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.     

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.     

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.     

Matrix metalloproteinases (MMPs) activity in cultured canine bone marrow stromal cells (BMSCs)

Autologous bone marrow stromal cells (BMSCs) infusion therapy improves the hepatic fibrosis. To investigate the mechanism of remission, we evaluated the matrix metalloproteinase (MMP)-2 and -9 activity in canine BMSCs and the effect of pro-inflammatory cytokines on their expression. The activity and the gene expression of MMPs were analyzed by gelatin zymography and quantitative RT-PCR, respectively. The specific gelatinase bands were indicative effect of MMP-2 and -9 in canine BMSCs. MMP-2 expression seemed to be increased by TNF-α and IL-1β while MMP-9 was enhanced by TNF-α and IL-6. These results suggested that remissive effect on liver fibrosis might be partly attributable to the MMP-2 and -9 activity in BMSCs under the inflammatory condition.     

大肠杆菌及脂多糖对奶牛乳腺上皮细胞基质金属蛋白酶表达的影响

为研究大肠杆菌(E.coli)及脂多糖(LPS)对奶牛乳腺上皮细胞(BMECs)基质金属蛋白酶(MMPs)表达的影响,以及MMPs与基质金属蛋白酶组织抑制因子(TIMPs)、尿激酶型纤溶酶原激活物(uPA)系统在调控细胞外基质(ECM)代谢中的作用,分别以106 CF U/mL热灭活E.coli菌液、7.5μg/mL ...     

Activity of matrix metalloproteinase-2 (MMP-2) in canine oronasal tumors

Activity of matrix metalloprotease-2 (MMP-2) and the expression of its related molecules were examined in spontaneous canine oronasal tumors. Tissue samples from melanoma and squamous cell carcinoma possessed higher MMP-2 activity, as shown in gelatin zymography, in comparison with acanthomatous epulis and nasal adenocarcinoma. Regional lymph node invasion and distant metastases were more frequently observed in the MMP-2 positive cases. There were no significant differences by RT-PCR examination in the expression of the genes encoding MMP-2, MT1-MMP and TIMP-2 among the tumor histological types. However, the MMP-2/TIMP-2 ratio showed a significantly higher level of the genes in the malignant oral melanoma and squamous cell carcinoma. The MMP-2/TIMP-2 ratio was also positively correlated with MMP-2 activity in gelatin zymography. These results indicate that the MMP-2/TIMP-2 ratio may be of value in evaluating the prognosis in canine oronasal cavity tumors.     

Canine cerebral leishmaniasis: Potential role of matrix metalloproteinase-2 in the development of neurological disease

Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.     

Immunohistochemical evaluation of MMP-2 and TIMP-2 in canine mammary tumours: a survival study

Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival.MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs.     

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.     

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.     

Tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine‐derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine‐derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8). CDV replication‐induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV‐induced cancer therapy.     

Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.     

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. OBJECTIVE: To identify components of organic dust responsible for elevated MMP levels in equine airways. METHODS: Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. RESULTS: HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. CONCLUSIONS: The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. POTENTIAL RELEVANCE: Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.     

The role of inflammation and matrix metalloproteinases in equine endometriosis

Equine endometriosis is a multifactorial disease considered to be a major cause of equine infertility. The purpose of this study was to evaluate the reliability of histomorphological grading for biopsy-like samples compared to entire uterine wall samples, to examine the association between the degree of endometriosis with animal age, and to investigate the role of inflammation in endometriosis and the expression of different matrix metalloproteinases in equine endometrium. Histomorphological lesions in 35 uterine samples were examined while comparing biopsy-like samples and entire-wall samples. Seventeen uterine samples were stained with antibodies against MMP-2, MMP-9, MMP-14, and TIMP-2. The morphologic evaluation results of the biopsy-like tissue and entire-wall samples were significantly correlated. Endometriosis in older mares (>12 years of age) was more severe than in young mares (2~4 years of age), confirming the positive correlation between animal age and disease severity, while inflammation was poorly related to the degree of endometriosis. MMP-2 and MMP-14 were detected in stromal cells, while MMP-9 and TIMP-2 were both found in stromal and glandular epithelial cells. There were no significant differences in MMPs expression between the two groups (young vs. old mares). Additional studies on the activity of MMPs could further define the role of these enzymes in equine endometriosis.     

Matrix metalloproteinase-2 and -9 involvement in canine tumors

Matrix metalloproteinases (MMPs) are a family of enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularization. They are also involved in pathologic processes such as tumor invasion and metastasis in experimental cancer models and in human malignancies. We used gelatin zymography and immunohistochemistry to determine whether MMP-2 and MMP-9 are present in canine tumors and normal tissues and whether MMP production correlates with clinicopathologic parameters of prognostic importance. High levels of pro-MMP-9, pro-MMP-2, and active MMP-2 were detected in most canine tumors. Significantly higher MMP levels were measured in canine tumors than in nontumors, malignancies had higher MMP levels than benign tumors, and sarcomas had higher active MMP-2 than carcinomas. Cartilaginous tumors produced higher MMP levels than did nonsarcomatous malignancies, benign tumors, and normal tissues, and significantly greater MMP-2 than osteosarcomas and fibrosarcomas. Pro-MMP-9 production correlated with the histologic grade of osteosarcomas. The 62-kd form of active MMP-2 was detected only in high-grade, p53-positive, metastatic malignancies. Zymography proved to be a sensitive and quantitative technique for the assessment of MMP presence but has the limitation of requiring fresh tissue; immunohistochemistry is qualitative and comparatively insensitive but could be of value in archival studies. MMP presence was shown in a range of canine tumors, and their link to tumor type and grade was demonstrated for the first time. This study will allow a substantially improved evaluation of veterinary cancer patients and provides baseline information necessary for the design of clinical trials targeting MMPs.     

Inhalation of organic dusts and lipopolysaccharide increases gelatinolytic matrix metalloproteinases (MMPs) in the lungs of heaves horses

We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the majority of gelatinolytic activity comprising pro- and active MMP-9. The 5 h duration hay/straw challenge increased BALF gelatinolytic MMP activity in heaves horses at 5 and 24 h after the start of this challenge, with activity returning to baseline by Day 4. In contrast to hay/straw and HDS challenges, LPS inhalation increased BALF gelatinolytic MMP activity in both groups. For all challenges, absolute BALF neutrophil counts were highly significantly correlated (P<0.0001) with levels of proMMP-9 and active MMP-9, but not with levels of MMP-2 (P>0.05). As gelatinolytic MMPs are pro-inflammatory agents, they may contribute to lung dysfunction and tissue destruction in heaves horses exposed to airborne organic stable dusts.