Dysregulation of Matrix Metalloproteinases and their Tissue Inhibitors by S100A4

The S100A4 protein as well as the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are associated with diseases such as arthritis and cancer. This mini review focuses on in vitro and in vivo studies indicating S100A4 involvement in regulation of MMPs and TIMPs, and the biological and pathobiological consequences of this regulation.     

转VEGF基因对血管损伤后修复过程中基质金属蛋白酶表达的影响

目的: 观察局部转染血管内皮生长因子(VEGF)基因对血管球囊拉伤后修复过程中基质金属蛋白酶(MMPs)及其组织抑制因子(TIMPs)表达的影响。方法: 90只新西兰大白兔随机分为3组,Ⅰ组单纯拉伤右髂动脉;Ⅱ组拉伤后局部转染真核表达质粒AdtrackCMV;Ⅲ组拉伤后局部转染pAdtrackCMV-VEGF165;每组按实验终点随机分为5个亚组,术前一周开始给予高脂饮食至实验终点。取拉伤段髂动脉用于总RNA提取、组织病理和免疫组化检测。结果: 3组动物血脂水平保持在正常的5-10倍,组间血管损伤程度评分相似(P＞0.05)。Ⅰ、Ⅱ组整个观察过程中均可检测到MMP2、TIMP1,2持续表达;而转VEGF基因组术后3d时可检测到MMP1,2,9、TIMP1,2表达,1周时达到高峰,8周时无表达。结论: 血管损伤后修复过程中存在MMPs和TIMPs表达失衡,致使基质堆积,并发生病理性重塑,而局部转染VEGF基因选择性改变局部MMPs表达,促进基质降解和适应性重塑。     

Chinese Medicinal Herbs in Treating Model Rats with Hepatic Fibrosis

The objective of this study was to examine the effects of Chinese medicine formula-Yu Zhang Dan (YZD, composed of Herba Lysimachiae, Rhizoma Polygoni Cuspidati, Radix Curcumae) on the model rats with hepatic fibrosis. Forty male Sprague-Dawley (SD) rats were used in the present study, and they were separated randomly into 4 groups: a normal control group (Group A, n=5), a model control (Group B, n=15), a high dose of YZD (Group C, n=10), and a low dose of YZD (Group D, n=10). Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl₄). The variation of the serum alanine transaminase (ALT), aspartate aminotransferase (AST), hyaluronate acid (HA), laminin (LN), type • • procollagen peptide (P• •NP), L-Glutathione (GSH) was respectively measured with radioimmunoassay (RIA) and detection of transforming growth factor-beta 1 (TGF-β1) and smooth muscle alpha actin (α-SMA) was conducted with immunohistochemistry. The ALT, AST HA, LN and PIII NP levels in the serum of the model control group were significantly higher than those of the normal control group (P<0.05), and both of the high dose of YZD and low dose of YZD significantly decreased the ALT, AST HA, LN and PIII NP levels of the model rats (P<0.05). The TGF-β1 and α-SMA levels of the model control group were significantly higher than those of the normal control group (P<0.05), and both of the high dose of YZD and low dose of YZD significantly decreased the TGF-β1 levels of the model rats (P<0.05) , and only the high dose of YZD significantly decreased the α-SMA levels of the model rats (P<0.05). The expression of TGF-β1 and α-SMA in the liver tissues of the rats were in the cytoplasm of the cells. It may be through decreasing the ALT, AST, HA, LN and PIII NP levels in the serum of the model rats and decreasing the expression of TGF-β1 and α-SMA in the liver tissues of the model rats that YZD significantly relieved the hepatic fibrosis.     

成骨细胞旁分泌影响软骨细胞中MMPs与TIMPs的表达

背景：细胞之间体外共培养能最大限度的模拟体内真实的微环境,细胞划痕实验及炎症因子白细胞介素1β刺激后基质金属蛋白酶及基质金属蛋白酶抑制剂之间的平衡可能破坏,从而导致关节软骨细胞外基质的降解,软骨细胞功能的失调,关节软骨的退变。目的：在成骨细胞上清液与软骨细胞体外共培养下,观察炎症因子白细胞介素1β对体外培养的软骨细胞的迁移、基质金属蛋白酶及组织金属蛋白酶抑制剂表达的影响。方法：实验分为软骨细胞单培养组﹑软骨细胞与成骨细胞上清液共培养组和软骨细胞与成骨细胞上清液共培养+白细胞介素1β组,划痕实验观察3组24 h软骨细胞的迁移变化;半定量PCR实验分析以上3组24 h软骨细胞中基质金属蛋白酶1,2,3,9及组织金属蛋白酶抑制剂1,2,3,4的变化情况。结果与结论：与单培养组比较,共培养组和共培养+白细胞介素1β组细胞迁移率显著增加(P < 0.01);与单培养组比较,共培养组中基质金属蛋白酶1,2,3,9基因表达明显增高(P < 0.05),共培养+白细胞介素1β组基质金属蛋白酶1,3,9基因表达明显增高(P < 0.01);与单培养组比较,共培养组和共培养+白细胞介素1β组中组织金属蛋白酶抑制剂1基因表达明显升高(P < 0.01),组织金属蛋白酶抑制剂3,4基因表达明显下降(P < 0.05)。提示成骨细胞上清液与软骨细胞共培养促进软骨细胞的迁移,增强软骨细胞中基质金属蛋白酶1,2,3,9的基因表达且调节组织金属蛋白酶抑制剂家族的基因表达。白细胞介素1β抑制共培养的软骨细胞迁移及组织金属蛋白酶抑制剂家族的基因表达。        中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300 mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1–10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.     

Role of metalloproteinases and tissue inhibitors of metalloproteinases during cardiopulmonary bypass in rats

Matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) regulate matrix remodeling in the heart. Changes in synthesis and release of MMPs and TIMPs are observed after extracorporeal circulation (ECC). Thus, MMPs and TIMPs are supposed to be involved in ECC-mediated cardiac dysfunction. The aim was to examine the role of MMPs and TIMPs in ECC-mediated cardiac dysfunction. Extracorporeal circulation was instituted in rats for 60 min at a flow rate of 120 ml/kg/min. Three groups (n = 10) were studied: group CAO: 60 min ECC without aortic cross-clamping, group CAC: 60 min ECC including 30 min aortic cross-clamping (crystalloid Inzolen(?) cardioplegia), and group CAB: 60 min ECC including 30 min aortic cross-clamping (blood cardioplegia). Left ventricular (LV) function was measured with conductance catheter. Matrix metalloproteinase-activity was determined by zymography and TIMP activity was determined by reverse zymography. Gene expression of MMPs and TIMPs was determined by real-time polymerase chain reaction. Sixty minutes after weaning from bypass, there was a preserved LV function in the CAO and CAB group and an impaired LV function in the CAC group. We observed an increased myocardial activity and an increased myocardial messenger RNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-4 in all ECC groups, when compared with sham animals. With regard to enzyme activity, there was an imbalance of MMP/TIMP ratio leading to an increased activity of MMP in the CAC group. In terms of gene expression, there was an imbalance of MMP-2/TIMP-4 ratio leading to an increased expression of MMP-2 in the CAC group. MMP-2 contributes to myocardial reperfusion injury in this in vivo model of ECC with cardioplegic arrest.     

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Pituitary Adenomas: Possible Markers of Neuroendocrine Cells

Matrix metalloproteinases (MMPs) are involved in remodeling processes and have been immunocytochemically localized in some endocrine glands and their tumors. Using anterior pituitary gland and pituitary adenomas, immunocytochemical localization of MMP-2 (gelatinase-A),-9 (gelatinase-B), tissue inhibitor of metalloproteinase (TIMP)-1 and-2 was performed. Normal anterior-pituitary cells all contain MMPs and lesser amount of TIMPs, whereas far fewer MMPs and TIMPs are identified in anterior pituitary adenomas. There is no correlation between pituitary hormone and MMPs-TIMPs localization, thus MMP-TIMP homeostasis may not be involved in hormone synthesis and secretion of anterior pituitary cells and their adenomas. Because MMPs and TIMPs are more abundantly and specifically localized in pituitary cells and their adenomas, MMPs and TIMPs may be included as markers for endocrine cells, including anterior-pituitary cells.     

Antifibrotic potential of a selective COX-2 inhibitor (celecoxib) on liver fibrosis in rats

Hepatic stellate cells (HSCs) play a critical role in the fibrogenesis of the liver, so they are the target cells of antifibrotic therapy. Activated HSCs, but not quiescent HSCs, express cyclooxygenase-2 (COX-2). The present study was designed to investigate the possible prophylactic and therapeutic effects of a selective COX-2 inhibitor (celecoxib) on liver fibrosis induced by thioacetamide (TAA) in rats. Forty-two male albino rats were divided into five groups: group I, negative control; group II, model of fibrosis; group III, preventive model before induction of fibrosis where celecoxib was given for 4 weeks before TAA; group IV, preventive model at the time of induction of fibrosis where celecoxib was given concomitantly with TAA for 6 weeks; group V, therapeutic model treated after induction of fibrosis. Liver function tests, serum TGF-β1 (ELISA), and histopathological examination of liver sections were performed. Both Metavir and Ishak fibrosis scoring systems were used for the evaluation of fibrosis. Groups III, IV, and V showed significant amelioration of liver function tests and a decrease in serum TGF-β1 as compared to the fibrosis model group (II). Histopathological examination showed the mildest degree of fibrosis in group III. Celecoxib had a hepatoprotective and therapeutic effect against liver damage produced by TAA but with different degrees. The highest efficacy of celecoxib was in the preventive group (III) before time of induction of liver fibrosis, followed by the therapeutic group (V) and then the preventive group (IV) at time of induction of liver fibrosis.     

Value of α-smooth muscle actin and glial fibrillary acidic protein in predicting early hepatic fibrosis in chronic hepatitis C virus infection

Introduction

α-Smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. The reports concerning GFAP expression in human liver are still conflicting. The aim of the study is investigation the utility of GFAP compared to α-SMA as an indicator of early activated HSCs, in predicting fibrosis in chronic hepatitis C (CHC) patients.

Material and methods

With immunohistochemistry and a semi-quantitative scoring system, the expressions of α-SMA and GFAP on HSCs in liver biopsies from patients with pure CHC (n = 34), hepatitis C virus-induced cirrhosis (n = 24), mixed CHC/schistosomiasis (n = 11) and normal controls (n = 10) were analysed.

Results

The immunoreactivity of α-SMA and GFAP in perisinusoidal, periportal and pericentral areas was assessed. α-Smooth muscle actin and GFAP-positive HSCs were significantly increased in all diseased groups compared with normal controls. In pure CHC with or without cirrhosis, perisinusoidal α-SMA-positive HSCs were predominant in relation to GFAP-positive cells. On the other hand, GFAP-positive cells were predominant in the group of schistosomiasis as compared with the other diseased groups. It was noticed that expression of GFAP on perisinusoidal HSCs in CHC patients sequentially decreased with the progression of fibrosis.

Conclusions

Glial fibrillary acidic protein could represent a more useful marker than α-SMA of early activation of HSCs in CHC patients and seems to be an early indicator of hepatic fibrogenesis.     

Integrin β1 Establishes Liver Microstructure and Modulates Transforming Growth Factor β during Liver Development and Regeneration

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin β1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin β1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell–cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor β (TGF-β) and widespread fibrosis. Targeted deletion of integrin β1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin β1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-β. Taken together, these findings identify integrin β1 as a key determinant of liver architecture with a critical role as a regulator of TGF-β secretion. These results suggest that disrupting the hepatocyte–extracellular matrix interaction is sufficient to drive fibrosis.

The homogeneous appearance of the liver belies a complex microstructure essential for proper function. A fenestrated endothelium provides minimal resistance to blood flow to maintain low portal pressure and allows filtered plasma to bathe hepatocytes, permitting nutrient exchange and release of synthetic products. At the apical hepatocyte surface, a specialized canalicular network conveys bile out of the liver for secretion into the intestine.Establishing precise microstructure is thus critical for liver function. The role of hepatocyte–extracellular matrix (ECM) interactions in the development of liver microstructure was therefore examined in the current study.Integrins play an essential role in hepatocyte–ECM interactions. They are single-pass transmembrane receptors that function as adhesion molecules on the cell surface and mediate cell–matrix and cell–cell interactions.¹ They exist on the hepatocyte surface as heterodimers of an α and β subunit and contain a short cytoplasmic tail responsible for chemical signaling and physical force transduction through links to the actin cytoskeleton.² Component subunits exhibit high selectivity in their interactions. In particular, integrin receptors on hepatocytes bind collagen I, laminin, and fibronectin.3, 4, 5Integrin signaling involves a multiprotein complex that is recruited to and becomes associated with the intracellular portion of the protein. Although heterogeneous, signaling often involves focal adhesion kinase (FAK) as a platform for various phosphorylation events, including autophosphorylation, as well as signaling involving Src-family kinase, integrin-linked kinases, and paxillin.6, 7, 8 Principal integrin pairs expressed by hepatocytes are α1β1, α5β1, and a9β1.^9,10 The α1β1 heterodimer predominantly binds collagen IV, the α5β1 receptor binds fibronectin, and the α9β1 receptor binds to a non-RGD (Arg-Gly-Asp) site on tenascin.^11,12 Data on specific roles for integrins in the liver are limited to a few reports. α5β1 integrins sense tauroursodeoxycholic acid and become active in response to cell swelling.¹³ Mice with transgene-mediated osteopontin expression in hepatocytes develop fibrotic livers.¹⁴ Disruption of integrin-linked kinase in hepatocytes leads to increased deposition of ECM.¹⁵ Integrin β1 is essential for liver regeneration, but its effect on fibrosis has not been determined.¹⁶ In humans, hepatitis C is associated with increased expression of various integrins, including β1, α1, α5, and α6, which reflect disease severity.¹⁷ Expression of integrin α6 occurs in a variety of chronic liver diseases.¹⁸A critical role for integrins after injury is also suggested by the known role of ECM during injury response. ECM remodeling via matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is critical in recreating normal liver architecture. MMP-2, MMP-3, and MMP-14 are expressed by stellate cells during activation.^19,20 MMP-9 is expressed in hepatocytes and Kupffer cells after setting of injury.²¹ TIMP-1 modulates liver MMPs, and both TIMP-1 and TIMP-2 are expressed in hepatocytes during injury.22, 23, 24To determine how hepatocyte integrins help establish liver microstructure and re-establish it after liver injury, models were created to assess the consequences of hepatocyte-specific deletion of integrin β1 from before birth and in the adult mice after injury. Hepatocyte size and shape, interactions between hepatocytes and other cells, and signaling pathways relevant to integrin signaling and fibrosis were investigated.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in some endocrine organs and their tumors

Matrix metalloproteinases (MMPs) are single-chain zinc-containing metalloenzymes. The MMP gene family currently includes more than 19 endopeptidases. Both MMP-2 and 9 are widely expressed by many stromal and endothelial cells. Tissue inhibitors of metalloproteinases (TIMPs) form complexes with MMPs, which in turn inhibit active MMPs. MMP and TIMP homeostasis has been implicated in many aspects of both physiological and pathological processes. The latter include tumor invasion and metastasis. Although ductal adenocarcinomas of pancreas were immunocytochemically faintly stained for MMPs and TIMPs, normal pancreatic islets in the normal adjacent pancreas were found to be strongly stained for MMPs and TIMPs. Five kinds of islet cell tumors, including insulinomas, gastrinomas, glucagonomas, pancreatic polypeptide- (PP) omas, and nonfunctioning islet cell tumor, were stained for MMPs and TIMPs. The tumor cells were relatively weakly stained for MMPs and TIMPs compared to normal islets. Similarly, weaker staining for MMPs and TIME’s was noted for medullary thyroid carcinomas (MTCs) and pituitary adenomas. There was no correlation between immunostaining intensity of protein hormones and MMPs and TIMPs. However parathyroid hyperplasia, adenoma, and carcinoma that stained for MMPs and TIMPs were weaker, which paralleled the weaker immunostaining for parathyroid hormone and chromogranin. This weaker staining for MMPs and TIMPs in endocrine tumors may imply a less significant role of tumor invasion and metastasis by MMP and TIMP homeostasis. At present, immunocytochemical staining for MMPs and TIMPs may well be used as new markers for neuroendocrine cells and their tumors.     

环孢霉素A诱导大鼠心肌纤维化和对基质金属蛋白酶及其抑制剂表达的影响

目的:研究环孢霉素A(CsA)诱导的大鼠心肌损伤及心肌纤维化的程度,检测基质金属蛋白酶(MMP) 2和MMP9及其组织抑制物(TIMP) 2和TIMP1表达的变化,为探寻CsA诱导心肌纤维化的发生机制提供理论依据。方法:64只健康雌性6～8周龄Wistar大鼠随机分为4组:对照组、低剂量CsA组、中剂量CsA组和高剂量CsA组,分别腹腔注射生理盐水、5 mg·kg~(-1)·d~(-1)CsA、12. 5 mg·kg~(-1)·d~(-1)CsA和25 mg·kg~(-1)·d~(-1)CsA,分别在第1、2、3周时处死大鼠。利用HE染色观察心肌组织形态结构,Masson染色观察心肌胶原沉积情况,免疫组织化学和Western blot法检测MMP2、MMP9、TIMP2和TIMP1蛋白的表达情况。结果:HE染色可见,随着CsA作用时间的延长和剂量的增加,心肌组织形态结构损伤严重,逐渐出现心肌灶状坏死及纤维化。Masson染色结果亦可见心肌纤维化程度随着时间和剂量的增加逐渐加重。免疫组织化学及Western blot检测结果显示,在CsA导致大鼠心肌纤维化的过程中,同一时点MMP2和TIMP2随CsA剂量增加表达显著增高(P 0. 05)。第1周MMP2高表达,随着时间的延长表达逐渐减低; TIMP2则随时间延长表达逐渐增高(P 0. 05)。与对照组相比MMP9和TIMP1表达则减低。各用药组MMP9第1周低表达,第2周表达增高,第3周表达减低(P 0. 05);同一时点,CsA对于MMP9表达变化的差异无统计学显著性。TIMP1随CsA剂量增加表达增高(P 0. 05)。结论:CsA可引起大鼠心肌损伤及间质纤维化,随着用药时间的延长和剂量的增加,纤维化程度逐渐加重。CsA诱导的大鼠心肌纤维化与MMPs/TIMPs表达的改变及MMPs/TIMPs的失衡有关。     

Tissue Inhibitor of Metalloproteinases 3 Regulates Resolution of Inflammation following Acute Lung Injury

Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits not only matrix metalloproteinases but also a disintegrin and metalloproteinase domain family members and thus contributes to controlling diverse processes mediated by proteolysis. We used Timp3^−/− mice to assess the role of this inhibitor in acute lung injury. After bleomycin-induced injury, inflammation, as indicated by the influx of neutrophils in bronchoalveolar lavage (BAL), peaked at 7 days post-injury in the wild-type mice and began to wane thereafter; however, in Timp3^−/− mice, inflammation persisted up to 28 days. Furthermore, although the level of chemokines in BAL and lung homogenate was similar in both genotypes, BAL from Timp3^−/− mice 7, 14, and 28 days post-injury had increased neutrophil chemotactic activity compared with wild-type BAL. At day 14, a higher percentage of apoptotic neutrophils were present in wild-type mice compared with Timp3^−/− mice, further suggesting that TIMP3 constrains continued neutrophil influx. In addition, total matrix metalloproteinase activity was increased in lungs from Timp3^−/− mice, and treatment of mice with a synthetic inhibitor of metalloproteinases rescued the enhanced neutrophilia phenotype. These data demonstrate that TIMP3 regulates neutrophil influx in the lung following injury through its ability to inhibit metalloproteinase activity and indicates that TIMP3 functions to promote the resolution of inflammation in the lung.Acute lung injury, which often results from severe pulmonary infection or trauma and can progress to acute respiratory distress syndrome, is characterized by profound inflammation and tissue injury.¹ Recovery from acute lung injury requires the proper regulation of wound repair, inflammation, and the deposition and remodeling of extracellular matrix.² Many of these processes are mediated by matrix metalloproteinases (MMPs), which in turn are balanced by their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).^2–4TIMP3, which is expressed and stored in the lung matrix, is unique from other TIMPs in that in addition to inhibiting MMPs, it is also an efficient inhibitor of multiple members of the a disintegrin and metalloproteinase (ADAM) domain family, including ADAM17 or tumor necrosis factor α (TNFα)-converting enzyme.³ The importance of TIMP3 in lung homeostasis is highlighted by the development of enlarged alveolar spaces in aging TIMP3-null (Timp3^−/−) mice.⁵ Additionally, adult Timp3^−/− mice have altered inflammatory responses in various tissues. For example, following partial hepatectomy or systemic LPS exposure, circulating levels of active TNFα are increased in Timp3^−/− mice compared with wild-type mice.^6,7 Furthermore, shedding of TNFα is increased in macrophages isolated from Timp3^−/− mice,⁷ suggesting that TIMP3 controls the proinflammatory activation of macrophages in a cell-autonomous manner.Several members of both the MMP and TIMP families have been demonstrated to control various processes in response to lung injury. For example, our group and others demonstrated that mice lacking matrilysin (MMP7) are protected from bleomycin-induced lung injury due to impaired neutrophil migration into the air space during the acute phase and to a later modest reduction in fibrosis.^8,9 Mice lacking TIMP1 reveal phenotypes in response to bleomycin injury that are nearly opposite to those seen in Mmp7^−/− mice, such as enhanced neutrophil influx into the alveolar space,^10,11 likely due to its ability to inhibit MMP7.^10,11 Overexpression of TIMP1 in the lung epithelium, however, does not protect against the deleterious effects of bleomycin,¹² suggesting that the endogenous levels of this inhibitor are well tuned to govern the activity of proteinases it targets.Recently, microarray analysis of BALB/c (which are resistant to bleomycin-induced lung injury) and C57BL/6 mice (which are susceptible to bleomycin-induced lung injury) revealed that Timp3 expression is reduced in C57BL/6 mice—compared with its elevated expression in BALB/c—during the initial, damaging inflammatory response.¹³ These data predict that TIMP3 functions to moderate the host response to tissue injury. Indeed, we report that whereas TIMP3 does seem to affect early response to injury, it is critical for proper resolution of inflammation and fibrosis at later stages. These findings suggest that this natural inhibitor functions to block the activity of metalloproteinases (both MMP and ADAM) mediating proinflammatory and fibrotic pathways.     

MMP-2, MMP-9 and their inhibitors TIMP-2 and TIMP-1 production by human monocytes in vitro in the presence of different forms of hydroxyapatite particles

After calcium-phosphates biomaterials based implantation like hydroxyapatite (HA) coating, particles are released in the periprosthetic tissues. Wear-debris induced fibrous membranes contain macrophage subsets that can produce metalloproteinases (MMPs), which are considered to be key enzymes in extra-cellular matrix turnover. Tissue inhibitors of metalloproteinases (TIMPs) are important regulator of MMPs activity. Interleukin-1 mainly produced by monocytes can also regulate MMPs production. In the present work, we have evaluated the effect of HA particles characteristics (size, shape and sintering temperature) on the MMP-2, -9 and their respective inhibitors TIMP-2, -1 production. Our results demonstrate that sintering temperature (that modify crystal size and surface area) have little effect on MMPs and TIMPs production. Non-phagocytable particles induced more MMP-9, although phagocytable particles induced more IL-1beta release. The shape of the particles was the most important factor since needle-shaped particles induced the most significant up-regulated expression of MMPs and IL-1beta.     

Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis.

Immunohistochemical and confocal microscopic studies of the localization of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen were made in lung tissues from patients with normal pulmonary histology (n = 3), diffuse alveolar damage (n = 14), and idiopathic pulmonary fibrosis (n = 12). Pretreatment with pepsin revealed otherwise undetectable MMP- and TIMP-immunoreactive sites. In normal lung, MMP-2, MMP-9, TIMP-1, and TIMP-2 were localized in ciliated cells, endothelial cells, pneumocytes, macrophages, and smooth muscle cells; fibroblasts showed a strong reaction only for MMP-2. Only TIMP-2 showed co-localization with type IV collagen. Myofibroblasts and epithelial cells expressed increased reactivity for MMPs and TIMPs in both disorders. The reactivities for MMPs and TIMPs were stronger in diffuse alveolar damage. MMP-2 showed focal co-localization in capillary endothelial and disrupted epithelial basement membranes, suggesting activation of collagenolysis. A protective effect against this lysis was suggested by the extensive co-localization of TIMP-2 with type IV collagen and fibrillar collagens. Alveolar buds showed increased reactivity for MMPs and TIMPs in their lining epithelial cells, myofibroblasts, and their basement membranes; however, their matrices were mostly unreactive. These findings emphasize the complexity of the roles of MMPs and TIMPs in collagen turnover in diffuse alvcolar damage and idiopathic pulmonary fibrosis.     

Metalloproteinases and their Tissue Inhibitors in Multiple Sclerosis

Matrix metalloproteinases (MMPs) comprise a family of proteolytic enzymes. MMPs are capable of disrupting the blood-brain barrier (BBB), mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in the processing of a variety of cell surface molecules, including the proinflammatory cytokine TNF-alpha. Each of these mechanisms are thought to be important in the pathogenesis of multiple sclerosis (MS). We investigated mRNA expression of MMP-3, MMP-9 and two tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in parallel in blood mononuclear cells (MNC) from patients with MS and controls, using in situ hybridization. Numbers of MMP-9 mRNA-expressing cells in blood were higher in patients with MS compared to other neurological diseases (OND), other inflammatory neurological diseases (OIND) and healthy subjects (P<0.0001 for all comparisons). Patients with MS had also higher levels of MMP-3 and TIMP-1 mRNA expressing blood MNC compared to patients with OND and healthy subjects. A positive correlation was observed for MMP-9 and TIMP-1 mRNA expression in MS. These results demonstrate that MMPs and TIMPs are upregulated in MS and may contribute to the pathogenesis of the disease.     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.