A functional polymorphism in THBS2 that affects alternative splicing and MMP binding is associated with lumbar-disc herniation

Lumbar-disc herniation (LDH), one of the most common musculoskeletal diseases, has strong genetic determinants. Recently, several genes that encode extracellular matrix (ECM) proteins in the intervertebral disc have been reported to associate with LDH. Thrombospondins (THBSs) 1 and 2 are good candidates for the LDH susceptibility gene: They are intervertebral disc ECM proteins that regulate the effective levels of matrix metalloproteinases (MMPs) 2 and 9, which are key effectors of ECM remodeling. Here, we report that THBS2 is associated with LDH in Japanese populations. An intronic SNP in THBS2 (IVS10-8C --> T; rs9406328) showed significant association (p = 0.0000028) with LDH in two independent Japanese populations. This SNP, located in a polypyrimidine tract upstream of the 3' splice site of intron 10, exerts allelic differences on exon 11 skipping rates in vivo, with the susceptibility allele showing increased skipping. Skipping of exon 11 results in decreased THBS2 interaction with MMP2 and MMP9. Further, a missense SNP in MMP9 (Q279R; rs17576) is also strongly associated with LDH in the Japanese population (p = 0.00049) and shows a combinatorial effect with THBS2 (odds ratio 3.03, 95% confidence interval 1.58-5.77). Thus, a splicing-affecting SNP in THBS2 and a missense SNP in MMP9 are associated with susceptibility to LDH. Our data indicate that regulation of intervertebral disc ECM metabolism by the THBS2-MMP system plays an essential role in the etiology and pathogenesis of LDH.     

Elevated levels of matrix metalloprotein-3 in patients with coronary aneurysm: A case control study

BACKGROUND: Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms through increased proteolysis of extracellular matrix proteins. Increased proteolysis due to elevated matrix degrading enzyme activity in the arterial wall may act as a susceptibility factor for the development of coronary aneurysms. The aim of this study was to investigate the association between MMPs and presence of coronary aneurysms. METHODS: Thirty patients with aneurysmal coronary artery disease and stable angina were enrolled into study (Group 1). Fourteen coronary artery disease patients with stable angina were selected as control group (Group 2). MMP-1, MMP-3 and C-reactive protein (CRP) were measured in peripheral venous blood and matched between the groups. RESULTS: Serum MMP-3 level was higher in patients with aneurismal coronary artery disease compared to the control group (20.23 +/- 14.68 vs 11.45 +/- 6.55 ng/ml, p = 0.039). Serum MMP-1 (13.63 +/- 7.73 vs 12.15 +/- 6.27 ng/ml, p = 0.52) and CRP levels (4.78 +/- 1.47 vs 4.05 +/- 1.53 mg/l, p = 0.13) were not significantly different between the groups. CONCLUSION: MMPs can cause arterial wall destruction. MMP-3 may play role in the pathogenesis of coronary aneurysm development through increased proteolysis of extracellular matrix proteins.     

Arachidonic acid and colorectal carcinogenesis

Vascular lesion development is associated with an accumulation of extracellular matrix proteins within the vessel wall. Matrix metalloproteinases (MMPs) degrade these proteins. Conversely, oxidized low density lipoprotein (LDL) is implicated in atherogenesis through, amongst other cellular effects, a stimulation of the deposition of collagen within the vascular lesion. The present study investigated the potential for an interaction between oxidized LDL and MMP levels. Within the vessel wall fibroblasts, smooth muscle, endothelial and infiltrating cells have been reported to secrete MMPs into the extracellular space to effect remodeling of the extracellular matrix. A consequence of angioplasty and atherosclerotic disease is the loss of endothelial cells or endothelial function, respectively. We have investigated the effects of chronic incubation of cultured vascular smooth muscle cells from rabbit thoracic aorta with oxidized LDL and its influence on MMP levels in the extracellular space. Our data indicate that a low concentration of minimally oxidized LDL (0.005 mg/mL) significantly depressed the levels of MMP-2 and MMP-9 present in the culture medium. Native LDL exerted the same effect but exhibited reduced potency. The effects were not attributable to cytotoxicity exerted by the oxidized LDL. The reduction in MMP secretion into the extracellular medium was a result of decreased enzyme synthesis within the smooth muscle cell. Our results demonstrate that an important atherogenic moiety, oxidized LDL, can reduce MMP activity and hence has the potential to increase the deposition of extracellular matrix proteins within SMC-rich vascular lesions.     

Family-based association study of matrix metalloproteinase-3 and -9 haplotypes with susceptibility to ischemic white matter injury

Susceptibility to ischemic damage to the subcortical white matter of the brain has a strong genetic basis. Dysregulation of matrix metalloproteinases (MMPs) contributes to loss of cerebrovascular integrity and white matter injury. We investigated whether sequence variation in the genes encoding MMP3 and MMP9 is associated with variation in leukoaraiosis volume, determined by magnetic resonance imaging, in non-Hispanic whites and African-Americans using family-based association tests. Seven hundred and fifty-six white and 671 African-American individuals from sibships ascertained through two or more siblings with hypertension were genotyped for 7 and 8 haplotype-tagging polymorphisms in the MMP3 and MMP9 genes, respectively. MMP3 sequence variation was significantly associated with variation in leukoaraiosis volume in Whites. Two common haplotypes with opposing relationships to leukoaraiosis volume were identified. MMP9 sequence variation was also significantly associated with variation in leukoaraiosis volume in both African-Americans and Whites. Different haplotypes contributed to these associations in the two racial groups. These findings add to the growing body of evidence from animal models and human clinical studies suggesting a role of MMPs in ischemic white matter injury. They provide the basis for further investigation of the role of these genes in susceptibility and/or progression to clinical disease.     

Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.     

Genetic and morphologic determinants of pneumothorax in lymphangioleiomyomatosis

Lymphangioleiomyomatosis, a multisystem disease affecting women, is characterized by proliferation of abnormal smooth muscle-like cells in the lungs, leading to cystic destruction of the parenchyma and recurrent pneumothoraces. Clinical characteristics of lymphangioleiomyomatosis patients were analyzed to determine the relationship of pneumothoraces to disease progression. Patients were genotyped for polymorphisms in genes of extracellular matrix proteins collagen, elastin, and matrix metalloproteinase-1 to assess their association with pneumothoraces. Clinical data and polymorphisms in the genes for types I and III collagen, elastin, and matrix metalloproteinase-1 were compared with the prevalence of pneumothorax. Of 227 patients, 57% reported having had at least one pneumothorax. Cyst size on high-resolution computed tomography scans was associated with pneumothorax; patients with a history of pneumothorax were more likely to have larger cysts than patients who had no pneumothoraces. In patients with mild disease, those with a history of pneumothorax had a faster rate of decline in forced expiratory volume in 1 s (FEV(1); P = 0.001, adjusted for age) than those without. Genotype frequencies differed between patients with and without pneumothorax for polymorphisms in the types I and III collagen and matrix metalloproteinase-1 genes. Larger cysts may predispose lymphangioleiomyomatosis patients to pneumothorax, which, in early stages of disease, correlates with a more rapid rate of decline in FEV(1). Polymorphisms in types I and III collagen and matrix metalloproteinase-1 genes may cause differences in lung extracellular matrix that result in greater susceptibility to pneumothorax.     

Matrix metalloproteinases in lung biology

Despite much information on their catalytic properties and gene regulation, we actually know very little of what matrix metalloproteinases (MMPs) do in tissues. The catalytic activity of these enzymes has been implicated to function in normal lung biology by participating in branching morphogenesis, homeostasis, and repair, among other events. Overexpression of MMPs, however, has also been blamed for much of the tissue destruction associated with lung inflammation and disease. Beyond their role in the turnover and degradation of extracellular matrix proteins, MMPs also process, activate, and deactivate a variety of soluble factors, and seldom is it readily apparent by presence alone if a specific proteinase in an inflammatory setting is contributing to a reparative or disease process. An important goal of MMP research will be to identify the actual substrates upon which specific enzymes act. This information, in turn, will lead to a clearer understanding of how these extracellular proteinases function in lung development, repair, and disease.     

Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus

A detailed analysis of the differential effects of estrogen (E) compared to raloxifene (Ral), a selective estrogen receptor modulator (SERM), following estrogen receptor (ER) binding in gynecological tissues was conducted using gene microarrays, Northern blot analysis, and matrix metalloproteinase (MMP) 2 activity studies. We profiled gene expression in the uterus following acute (1 day) and prolonged daily (5 wk) treatment of E and Ral in ovariectomized rats. Estrogen regulated twice as many genes as Ral, largely those associated with catalysis and metabolism, whereas Ral induced genes associated with cell death and negative cell regulation. Follow-up studies confirmed that genes associated with matrix integrity were differentially regulated by Ral and E at various time points in uterine and vaginal tissues. Additional experiments were conducted to determine the levels of MMP2 activity in uterus explants from ovariectomized rats following 2 wk of treatment with E, Ral, or one of two additional SERMs: lasofoxifene, and levormeloxifene. Both E and lasofoxifene stimulated uterine MMP2 activity to a level twofold that of Ral, whereas levormeloxifene elevated MMP2 activity to a level 12-fold that of Ral. These data show that one of the significant differences between E and Ral signaling in the uterus is the regulation of genes and proteins associated with matrix integrity. This may be a potential key difference between the action of SERMs in the uterus of postmenopausal women.     

Matrix metalloproteinases: Expression,regulation and role in the immunopathology of tuberculosis

Mycobacterium tuberculosis (Mtb) leads to approximately 1.5 million human deaths every year. In pulmonary tuberculosis (TB), Mtb must drive host tissue destruction to cause pulmonary cavitation and dissemination in the tissues. Matrix metalloproteinases (MMPs) are endopeptidases capable of degrading all components of pulmonary extracellular matrix (ECM). It is well established that Mtb infection leads to upregulation of MMPs and also causes disturbance in the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs), thus altering the extracellular matrix deposition. In TB, secretion of MMPs is mainly regulated by NF‐κB, p38 and MAPK signalling pathways. In addition, recent studies have demonstrated the immunomodulatory roles of MMPs in Mtb pathogenesis. Researchers have proposed a new regimen of improved TB treatment by inhibition of MMP activity to hinder matrix destruction and to minimize the TB‐associated morbidity and mortality. The proposed regimen involves adjunctive use of MMP inhibitors such as doxycycline, marimastat and other related drugs along with front‐line anti‐TB drugs to reduce granuloma formation and bacterial load. These findings implicate the possible addition of economical and well‐tolerated MMP inhibitors to current multidrug regimens as an attractive mean to increase the drug potency. Here, we will summarize the recent advancements regarding expression of MMPs in TB, their immunomodulatory role, as well as their potential as therapeutic targets to control the deadly disease.     

Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: identification of MMP16 as a new player in lung development

Backgound

Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD).

Methods and Findings

Two hundred and eighty-four consecutive neonates with a gestational age of <28 weeks were included in this prospective study. Forty-five neonates developed BPD. Nine single-nucleotide polymorphisms (SNPs) were sought in the MMP2, MMP14 and MMP16 genes. After adjustment for birth weight and ethnic origin, the TT genotype of MMP16 C/T (rs2664352) and the GG genotype of MMP16 A/G (rs2664349) were found to protect from BPD. These genotypes were also associated with a smaller active fraction of MMP2 and with a 3-fold-lower MMP16 protein level in tracheal aspirates collected within 3 days after birth. Further evaluation of MMP16 expression during the course of normal human and rat lung development showed relatively low expression during the canalicular and saccular stages and a clear increase in both mRNA and protein levels during the alveolar stage. In two newborn rat models of arrested alveolarization the lung MMP16 mRNA level was less than 50% of normal.

Conclusions

MMP16 may be involved in the development of lung alveoli. MMP16 polymorphisms appear to influence not only the pulmonary expression and function of MMP16 but also the risk of BPD in premature infants.     

Matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathy in vivo in transgenic mice

Cerebral amyloid angiopathy (CAA), characterized by extracellular β-amyloid peptide (Aβ) deposits in vessel walls, is present in the majority of cases of Alzheimer's disease and is a major cause of hemorrhagic stroke. Although the molecular pathways activated by vascular Aβ are poorly understood, extracellular matrix metalloproteinases (MMP) and Aβ-induced oxidative stress appear to play important roles. We adapted fluorogenic assays for MMP activity and reactive oxygen species generation for use in vivo . Using multiphoton microcopy in APPswe/PS1dE9 and Tg-2576 transgenic mice, we observed strong associations between MMP activation, oxidative stress, and CAA deposition in leptomeningeal vessels. Antioxidant treatment with α-phenyl- N -tert-butyl-nitrone reduced oxidative stress associated with CAA (∼50% reduction) without affecting MMP activation. Conversely, a selection of agents that inhibit MMP by different mechanisms of action, including minocycline, simvastatin, and GM6001, reduced not only CAA-associated MMP activation (∼30–40% reduction) but also oxidative stress (∼40% reduction). The inhibitors of MMP did not have direct antioxidant effects. Treatment of animals with α-phenyl- N -tert-butyl-nitrone or minocycline did not have a significant effect on CAA progression rates. These data suggest a close association between Aβ-related MMP activation and oxidative stress in vivo and raise the possibility that treatment with MMP inhibitors may have beneficial effects by indirectly reducing the oxidative stress associated with CAA.     

Differential expression of extracellular matrix components in the Fallopian tubes throughout the menstrual cycle

ABSTRACT: BACKGROUND: One of the unique characteristics of the female genital tract is the extensive tissue remodeling observed throughout the menstrual cycle. Multiple components of the extracellular matrix take part in this tissue rebuilding; however, the individual components involved have not been identified. METHODS: In the present study, the expression of extracellular matrix proteins and selected matrix metalloproteinase (MMP) activities in Fallopian tubes (FT) throughout the menstrual cycle were examined by PCR array, immunocytochemistry, zymography and bioinformatics. RESULTS: Of the eighty-four genes analyzed, eighty-three were expressed in the FT during at least one stage of the menstrual cycle. We observed a significant increase (>/=2-fold) in ADAMTS1, ADAMTS13, COL7A1, MMP3, MMP9, PECAM1, and THBS3 in the periovulatory phase compared to the follicular phase. Meanwhile, we observed a significant decrease (>/= 2-fold) in COL7A1, ICAM1, ITGA8, MMP16, MMP9, CLEC3B, SELE and TIMP2 in the lutheal phase compared to the periovulatory phase. Immunocytochemistry showed that MMP-3 and MMP-9 were localized in the endosalpinx during all phases of the menstrual cycle. Gelatin zymograms detected non-cycle-dependent protease activity. CONCLUSIONS: Several extracellular matrix components were regulated throughout the menstrual cycle in a cyclic pattern, suggesting a possible steroid regulation and a role in tissue remodeling and FT functions.     

Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate proteolysis of the extracellular matrix and other extracellular proteins, including growth factors and their receptors. The aberrant expression of these genes is common in most cancers. We profiled the RNA levels of every human MMP and TIMP in a variety of cell types (fibroblast, endothelial, hematopoietic, carcinoma, melanoma, and glioma) using quantitative PCR, with the aim of identifying novel expression patterns. Almost all members of the membrane-type (MT-) MMP and TIMP families were elevated in glioma lines compared to carcinomas. In clinical glioma specimens, there were positive correlations between glioma grade and RNA levels of MT-1, MT-2, and MT-6 MMP, TIMP-1 and TIMP-2, and for several growth factors and receptors. These findings suggest that advanced malignant gliomas have elevated levels of membrane-associated MMPs and TIMPs, which may potentially regulate vascularization and invasion. Concurrent elevation of signaling molecules suggests potential bidirectional relationships that enhance tumor aggressiveness.     

IGF-1 modulates gene expression of proteins involved in inflammation,cytoskeleton, and liver architecture

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1’s effects on liver by comparing wild-type controls, heterozygous igf1^+/?, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.