Decreased expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in the kidneys of hereditary nephrotic (ICGN) mice

Matrix metalloporoteinases (MMPs), which are dominantly regulated by tissue inhibitors of metalloproteinase (TIMPs), play important roles in extracellular matrix (ECM) degradation and are involved in the progression of kidney diseases. In glomeruli and tubulointerstitum of hereditary nephrotic (ICR-derived glomerulonephritis: ICGN) mouse kidneys, hyper-accumulation of ECM components occurred, and MMP activity decreased. In the present study, because lower levels of MMP activity may contribute to the progression of renal fibrosis in ICGN mice, Western blotting analysis and immunohistochemical staining for MMPs and TIMPs were performed to verify the expression levels of these proteins. Levels of MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 in the kidneys were decreased in ICGN mice in comparison with normal ICR mice. These results indicate that small amounts and low levels of activity of MMPs cause the progression of renal fibrosis in ICGN mice.     

Seizure activity in dogs is associated with enhanced TIMP-2 expression of microglia

In the pathogenesis of epilepsy aberrant synaptic plasticity plays an important role. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are responsible for nervous tissue remodelling resulting in synaptic plasticity in the central nervous system (CNS) and might therefore be crucially involved in epileptogenesis. To assess the potential pathogenetic role of microglial MMPs and TIMPs in seizure induction, twenty-four dogs suffering from different intracranial diseases with and without seizure activity were comparatively examined. Microglial cells were isolated by density gradient centrifugation and their expression profiles of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) were examined via quantitative real-time PCR (qPCR). Interestingly, a significant up-regulation of TIMP-2 expression was found for the first time in dogs suffering from seizures. In conclusion, microglial TIMP expression might be involved in seizure generation.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.     

Variations on brain microglial gene expression of MMPs, RECK, and TIMPs in inflammatory and non-inflammatory diseases in dogs

Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloproteinases), and the membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) contribute to the pathogenesis of many CNS diseases. To assess the potential pathogenetic roles of microglial MMP, TIMP, and RECK generation in extracellular matrix breakdown, opening of the blood brain barrier (BBB) and subsequent recruitment of leukocytes in the CNS, twenty-four dogs suffering from spontaneously occurring different intracranial and extracranial (control group) diseases were examined. Microglia cells were isolated ex vivo by density gradient centrifugation and their expressions of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK were examined via quantitative real-time polymerase chain reaction (qPCR). Zymography on CNS tissues in selected cases was performed to assess differences at the protein level. Dogs were grouped in different disease categories according to histopathological examinations, in groups with or without inflammatory reactions, and in groups with/without contrast enhancement in advanced diagnostic imaging as a function of BBB breakdown. The results showed a significant up-regulation of MMP-9 in dogs with inflammation in the nervous system compared to dogs with non-inflammatory diseases. An increased expression of MMP-9 might lead to a facilitated invasion of white blood cells. Furthermore, down-regulation of MMP-13 was found in dogs with contrast enhancement. Zymographical data reflected MMP-2 qPCR data. In conclusion, differential expression of MMPs and their inhibitors, but not of RECK, which might crucially influence the pathogenesis of a given disease, could be demonstrated in canine microglia. This reflects a further pathway in the microglial repertoire to respond to various disease conditions in the CNS, a characteristic that might be of particular relevance as a target for specific treatments.     

Investigation of the composition, turnover, and thermal properties of ruptured cranial cruciate ligaments of dogs

OBJECTIVE: To assess different components of the extracellular matrix with regard to their thermal properties, composition, and turnover in ruptured cranial cruciate ligaments (CCLs) of dogs, compared with components of intact CCLs from a breed predisposed to CCL failure. SAMPLE POPULATION: Ruptured CCLs obtained from 8 dogs of breeds predisposed to ruptured CCLs and intact CCLs from 12 cadaveric Labrador Retrievers. PROCEDURE: Ruptured and intact CCLs were analyzed for water content; collagen content and collagen cross-links were evaluated via hydroxyproline and amino-acid analyses, respectively. Glycosaminoglycan (GAG) content was analyzed via dimethylmethylene blue and uronic acid assays. Matrix metalloproteinases (MMPs)-2 and -9 and the tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 were detected via gelatin SDS-PAGE zymography and reverse gelatin zymography. Thermal analysis of ligaments was performed by use of differential scanning calorimetry. RESULTS: Ruptured CCLs had significantly higher lamounts of immature cross-links, total and sulfated GAGs, and water content, compared with that of the intact ligaments. Compared with intact CCLs, concentration of pro-MMP-2 was significantly higher in ruptured CCLs; the maximum temperature of collagen denaturation was significantly lower in the ruptured CCLs. CONCLUSIONS AND CLINICAL RELEVANCE: The extracellular matrix of ruptured CCLs had an increased matrix turnover indicated by increased collagen and GAG synthesis, compared with that of intact CCLs. Although the extracellular matrix changes may have occurred before ligament rupture, it is possible that these observed changes may be part of a reparative process after rupture.     

Detection of the Matrix Metalloproteinases MMP‐2 and MMP‐9 and Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐2 in Llama (Lama glama) Oviduct

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in several reproductive events like oocyte–spermatozoa interaction and semen liquefaction. In order to study their role in the llama oviductal reproductive process, MMP activity in oviductal fluid (OF) was assayed. Considering that llama genome sequences are partially known, a strategy to procure cDNA sequences of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 was designed. Afterwards, their expression patterns in the different llama oviductal segments were assayed. Gelatine zymograms detected 62 and 94 kDa protease activities that matched MMP‐2 and pro‐MMP‐9, respectively. Expression pattern analysis showed that MMP and TIMP mRNAs were present in ampulla, isthmus, utero‐tubal junction (UTJ) and papilla. Altogether, these findings support the argument that MMPs/TIMPs are produced in the oviduct and secreted into the oviductal lumen. Our results encourage further studies to elucidate the role of these proteins in reproductive oviductal events.     

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

Influence of persistent canine distemper virus infection on expression of RECK, matrix-metalloproteinases and their inhibitors in a canine macrophage/monocytic tumour cell line (DH82)

A morbillivirus infection of tumour cells is known to exert oncolytic activity, but the mechanism of this inhibitory action has not been well defined. Matrix metalloproteinases (MMPs) are important enzymes degrading the extracellular matrix and are often upregulated in malignant neoplasms. Recent studies have demonstrated that RECK may potently suppress MMP-2 and -9 activity, thus inhibiting angiogenesis and metastasis. In this study, real time quantitative polymerase chain reaction (RT-qPCR) was used to determine the effect of persistent infection with canine distemper virus (CDV) infection on the expression of MMPs and their inhibitors (TIMPS) in a canine macrophage/monocytic tumour cell line (DH82). The activity of proMMP-2 and proMMP-9 was also verified zymographically. Following CDV infection, MMP-2, TIMP-1 and TIMP-2 were down-regulated, while RECK was upregulated. These findings suggest that CDV infection restores RECK expression in tumour cells and may interfere with the intracellular processing of MMPs and TIMPs, thus possibly influencing tumour cell behaviour beneficially for the host. However, this needs to be verified in in vivo studies.     

Canine TIMP-2: purification, characterization and molecular detection

Matrix metalloproteinases (MMPs), which degrade tissues in health and disease are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP-2 is particularly important for control of MMP-2 and both have been implicated in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovial fibroblasts and three tumour-derived canine cell lines, K1, K6 and DH82. Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fibroblasts and the three cells lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was purified and n-terminal amino acid sequencing showed it to be highly homologous to equine and human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Polymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sources and proved positive in all cases. This work highlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, therefore, opens the possibilities of targeting TIMP-2 for therapeutic intervention against connective amino acid tissue degradation in a range of diseases.     

Roles of an extracellular matrix (ECM) receptor and ECM processing enzymes in demyelinating canine distemper encephalitis

Canine distemper virus (CDV) belongs to the genus Morbillivirus of the Paramyxoviridae family. Due to the central nervous system (CNS) tropism of the virus and associated neuropathological changes, demyelinating canine distemper encephalitis (CDE) represents a relevant model for human demyelinating diseases like multiple sclerosis. The present review decribes the role of CD44 antigen (CD44), the principle cell surface receptor for hyaluronate and extracellular matrix (ECM) processing enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (TIMPs) in the pathogenesis of demyelination. In acute and subacute CDE, a plaque-associated CD44 up-regulation is found that parallels astrocyte activation. Likewise, MMPs and TIMPs are prominently up-regulated in these lesions and are expressed mostly by astrocytes and microglia. In chronic lesions, CD44 expression declines together with the number of glial fibrillary acidic protein (GFAP) positive astrocytes. In addition, in this plaque type, CD44 is expressed on the cell membrane of perivascular mononuclear cells. In this phase, a decrease of MMP and TIMP expressions apart from MMP-11, -12, and -13 is obvious. In summary, CD44 and MMPs might be associated with the onset of demyelination and may interact to initiate ECM disturbances. Ligation of CD44 in the early phase may induce chemokines and cytokines and hence initiate and perpetuate the inflammatory process. In the chronic phase, it is conceivable that a MMP-TIMP imbalance may be the motor for lesion progression with a simultaneous influx of CD44-positive activated immune cells.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

Matrix metalloproteinases-2 and -9 activities in bovine follicular fluid of different-sized follicles: relationship to intra-follicular inhibin and steroid concentrations

Matrix metalloproteinases (MMPs) play very important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, ovulation and atresia. The aim of the present study was to determine the content of gelatinases in follicular fluid in various sized bovine follicles. Bovine ovaries were collected from local slaughterhouse and follicular fluid from follicles of 2 to over 25 mm in diameter was collected. Gelatinase activity within the follicular fluid was analyzed by gelatin zymography. The concentration of inhibin in the follicular fluid was also measured by immunoblot analysis. The proMMP-2 and alpha-subunit (alphaN) inhibin was detected in all follicles regardless of their size. The abundance of proMMP-2 varied with follicular size, while alphaN inhibin increased significantly (P<0.01) in follicles of 10-14 and 15-20 mm in size. There was a positive and negative correlation between estradiol (E(2)) and progesterone (P(4)) concentrations with abundance of proMMP-2, respectively. Follicles of diameter over 25 mm had greater proMMP-9 activity than other follicles. These same follicles had significantly (P<0.01) lower inhibin levels than follicles of 10-14 and 15-20 mm in size. In conclusion, these results suggest a significant role of these proteases in growth and development of bovine follicle, particularly proMMP-2 and active MMP-2 activities in the follicular fluid could serve as markers of follicular health while abundance of proMMP-9 may possibly denote a follicular cyst.     

Effects of autologous stem cells on immunohistochemical patterns and gene expression of metalloproteinases and their tissue inhibitors in doxorubicin cardiomyopathy in a rabbit model

This study aims to investigate the expression of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in chronic doxorubicin cardiomyopathy in a rabbit model and to evaluate the effects of bone marrow-derived mesenchymal stem cell (MSC) transplantation in this disease. Thirty-nine 3-month-old New Zealand rabbits were divided into 4 groups: group 1 (n = 9) was the untreated control. Groups 2-4 were treated with 6 weeks of doxorubicin (3 mg/kg). Group 2 (n = 6) received no further treatment. In group 3 (n = 9), animals were treated with culture medium (CM) alone. In group 4 (n = 15), autologous MSCs (1.5-2.0 x 10(6)/ml) were injected in the left ventricular (LV) wall. Hearts were stained with HE and picrosirius red. MMP-1, -2, -3 and -9 and TIMP-2 and -3 were detected immunohistochemically. The mRNA levels were determined by real-time polymerase chain reaction. The results confirmed that doxorubicin treatment resulted in minimal myocardial fibrosis and showed that expression of MMPs increased and TIMP-3 decreased. The injection procedure resulted in increased myocardial fibrosis in groups 3 and 4. After MSC injection, MMP-1, MMP-2, and TIMP-3 expression was higher than that in group 2. CM injection led to more fibrosis, elevated TIMP-3, but diminished MMP-1 and MMP-2 expression compared with MSC injection. The mRNA levels of MMPs and TIMPs were not significantly different among all groups. In conclusion, chronic doxorubicin cardiomyopathy was characterized by increased MMP and decreased TIMP-3 expression. MSCs injection into the LV resulted in marked differences of collagen content and MMP/TIMP expression in the whole heart, although significant numbers of living MSCs were not detected after 4 weeks.     

Collagenase-1 (MMP-1) activity in equine synovial fluid: influence of age, joint pathology, exercise and repeated arthrocentesis

REASONS FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs) are considered candidate biomarkers for both physiological and pathological tissue remodelling because of their key role in articular cartilage homeostasis. As disruption of the collagenous architecture is thought to be pivotal in chronic degenerative diseases such as osteoarthritis (OA), the collagenases form an interesting subset of the MMPs. The significance of any biomarker in synovial fluid (SF) can be assessed properly only when fluctuations in patterns induced by physiological processes such as development and growth, and by external influences and interventions such as exercise and repeated arthrocentesis, are known and taken into account. OBJECTIVES: To investigate the activity of MMP-1 in equine SF at different stages of development and in joints affected by OA, and the influence of exercise and repeated arthrocentesis thereon. METHODS: MMP-1 activity was determined in SF of normal joints of fetal, juvenile and mature horses, and in SF of horses suffering from OA, using an internally quenched fluorogenic peptide substrate. MMP-1 activity was also measured in SF from horses subjected to an exercise regimen and those subjected to repeated arthrocentesis. RESULTS: An age-related decline in the SF levels of active MMP-1 was observed. MMP-1 activity was 15-fold higher in fetal than in juvenile animals, which showed significantly higher MMP-1 activity levels than mature horses. In SF of OA joints, MMP-1 activity was increased. Exercise did not affect MMP-1 activity in SF, but repeated arthrocentesis (within 60 h) increased MMP-1 activity significantly. CONCLUSIONS: The high MMP-1 activity in SF of young individuals parallels the high metabolic activity occurring during rapid growth and differentiation at early age. The elevated MMP-1 activity in SF of OA joints probably reflects pathological matrix degradation, confirming the potential of MMP-1 to serve as a biochemical marker for early joint disease. Moderate exercise is not likely to influence the outcome of MMP-1 activity measurements in equine SF, but arthrocentesis should be taken into account as a possible confounding factor. POTENTIAL RELEVANCE: Given the crucial role of the collagen matrix for tissue integrity, MMP-1 activity may be a useful tool in diagnostic, therapeutic or prognostic studies in horses suspected of OA. However, care should be taken to exclude fluctuations in MMP-1 activity induced by physiological processes such as development and growth, and by interventions such as repeated arthrocentesis.     

Matrix metalloproteinase activity in tumor, stromal tissue, and serum from cats with malignancies

Matrix metalloproteinases (MMPs) are enzymes that play key roles in angiogenesis, tumor invasion, and metastasis in a wide variety of species. The purpose of this study was to evaluate pro and active MMP 2 and 9 concentrations in tumor, normal stromal tissue, and serum from tumor-bearing cats. We hypothesized that serum concentrations of pro and active forms of MMPs 2 and 9 would be predictive of MMP concentrations in tumor tissue and that these MMP concentrations would correlate with the histopathologic grade of the malignancies. Pro and active forms of MMPs 2 and 9 were determined by gelatin zymography and subsequent computerized densitometry from tumor and nearby stromal tissue and serum from 49 cats with various malignancies. The serum concentrations of MMPs from these tumor-bearing cats were compared with serum concentrations of MMPs from 44 normal cats of similar age and gender. Measurable concentrations of MMPs 2 and 9 were found within tumor, stromal, and serum samples. Mean concentrations of total pro and active MMPs 2 and 9 within tumor tissue were significantly higher (P values <.0001, .0031, <.001, and .0064, respectively) when compared with stromal tissue from the same animals. Serum MMP concentrations from tumor-bearing cats were higher than those from normal cats. Poor correlation was found between serum MMP concentrations and tissue MMP concentrations of increasing histologic grades of malignancies.