). We also determined the expression of the tissue inhibitors of MMP (TIMP-1, -2). Cells were grown on plastic, collagen-I, collagen-IV, or gelatin and in either starving medium (0.1% serum) or growth medium (5% serum), and were subjected to normoxia or hypoxia (1% O(2)). Collagenases expression was determined by zymography. TIMP-1, -2 expression was assessed by Western blotting and RT-PCR. Depending on serum concentration human lung cells expressed pro-MMP-2 on all substrates. Hypoxia increased pro-MMP-2 expression, on collagen type I or type IV further via Erk1/2 and p38 MAP kinase signaling. MMP-9 was only expressed when cells were grown on collagen type IV and increased with serum concentration, and by hypoxia. TIMP-1 expression was only expressed when cells were grown on collagen type I and was significantly increased by hypoxia, while TIMP-2 expression was unchanged. We demonstrated that the hypoxia, ECM composition, and nutrition, rather than one of these conditions alone, modulate the expression and activity of collagenases and their inhibitors in primary human lung fibroblasts.     

Plaque oxysterols induce unbalanced up-regulation of matrix metalloproteinase-9 in macrophagic cells through redox-sensitive signaling pathways: Implications regarding the vulnerability of atherosclerotic lesions

An imbalance in the matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs) contributes to atherosclerotic plaque destabilization and rupture. Here we determined whether oxysterols accumulating in advanced atherosclerotic lesions play a role in plaque destabilization. In human promonocytic U937 cells, we investigated the effects of an oxysterol mixture of composition similar to that in advanced human carotid plaques on the expression and synthesis of MMP-9 and its endogenous inhibitors TIMP-1 and TIMP-2. A marked increment of MMP-9 gene expression, but not of its inhibitors, was observed by real-time RT-PCR; MMP-9 gelatinolytic activity was also found increased by gel zymography. Consistently, a net increment of MMP-9 protein level was also observed by immunoblotting. Using antioxidants or specific inhibitors or siRNAs, we demonstrated that the oxysterol mixture induces MMP-9 expression through: (i) overproduction of reactive oxygen species, probably by NADPH-oxidase and mitochondria; (ii) up-regulation of mitogen-activated protein kinase signaling pathways via protein kinase C; and (iii) up-regulation of activator protein-1- and nuclear factor-κB-DNA binding. These results suggest, for the first time, that oxysterols accumulating in advanced atherosclerotic lesions significantly contribute to plaque vulnerability by promoting MMP-9/TIMP-1/2 imbalance in phagocytic cells.     

The Inhibitory Effect of C-phycocyanin Containing Protein Extract (C-PC Extract) on Human Matrix Metalloproteinases (MMP-2 and MMP-9) in Hepatocellular Cancer Cell Line (HepG2)

Spirulina platensis :have been studied for several biological activities. In the current study C-phycocyanin containing protein extract (C-PC extract) of Spirulina platensis have been studied for its effect on human matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and tissue inhibitors of MMPs (TIMP-1 and TIMP-2). In the present study, breast cancer cell line (MDA-MB 231) and hepatocellular cancer cell line (HepG2) were examined for inhibition of MMPs at different levels of expression after C-PC extract treatment. Herein, we have demonstrated that C-PC extract significantly reduced activity of MMP-2 by 55.13% and MMP-9 by 57.9% in HepG2 cells at 15 μg concentration. Additionally, the treatment has reduced mRNA expression of MMP-2 and MMP-9 at 20 μg concentration by 1.65-folds and 1.66-folds respectively. The C-PC extract treatment have also downregulated a mRNA expression of TIMP-2 by 1.12 folds at 20 μg concentration in HepG2 cells. Together, these results indicate that C-PC, extract successfully inhibited MMP-2 and -9 at different levels of expression and TIMP-2 at a mRNA expression level; however, extract did not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells as well as the exact mechanism of inhibition of MMP-2, MMP-9 and TIMP-2 remained unclear.     

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.     

Nampt及高糖高胰岛素微环境对人顺铂耐药肺癌细胞株增殖和转移的影响

摘要 目的：探究烟酰胺磷酸核糖转移酶（Nampt）及高糖高胰岛素（HG+HI）微环境对人顺铂耐药肺癌细胞增殖和转移的影响。方法：将人顺铂耐药细胞株A549/DDP分为6组（n=6）：对照组（control）、高糖高胰岛素干预组（HH，使用添加30 mmol/L的葡萄糖和500 mU/L的胰岛素的培养基培养72 h）、分别转染sh-NC和sh-Nampt组（sh-NC和sh-Nampt，使用Lipofectamine 2000将sh-NC和sh-Nampt分别转染到细胞中，转染时间为48 h）、HH干预sh-NC和sh-Nampt组（HH+sh-NC和HH+sh-Nampt）。每组6个重复样本。qRT-PCR检测转染效率，MTT法检测细胞增殖，流式细胞仪检测细胞凋亡，Transwell检测细胞迁移和侵袭，qRT-PCR检测Nampt mRNA，Western blot检测Nampt、Bcl-2、Bax、MMP-2、MMP-9、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果：与对照组和sh-NC组比较，sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，而细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。与对照组和sh-NC组比较，HH组和HH+sh-NC组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平升高，Bax蛋白表达水平降低（P<0.005）。与HH组和HH+sh-NC组比较，HH+sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。结论：高糖高胰岛素微环境可能通过上调Nampt/PI3K/AKT信号通路诱导人顺铂耐药肺癌细胞的增殖和转移。     

基于JAK2/STAT3信号通路探讨白藜芦醇对骨肉瘤MG-63细胞凋亡及迁移的影响

摘要 目的：研究白藜芦醇(RES)通过蛋白酪氨酸激酶2/信号转导子与激活子3（JAK2/STAT3）信号通路对人骨肉瘤体外细胞株MG-63细胞凋亡、侵袭和迁移的影响。方法：体外培养MG-63细胞,以不同浓度的RES作用于MG-63细胞。Annexin V-FITC/PI双染流式细胞术检测不同时间和不同浓度的RES对MG-63细胞凋亡的影响。划痕实验和Transwell实验检测不同时间和不同浓度的RES对MG-63细胞侵袭和迁移能力的影响。免疫印迹实验检测不同时间和不同浓度的RES对MG-63细胞磷酸化蛋白酪氨酸激酶2（p-JAK2）、磷酸化信号转导子与激活子3（p-STAT3）、凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2家族促凋亡蛋白（Bax）及基质金属蛋白酶（MMP）-2、MMP-9表达的影响。结果：RES浓度越高,时间越久,MG-63细胞凋亡率越高（P<0.05）。RES浓度越高,MG-63细胞迁移和侵袭能力越弱(P<0.05)。RES处理MG-63细胞后其p-JAK2、p-STAT3、Bcl-2以及MMP-2、MMP-9的表达明显降低,而Bax蛋白表达明显升高,且p-JAK2、p-STAT3、Bax、Bcl-2以及MMP-2、MMP-9的表达水平变化具有RES浓度依赖性（P<0.05）。结论：RES可能通过调控JAK2/STAT3信号通路促使人骨肉瘤MG-63细胞凋亡,并抑制MG-63细胞侵袭和迁移。     

Interstitial collagenase, gelatinases A and B and their endogenous inhibitors in squamous cell cervical carcinomas

Interstitial collagenase and gelatinases are matrix metalloproteinases (MMP), which play the key role in tumor invasion and metastasis determining tumor malignancy. The aim of this study was to elucidate peculiarities of expression of interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9) and their endogenous tissue inhibitors TIMP-1 and TIMP-2 as invasive factors of squamous cell carcinomas (SCC) of human cervical cancer. The study was carried out using 24 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to tumor. All carcinoma specimens expressed the E7 HPV-16 gene. Results obtained indicate that the increased expression of MMP-1 and MMP-9 and low expression of TIMP-1 and TIMP-2 make the main contribution to the destructive (invasive) potential of SCC. Changes in MMP-2 expression are less important. In the specimens of morphologically normal tissue adjacent to the tumor, substantial expression of MMP-1, MMP-2 and MMP-9 was registered. This expression appears to make additional contribution to the tumor destructive potential.     

PTEN蛋白表达和明胶酶及其抑制因子在乳腺癌侵袭转移中的关系

目的研究乳腺癌中明胶酶(MMP-2、MMP-9)及其抑制物(TIMP-1)与抑癌基因PTEN产物PTEN蛋白的表达在肿瘤侵袭转移中的关系.方法应用免疫组织化学S-P法检测98例浸润性乳腺癌组织MMP-2、MMP-9及TIMP-1和PTEN蛋白的表达.结果乳腺癌MMP-2、MMP-9与PTEN表达呈显著负相关,而TIMP-1与PTEN蛋白表达呈显著正相关(P<0.05).在PTEN低表达组中,MMP-2、MMP-9的表达与腋窝淋巴结受累呈正相关;在PTEN高表达组中,MMP-2、MMP-9的表达与腋窝淋巴结受累呈正相关,TIMP-1与腋窝淋巴结受累呈负相关.结论在乳腺癌侵袭转移中MMP-2、MMP-9、TIMP-1与PTEN蛋白表达显著相关.MMP-2、MMP-9和TIMP-1对肿瘤细胞侵袭转移的作用可能在一定程度上受到PTEN的调控.     

Apoptosis in human term placenta is not increased during labor but can be massively induced in vitro.

Apoptosis in human placental villi is reported to increase until close to delivery. However, the involvement of the apoptotic process in the initiation of labor, and more particularly in relation to the decrease in placental perfusion during uterine contractions, remains unknown. The purpose of the study was to examine the reactivity of the apoptotic machinery in term placentae obtained before or after the onset of labor and after in vitro incubations. The incidence of apoptotic nuclei (< 1%) as evidenced by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, and the histological distribution of immunoreactive Bcl-2, Bax, and Bcl-x proteins, were similar in placentae collected after delivery and before the onset of labor and in placental explants maintained overnight at 4 degrees C in a minimal salt-Hepes medium. By contrast, 28% of nuclei contained fragmented DNA when placental explants were incubated overnight at 37 degrees C. This marked increase was associated with a decrease in the intensity of the Bcl-2 immunostaining and an increase in the intensity of Bax and Bcl-x immunostaining. In conclusion, the present study clearly evidences the presence of an active apoptotic machinery in term placental cells that is not involved in normal parturition.     

Opposite effects of high glucose on MMP-2 and TIMP-2 in human endothelial cells

Diabetes mellitus (DM) is a major risk factor for atherosclerosis and causes multiple cardiovascular complications. Although high glucose can induce matrix metalloproteinases (MMPs), its inhibitors and cell apoptosis, little is known about the roles of MMPs in regulating cell apoptosis in response to high glucose. To address this issue, we elucidated the relationship between MMPs, its inhibitors and cell apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with medium containing 5.5 mM or 33 mM of glucose in the presence or the absence of ascorbic acid and MMP inhibitors (GM6001 and endogenous tissue inhibitors of MMPs, TIMP-1, and TIMP-2). For detection of cell apoptosis, the cell death detection ELISA assay was used. The results revealed that high glucose-induced apoptosis could be suppressed by ascorbic acid, GM6001 and TIMP-2, but not by TIMP-1. The activities of MMP-2, MMP-9 and its inhibitors, TIMP-1, TIMP-2 after high glucose treatment, were also detected by ELISA method. We found that the activated form of MMP-2, but not MMP-9, was increased, while the level of TIMP-2, but not TIMP-1, was decreased. In Western blot and RT-PCR analysis, the expression of TIMP-2, but not TIMP-1, after high glucose treatment was downregulated, whereas the levels of MMP-2 and -9 proteins and mRNA were not changed. The present study indicated that oxidative stress induced by high glucose might be involved in the opposite effects on MMP-2 activation and TIMP-2 downregulation. This reactive oxygen species (ROS)-dependent MMP-2 activation in turn mediates high glucose-induced cell apoptosis in HUVECs.     

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN’s apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.     

Early establishment of epithelial apoptosis in the developing human small intestine

In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.