Absence of tissue inhibitor of metalloproteinases 3 disrupts alveologenesis in the mouse

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) throughout lung development. We examined lungs from TIMP3 null mice and found significant air space enlargement compared with wild type (WT) animals during a time course spanning early alveologenesis (post‐partum days 1, 5, 9 and 14). Trichrome staining revealed a similar pattern of collagen distribution in the walls of nascent alveoli; however, the alveolar walls of TIMP3 mutant mice appeared to be thinner than controls. Assessment of MMP2 and MMP9 activities by gelatin zymography demonstrated a significant elevation in the active form of MMP2 at post‐partum days 1 and 5. Treatment of null pregnant dams with a broad spectrum synthetic metalloproteinase inhibitor, GM6001, on embryonic day 16.5 enhanced the formation of primitive alveoli during the saccular stage of lung development as evidenced by a partial, but significant, rescue of alveolar size in post‐partum day 1 animals. We propose that increased MMP activity in the absence of TIMP3 enhances ECM proteolysis, upsetting proper formation of primitive alveolar septa during the saccular stage of alveologenesis. Therefore, TIMP3 indirectly regulates alveolar formation in the mouse. To our knowledge, ours is the first study to demonstrate that in utero manipulation of the TIMP/MMP proteolytic axis, to specifically inhibit proteolysis, significantly affects lung development.     

Cellular localization of gelatinases and tissue inhibitors of metalloproteinases during follicular growth, ovulation, and early luteal formation in the rat

The matrix metalloproteinase (MMP) system consists of a proteolytic component, the metalloproteinases, and an associated class of tissue inhibitors of metalloproteinases (TIMPs). We investigated the cellular localization of the TIMPs and the gelatinase family of MMPs throughout the latter stages of follicular growth and during the periovulatory period. Immature female rats were injected with eCG, and ovaries were collected at the time of eCG administration (0 h) and at 6, 12, 24, or 36 h after eCG injection (i.e., follicular development group). A second group of animals (periovulatory) was injected with eCG followed by hCG 48 h later, and ovaries were collected at 0, 12, and 24 h after hCG. Ovaries were processed for the cellular localization of gelatinase or TIMP mRNA or gelatinolytic activity. Gelatinase mRNA (MMP-2 and MMP-9) was localized to the theca of developing follicles and to the stroma. Following a hCG stimulus, MMP-2 mRNA increased as the granulosa cells of preovulatory follicles underwent luteinization during formation of the corpus luteum (CL). MMP-9 mRNA remained predominantly in the theca during this period. In situ zymography for gelatinolytic activity demonstrated a pattern of activity that corresponded with the localization of MMP-2 and MMP-9 mRNA around developing follicles. Gelatinolytic activity was observed at the apex of preovulatory follicles and throughout the forming CL. The mRNA for TIMP-1, -2, and -3 was localized to the stroma and theca of developing follicles. TIMP-3 mRNA was present in the granulosa cells of certain follicles but was absent in granulosa cells of adjacent follicles. At 12 h after hCG, luteinizing granulosa cells expressed TIMP-1 and TIMP-3 mRNA, but TIMP-2 mRNA was at levels equivalent to the background. In the newly forming CL at 24 h after hCG administration, the luteal cells expressed TIMP-1, -2, and -3 mRNA, although the pattern of cellular expression was unique for each of the TIMPs. These findings demonstrate that the MMPs and TIMPs are in the cellular compartments appropriate for impacting the remodeling of the extracellular matrix as the follicle grows, ovulates, and forms the CL.     

Complex role of matrix metalloproteinases in angiogenesis

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a significant role in regulating angiogenesis,the process of new blood vessel formation.Interstitial collagenase (MMP-1),72kDa gelatinase A/type IV collagenase (MMP-2),and 92 kDA gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and promote angiogenesis.TIMP-1,TIMP-2,TIMP-3,and possibly,TIMP-4 inhibit neovascularization.A new paradign is emerging that matrilysin (MMP-7),MMP-9,and metalloelastase (MMP-12) may block angiogenesis by converting plasminogen to angiostatin,which is one of the most potent angiogenesis antagonists.MMPs and TIMPs play a complex role in regulating angiogenesis.An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide important information to allow the control of angiogenesis,e.g.the stimulation of angiogenesis for coronary collateral circulation formation;while the inhibition for treating arthritis and cancer.     

Type‐specific dysregulation of matrix metalloproteinases and their tissue inhibitors in end‐stage heart failure patients: relationship between MMP‐10 and LV remodelling

Although past studies observed the changes of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in end‐stage heart failure (HF) patients, a consistent and clear pattern of type‐specific MMPs and/or TIMPs has yet to be further defined. In this study, proteomic approach of human protein antibody arrays was used to compare MMP and TIMP expression levels of left ventricular (LV) myocardial samples from end‐stage HF patients due to dilated cardiomyopathy (DCM) with those from age‐ and sex‐ matched non‐failing patients. Western blot analysis, immunohistochemistry and ELISA were used for validation of our results. We observed that MMP‐10 and ‐7 abundance increased, accompanied by decreased TIMP‐4 in DCM failing hearts (n= 8) compared with non‐failing hearts (n= 8). The results were further validated in a cohort of 34 end‐stage HF patients derived from three forms of cardiomyopathies. Cardiac and plasma MMP‐10 levels were positively correlated with the LV end‐diastolic dimension in this HF cohort. In addition, we observed that insulin‐like growth factor‐2 promoted MMP‐10 production in neonatal rat cardiomyocytes. In conclusion, this study demonstrated a selective up‐regulation of MMP‐10 and ‐7 along with a discordant change of TIMP‐4, and a positive correlation between MMP‐10 levels and the degree of LV dilation in end‐stage HF patients. Our findings suggest that type‐specific dysregulation of MMPs and TIMPs is associated with LV remodelling in end‐stage HF patients, and MMP‐10 may act as a novel biomarker for LV remodelling.     

Ovarian matrix metalloproteinases are differentially regulated during the estrous cycle but not during short photoperiod induced regression in Siberian hamsters (<Emphasis Type="Italic">Phodopus sungorus</Emphasis>)

Background  

Matrix metalloproteinases (MMPs) are implicated as mediators for ovarian remodeling events, and are involved with ovarian recrudescence during seasonal breeding cycles in Siberian hamsters. However, involvement of these proteases as the photoinhibited ovary undergoes atrophy and regression had not been assessed. We hypothesized that 1) MMPs and their tissue inhibitors, the TIMPs would be present and differentially regulated during the normal estrous cycle in Siberian hamsters, and that 2) MMP/TIMP mRNA and protein levels would increase as inhibitory photoperiod induced ovarian degeneration.     

Matrix metalloproteinases and their inhibitors,modulators of neuro-immune interactions and of pathophysiological processes in the nervous system

The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), that regulate or degrade by proteolytic cleavage protein components of the extracellular matrix, cytokines, chemokines, cell adhesion molecules and a variety of membrane receptors. MMP activity is counterbalanced by their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs), a family of 4 secreted multifunctional proteins that have growth promoting activities. In physiological conditions MMP activity is tightly regulated and altered MMP regulation is associated with pathological processes including inflammation, cell proliferation, cell death and tissue remodeling. The MMP/TIMP system is involved in the development and function of cells of the immune system by promoting their differentiation, activation, migration across basement membranes and tissues. In the last years, data has accumulated indicating that the MMP/TIMP system is expressed in the nervous system where it regulates neuro-immune interactions and plays a major role in pathophysiological processes. In this review, we present recent in vivo and in vitro studies that highlight the contribution of the MMP/TIMP system to various diseases of the nervous system, involving blood brain barrier breakdown, neuroinflammation, glial reactivity, neuronal death, reactive plasticity, and to developmental and physiological processes including cell migration, axonal sprouting and neuronal plasticity. This review also alludes to the beneficial effects of synthetic MMP inhibitors in different animal models of neuropathology. In all, a further understanding of the role of MMPs and TIMPs in the nervous system should contribute to unravel mechanisms of neuronal plasticity and pathology and set the basis of new therapeutic strategies in nervous system disorders based on the development of synthetic MMP inhibitors.     

Expression and regulation of tissue inhibitors of metalloproteinases (TIMP1 and TIMP3) in goat oviduct

Tissue inhibitors of metalloproteinases (TIMPs) are associated with several reproductive processes, such as mammalian follicular growth, ovulation, CL formation, and embryonic development. However, the expression and function of TIMPs in goat oviducts remain unclear. This work aimed to identify TIMP1 and TIMP3 expression in the goat oviduct during the estrous cycle via immunohistochemistry, real-time polymerase chain reaction (PCR), and functional studies in cultured goat oviductal epithelial cells. Real-time PCR results demonstrated that TIMP1 and TIMP3 messenger RNAs were expressed in all goat oviductal regions at all stages of the estrous cycle. TIMP1 and TIMP3 proteins were also highly expressed in oviductal epithelial cells with very limited expression in other cell types. Oviductal epithelial cells were treated in vitro with various estradiol concentrations (1–100 nM) for 24 hours. The findings showed that TIMP1 expression increased up to 20 nM but then gradually decreased, whereas no significant effects existed among TIMP3 messenger RNA levels. Time-course studies indicated that estradiol significantly increased TIMP1 expression in a time-dependent manner from 8 hours to 24 hours. By contrast, TIMP3 expression was transiently induced in oviductal epithelial cells at 2 and 4 hours after estradiol treatment. Furthermore, treatment with TIMP1 functionally increased the viability of cultured oviductal epithelial cells. Overall, the results suggested that the differential regulation and function between TIMP1 and TIMP3 might be associated with their unique roles in fertilization and early embryonic development.     

Suppression of tissue inhibitors of metalloproteinases may reverse severe pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary vascular resistance and vascular remodeling leading to right heart failure and early death. The pathology of PAH is associated with endothelium dysfunction and vascular remodeling in pulmonary arteries. In diseased pulmonary arteries, the balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) is broken down. In this process, TIMP are up-regulated, which inhibits MMP, promotes extracellular matrix (ECM) deposition and finally leads to vascular remodeling. So, what would happen to PAH if the expression of TIMP was down-regulated in diseased pulmonary vessels? We hypothesize that the attenuation of TIMP at the advanced stage of PAH might reverse severe PAH, via ameliorating vascular remodeling and endothelium repair.     

Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha.

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Engineering of tissue inhibitor of metalloproteinases mutants as potential therapeutics

Matrix metalloproteinases (MMPs) play a central role in many biological processes such as development, morphogenesis and wound healing, but their unbalanced activities are implicated in numerous disease processes such as arthritis, cancer metastasis, atherosclerosis, nephritis and fibrosis. One of the key mechanisms to control MMP activities is inhibition by endogenous inhibitors called tissue inhibitors of metalloproteinases (TIMPs). This review highlights the structures and inhibition mechanism of TIMPs, the biological activities of TIMPs, the unique properties of TIMP-3, and the altered specificity towards MMPs achieved by mutagenesis. A potential therapeutic use of TIMP variants is discussed.     

Activity of matrix metallo proteinases (MMPs) and the tissue inhibitor of MMP (TIMP)-1 in electromagnetic field-exposed THP-1 cells

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are the main determinants of tissue remodeling in both physiological and pathological processes. Metabolic processes, which generate oxidants and antioxidants can be influenced by environmental factors such as electromagnetic fields (EMF). We analyzed the effects of EMF on the activity and expression of MMPs in THP‐1 cells. Cells were exposed to a 50 Hz, 1 mT EMF for 24 h and incubated with or without LPS. Our data indicate that THP‐1 cells exposed to EMF causes a reduction of anti‐oxidant enzyme activity and an enhancement of nitrogen intermediates involving the iNOS pathway. We then analyzed the role of nitration of TIMP‐1 in increasing the activity of MMPs in EMF exposed cells. Molecular modeling tools were employed to identify the most plausible sites in the active conformation of TIMP‐1; at least two protein sites, Y120 and Y38 and/or Y72 were identified. Reactive nitrogen species (RNS) may affect protein targets, such as TIMP‐1, which are crucial for the regulation of MMP activities by oxidation of sulfydryl groups, or by nitration of tyrosine residues. These results may suggest a pathway connecting an imbalance of MMPs and their cognate inhibitor TIMP‐1. J. Cell. Physiol. 227: 2767–2774, 2012. © 2011 Wiley Periodicals, Inc.     

Altered circulating levels of matrix metalloproteinases and inhibitors associated with elevated type 2 cytokines in lymphatic filarial disease

Background

Infection with Wuchereria bancrofti can cause severe disease characterized by subcutaneous fibrosis and extracellular matrix remodeling. Matrix metalloproteinases (MMPs) are a family of enzymes governing extracellular remodeling by regulating cellular homeostasis, inflammation, and tissue reorganization, while tissue-inhibitors of metalloproteinases (TIMPs) are endogenous regulators of MMPs. Homeostatic as well as inflammation-induced balance between MMPs and TIMPs is considered critical in mediating tissue pathology.

Methods

To elucidate the role of MMPs and TIMPs in filarial pathology, we compared the plasma levels of a panel of MMPs, TIMPs, other pro-fibrotic factors, and cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag−) active infection to those with clinically asymptomatic infections (INF) and in those without infection (endemic normal [EN]). Markers of pathogenesis were delineated based on comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag− compared to EN).

Results and Conclusion

Our data reveal that an increase in circulating levels of MMPs and TIMPs is characteristic of the filarial disease process per se and not of active infection; however, filarial disease with active infection is specifically associated with increased ratios of MMP1/TIMP4 and MMP8/TIMP4 as well as with pro-fibrotic cytokines (IL-5, IL-13 and TGF-β). Our data therefore suggest that while filarial lymphatic disease is characterized by a non-specific increase in plasma MMPs and TIMPs, the balance between MMPs and TIMPs is an important factor in regulating tissue pathology during active infection.     

Association of metalloproteinases,tissue inhibitors of matrix metalloproteinases,and proteoglycans with development,aging, and osteoarthritis processes in mouse temporomandibular joint

The temporomandibular joint (TMJ) is an important growth and articulation center in the craniofacial complex. In aging it develops spontaneous degenerative osteoarthritic (OA) lesions. Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) play key roles in extracellular matrix remodeling and degradation. Gelatinase activities and immunohistochemical localization of MMP-2, -3, -8, -9, and -13 and TIMP-1 and -2 were examined in mandibular condyle cartilage of neonatal mice up to 18 months old. The most intense immunostaining for all enzymes and TIMPs and the peak of gelatinase activities were found in animals in the stages of early growth (1 week to 3 months) followed by a decrease during maturation and aging. However, clusters of positively immunoreactive chondrocytes were detected in cartilages of old animals displaying OA lesions. Positive safranin-O staining, indicative of sulfated proteoglycans (PGs), was prominent in the TMJ of newborn mice up to 3 months old followed by reduction during maturation and aging, except in regions displaying OA lesions. Temporal codistribution of PGs, MMPs, and TIMPs during skeletal maturation reflected an active growth phase, whereas their reduction coincided with the more quiescent articulating and maintenance phase in the joint cartilage. Osteoarthritic lesions were associated with both increased PG synthesis and MMP immunoreactivity, indicating limited repair activity during initial stages of osteoarthritis.     

Matrix metalloproteinases and their endogenous inhibitors in neuronal physiology of the adult brain

More than 20 matrix metalloproteinases (MMPs) and four of their endogenous tissue inhibitors (TIMPs) act together to control tightly temporally restricted, focal proteolysis of extracellular matrix. In the neurons of the adult brain several components of the TIMP/MMP system are expressed and are responsive to changes in neuronal activity. Furthermore, functional studies, especially involving blocking of MMP activities, along with the identification of MMP substrates in the brain strongly suggest that this enzymatic system plays an important physiological role in adult brain neurons, possibly being pivotal for neuronal plasticity.