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.

TIMP-3 deficiency leads to dilated cardiomyopathy.

BACKGROUND: Despite the mounting clinical burden of heart failure, the biomolecules that control myocardial tissue remodeling are poorly understood. TIMP-3 is an endogenous inhibitor of matrix metalloproteinases (MMPs) that has been found to be deficient in failing human myocardium. We hypothesized that TIMP-3 expression prevents maladaptive tissue remodeling in the heart, and accordingly, its deficiency in mice would alone be sufficient to trigger progressive cardiac remodeling and dysfunction similar to human heart failure. METHODS AND RESULTS: Mice with a targeted timp-3 deficiency were evaluated with aging and compared with age-matched wild-type littermates. Loss of timp-3 function triggered spontaneous LV dilatation, cardiomyocyte hypertrophy, and contractile dysfunction at 21 months of age consistent with human dilated cardiomyopathy. Its absence also resulted in interstitial matrix disruption with elevated MMP-9 activity, and activation of the proinflammatory tumor necrosis factor-alpha cytokine system, molecular hallmarks of human myocardial remodeling. CONCLUSIONS: TIMP-3 deficiency disrupts matrix homeostasis and the balance of inflammatory mediators, eliciting the transition to cardiac dilation and dysfunction. Therapeutic restoration of myocardial TIMP-3 may provide a novel approach to limit cardiac remodeling and the progression to failure in patients with dilated cardiomyopathy.

Contribution of alveolar macrophages to the response of the TIMP-3 null lung during a septic insult.

Mice deficient in tissue inhibitor of metalloproteinase-3 (TIMP-3) develop an emphysema-like phenotype involving increased pulmonary compliance, tissue degradation, and matrix metalloproteinase (MMP) activity. After a septic insult, they develop a further increase in compliance that is thought to be a result of heightened metalloproteinase activity produced by the alveolar macrophage, potentially modeling an emphysemic exacerbation. Therefore, we hypothesized that TIMP-3 null mice lacking alveolar macrophages would not be susceptible to the altered lung function associated with a septic insult. TIMP-3 null and wild-type (WT) mice were depleted of alveolar macrophages before the induction of a septic insult and assessed for alteration in lung mechanics, alveolar structure, metalloproteinase levels, and inflammation. The results showed that TIMP-3 null mice lacking alveolar macrophages were protected from sepsis-induced alterations in lung mechanics, particularly pulmonary compliance, a finding that was supported by changes in alveolar structure. Additionally, changes in lung mechanics involved primarily peripheral tissue vs. central airways as determined using the flexiVent system. From investigation into possible molecules that could cause these alterations, it was found that although several proteases and inflammatory mediators were increased during the septic response, only MMP-7 was attenuated after macrophage depletion. In conclusion, the alveolar macrophage is essential for the TIMP-3 null sepsis-induced compliance alterations. This response may be mediated in part by MMP-7 activity but occurs independently of inflammatory cytokine and/or chemokine concentrations.

Lung mechanics in the TIMP3 null mouse and its response to mechanical ventilation.

Tissue inhibitor of metalloproteinase-3 (TIMP3) null mice develop emphysema-like airspace enlargement due to an enzymatic imbalance. This study investigates how these abnormalities alter lung mechanics and the response to 2 different mechanical ventilation strategies. Phenotypically, TIMP3 null mice had increased compliance, and decreased resistance, tissue damping, and tissue elastance over wild-type controls. Decreased compliance and increased resistance were observed following the injurious ventilation strategy; however, the TIMP3 null response to both ventilation strategies was similar to wild-type mice. In conclusion, TIMP3 null mice have significant alterations in lung mechanics; however, this does not affect their response to ventilation.

Tissue inhibitor of metalloproteinases 3 regulates extracellular matrix--cell signaling during bronchiole branching morphogenesis.

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) throughout embryogenesis. We examined lungs from TIMP3 null mice and found decreased bronchiole branching, enhanced activity of MMPs and enhanced fibronectin degradation throughout lung development compared to controls. Activation of focal adhesion kinase (FAK) was also reduced from embryonic days 12.5 through 14.5 in TIMP3 null lungs. Treatment with a synthetic MMP inhibitor, GM6001, in utero enhanced the branching pattern in both wild type and null lungs accompanied by a restoration of fibronectin localization, signaling through FAK and epithelial cell proliferation in null lungs. Direct down-regulation of FAK abundance in WT lung organ culture by siRNA targeting resulted in reduced bronchiole branching, phenocopying the TIMP3 defect. We propose that enhanced MMP activity in the absence of TIMP3 interferes with focal ECM proteolysis, perturbing the intracellular signaling necessary for correct pattern formation of the bronchiole tree during bronchiole branching morphogenesis. Thus, TIMP3 can indirectly regulate epithelial cell proliferation via MMP inhibitory activity. While others have demonstrated this function for MMPs, and there is in vitro evidence that TIMP3 controls proliferation, to our knowledge this is the first evidence of TIMP3 regulating proliferation in vivo.

Differential response of TIMP-3 null mice to the lung insults of sepsis, mechanical ventilation, and hyperoxia.

An imbalance in matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) leads to excessive or insufficient tissue breakdown, which is associated with many disease processes. The TIMP-3 null mouse is a model of MMP/TIMP imbalance, which develops air space enlargement and decreased lung function. These mice responded differently to cecal ligation and perforation (CLP)-induced septic lung injury than wild-type controls. The current study addresses whether the TIMP-3 knockout lung is susceptible to different types of insults or only those involving sepsis, by examining its response to lipopolysaccharide (LPS)-induced sepsis, mechanical ventilation (MV), and hyperoxia. TIMP-3 null noninjured controls of each insult consistently demonstrated significantly higher compliance vs. wild-type mice. Null mice treated with LPS had a further significantly increased compliance compared with untreated controls. Conversely, MV and hyperoxia did not alter compliance in the null lung. MMP abundance and activity increased in response to LPS but were generally unaltered following MV or hyperoxia, correlating with compliance alterations. All three insults produced inflammatory cytokines; however, the response of the null vs. wild-type lung was dependent on the type of insult. Overall, this study demonstrated that 1) LPS-induced sepsis produced a similar response in null mice to CLP-induced sepsis, 2) the null lung responded differently to various insults, and 3) the null susceptibility to compliance changes correlated with increased MMPs. In conclusion, this study provides insight into the role of TIMP-3 in response to various lung insults, specifically its importance in regulating MMPs to maintain compliance during a sepsis.

A null mutation for tissue inhibitor of metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis.

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs). We have examined the role of TIMP-3 on ECM homeostasis and bronchiole branching morphogenesis during murine embryogenesis. Employing an in vitro organ culture system, we found decreased bronchiolar branching in null lungs when compared with wild type (WT) counterparts after 2 days in culture. When a synthetic inhibitor of MMPs at low dose was added to the culture system, branching was augmented regardless of genotype. Gelatin and in situ zymography revealed that null lungs exhibited enhanced activation of MMPs throughout lung development. We analysed the impact of increased MMP activity on a number of ECM molecules by Western blot analysis, but found that only fibronectin abundance was consistently reduced in the null lungs throughout development. To confirm that our observed defect in culture was not simply a developmental delay in the null lung, we examined null and WT lungs from newborn pups. Here, we found not only a reduced number of bronchioles in the null, but also that the bronchiole tubes were dilated compared with controls and that alveologenesis was attenuated. We propose that the deletion of TIMP-3 disrupts the exquisite TIMP/MMP balance required for proper focal ECM proteolysis, which leads to correct bronchiole branching morphogenesis in the developing mouse lung.

Matrix metalloproteinases mediate the dismantling of mesenchymal structures in the tadpole tail during thyroid hormone-induced tail resorption.

It has been suggested that a family of tissue remodelling enzymes called matrix metalloproteinases (MMPs) play a causal role in the process of tail resorption during thyroid hormone-induced metamorphosis of the anuran tadpole; however, this hypothesis has never been directly substantiated. We cloned two new Xenopus MMPs, gelatinase A (MMP-2) and MT3-MMP (MMP-16), and the MMP inhibitor TIMP-2. These clones were used along with several others to perform a comprehensive expression study. We show that all MMPs and TIMP-2 are dramatically induced in the resorbing tail during spontaneous metamorphosis and are spatially coexpressed, primarily in the remodelling mesenchymal tissues. By Northern blotting, we show that all the examined MMPs/TIMP-2 are also induced by treatment of organ-cultured tails with thyroid hormone (T(3)). Using the organ culture model, we provide the first direct evidence that MMPs are required for T(3)-induced tail resorption by showing that a synthetic inhibitor of MMP activity/expression can specifically retard the resorption process. By gelatin zymography, we also show T(3) induction of a fifth MMP, preliminarily identified as gelatinase B (GelB; MMP-9). Moreover, T(3) not only induces MMP/TIMP expression but also MMP activation, and we provide evidence that TIMP-2 participates in the latter process. These findings suggest that MMPs and TIMPs act in concert to effect the dismantling of mesenchymal structures during T(3)-induced metamorphic tadpole tail resorption.

Negative impact of tissue inhibitor of metalloproteinase-3 null mutation on lung structure and function in response to sepsis.

Matrix metalloproteinases (MMPs) are degradative enzymes, which act to remodel tissue. Their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). An imbalance in the degradation/inhibition activities has been associated with many diseases, including sepsis. We have previously shown that TIMP-3 knockout animals develop spontaneous, progressive air space enlargement. The objectives of this study were to determine the effects of a septic lung stress induced by cecal ligation and perforation (CLP) on lung function, structure, pulmonary surfactant, and inflammation in TIMP-3 null mice. Knockout and wild-type animals were randomized to either sham or CLP surgery, allowed to recover for 6 h, and then euthanized. TIMP-3 null animals exposed to sham surgery had a significant increase in lung compliance when compared with sham wild-type mice. Additionally, the TIMP-3 knockout mice showed a significant increase in compliance following CLP. Rapid compliance changes were accompanied by significantly decreased collagen and fibronectin levels and increased gelatinase (MMP-2 and -9) abundance and activation. Additionally, in situ zymography showed increased airway-associated gelatinase activity in the knockout animals enhanced following CLP. In conclusion, exposing TIMP-3 null animals to sepsis rapidly enhances the phenotypic abnormalities of these mice, due to increased MMP activity induced by CLP.

Identification of an initiator-like element essential for the expression of the tissue inhibitor of metalloproteinases-4 (Timp-4) gene.

We have used real-time quantitative reverse transcriptase PCR (TaqMan) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5'-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.