Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.     

Differential expression of oxidative stress and extracellular matrix remodeling genes in low- or high-dose-rate photon-irradiated skin

Changes in the expression of genes implicated in oxidative stress and in extracellular matrix (ECM) remodeling and selected protein expression profiles in mouse skin were examined after exposure to low-dose-rate or high-dose-rate photon irradiation. ICR mice received whole-body γ rays to total doses of 0, 0.25, 0.5 and 1 Gy at dose rates of 50 cGy/h or 50 cGy/min. Skin tissues were harvested for characterization at 4 h after irradiation. For oxidative stress after low-dose-rate exposure, 0.25, 0.5 and 1 Gy significantly altered 27, 23 and 25 genes, respectively, among 84 genes assessed (P < 0.05). At doses as low as 0.25 Gy, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly altered, with changes >2-fold compared to 0 Gy. For an ECM profile, 18-20 out of 84 genes were significantly up- or downregulated after low-dose-rate exposure. After high-dose-rate irradiation, of 84 genes associated with oxidative stress, 16, 22 and 22 genes were significantly affected after 0.25, 0.5 and 1 Gy, respectively. Compared to low-dose-rate radiation, high-dose-rate exposure resulted in different ECM gene expression profiles. The most striking changes after low-dose-rate or high-dose-rate exposure on ECM profiles were on genes encoding matrix metalloproteinases (MMPs), e.g., Mmp2 and Mmp15 for low dose rate and Mmp9 and Mmp11 for high dose rate. Immunostaining for MMP-2 and MMP-9 proteins showed radiation dose rate-dependent differences. These data revealed that exposure to low total doses with low-dose-rate or high-dose-rate photon radiation induced oxidative stress and ECM-associated alterations in gene expression profiles. The expression of many genes was differentially regulated by different total dose and/or dose-rate regimens.     

Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13-/- mice is intrinsic to cartilage and bone. Mmp13-/- mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.     

Transforming growth factor-beta signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-beta is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-beta signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O(2)) or air from birth to 14 days of age. Expression of DNTGFbetaRII was induced by 20 microg/g ZnSO(4) given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-beta and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFbetaRII mice given ZnSO(4) compared with WT control mice and DNTGFbetaRII mice not given ZnSO(4). The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFbetaRII mice given ZnSO(4). These data support the conclusion that TGF-beta plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.     

Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation

Membrane type 1-matrix metalloprotease (MT1-MMP or MMP-14) is a major activator of pro-MMP-2 and is essential for skeletal development. We show here that it is required for branching morphogenesis of the submandibular gland but not the lung. Instead, in the lung, it is essential for postnatal development of alveolar septae. Lung development in Mmp14-/- mice is arrested at the prealveolar stage with compensatory hyperinflation of immature saccules. Mmp2-/- mice lacked comparable defects in the lung and submandibular gland, suggesting that MT1-MMP acts via mechanisms independent of pro-MMP-2 activation. Since the developmental defects in the lung are first manifest around the time of initial vascularization (E16.5), we investigated the behavior of pulmonary endothelial cells from Mmp14+/+ and Mmp14-/- mice. Endothelial cells from lungs of 1-week-old Mmp14-/- mice show reduced migration and formation of three-dimensional structures on Matrigel. Since pulmonary septal development requires capillary growth, the underlying mechanism of pulmonary hypoplasia in Mmp14-/- mice may be defective angiogenesis, supporting a model in which angiogenesis is a critical rate-limiting step for acquisition of pulmonary parenchymal mass.     

Role of matrix metalloprotease-9 in hyperoxic injury in developing lung

Matrix metalloprotease-9 (MMP-9) is increased in lung injury following hyperoxia exposure in neonatal mice, in association with impaired alveolar development. We studied the role of MMP-9 in the mechanism of hyperoxia-induced functional and histological changes in neonatal mouse lung. Reduced alveolarization with remodeling of ECM is a major morbidity component of oxidant injury in developing lung. MMP-9 mediates oxidant injury in developing lung causing altered lung remodeling. Five-day-old neonatal wild-type (WT) and MMP-9 (-/-) mice were exposed to hyperoxia for 8 days. The lungs were inflation fixed, and sections were examined for morphometry. The mean linear intercept and alveolar counts were evaluated. Immunohistochemistry for MMP-9 and elastin was performed. MMP-2, MMP-9, type I collagen, and tropoelastin were measured by Western blot analysis. Lung quasistatic compliance was studied in anaesthetized mice. MMP-2 and MMP-9 were significantly increased in lungs of WT mice exposed to hyperoxia compared with controls. Immunohistochemistry showed an increase in MMP-9 in mesenchyme and alveolar epithelium of hyperoxic lungs. The lungs of hyperoxia-exposed WT mice had less gas exchange surface area and were less compliant compared with room air-exposed WT and hyperoxia-exposed MMP-9 (-/-) mice. Type I collagen and tropoelastin were increased in hyperoxia-exposed WT with aberrant elastin staining. These changes were ameliorated in hyperoxia-exposed MMP-9 (-/-) mice. MMP-9 plays an important role in the structural changes consequent to oxygen-induced lung injury. Blocking MMP-9 activity may lead to novel therapeutic approaches in preventing bronchopulmonary dysplasia.     

MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13(-/-)) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13(-/-) mice. Granulation tissue in Mmp13(-/-) mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13(-/-) mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13(-/-) mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13(-/-) granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13(-/-) mice compared to WT mice. Mmp13(-/-) mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.     

Sulfatases are determinants of alveolar formation

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1(-/-) to study the importance of proteoglycan desulfation in lung development. The Sumf1(-/-) mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1(-/-) as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1(-/-) mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1(-/-) mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.     

A matrix metalloproteinase mediates airway remodeling in Drosophila

Organ size typically increases dramatically during juvenile growth. This growth presents a fundamental tension, as organs need resiliency to resist stresses while still maintaining plasticity to accommodate growth. The extracellular matrix (ECM) is central to providing resiliency, but how ECM is remodeled to accommodate growth is poorly understood. We investigated remodeling of Drosophila respiratory tubes (tracheae) that elongate continually during larval growth, despite being lined with a rigid cuticular ECM. Cuticle is initially deposited with a characteristic pattern of repeating ridges and valleys known as taenidia. We find that for tubes to elongate, the extracellular protease Mmp1 is required for expansion of ECM between the taenidial ridges during each intermolt period. Mmp1 protein localizes in periodically spaced puncta that are in register with the taenidial spacing. Mmp1 also degrades old cuticle at molts, promotes apical membrane expansion in larval tracheae, and promotes tube elongation in embryonic tracheae. Whereas work in other developmental systems has demonstrated that MMPs are required for axial elongation occurring in localized growth zones, this study demonstrates that MMPs can also mediate interstitial matrix remodeling during growth of an organ system.     

Bone marrow-derived matrix metalloproteinase-9 is associated with fibrous adhesion formation after murine flexor tendon injury

The pathogenesis of adhesions following primary tendon repair is poorly understood, but is thought to involve dysregulation of matrix metalloproteinases (Mmps). We have previously demonstrated that Mmp9 gene expression is increased during the inflammatory phase following murine flexor digitorum (FDL) tendon repair in association with increased adhesions. To further investigate the role of Mmp9, the cellular, molecular, and biomechanical features of healing were examined in WT and Mmp9(-/-) mice using the FDL tendon repair model. Adhesions persisted in WT, but were reduced in Mmp9(-/-) mice by 21 days without any decrease in strength. Deletion of Mmp9 resulted in accelerated expression of neo-tendon associated genes, Gdf5 and Smad8, and delayed expression of collagen I and collagen III. Furthermore, WT bone marrow cells (GFP(+)) migrated specifically to the tendon repair site. Transplanting myeloablated Mmp9(-/-) mice with WT marrow cells resulted in greater adhesions than observed in Mmp9(-/-) mice and similar to those seen in WT mice. These studies show that Mmp9 is primarily derived from bone marrow cells that migrate to the repair site, and mediates adhesion formation in injured tendons. Mmp9 is a potential target to limit adhesion formation in tendon healing.     

Global expression profiles from C57BL/6J and DBA/2J mouse lungs to determine aging-related genes.

This study identified gene expression profiles that provided evidence for genomic mechanisms underlying the pathophysiology of aging lung. Aging lungs from C57BL/6 (B6) and DBA/2 (D2) mouse strains differ in physiology and morphometry. Lungs were harvested from B6 mice at 2, 18, and 26 mo and from D2 mice at 2 and 18 mo of age. Purified RNA was subjected to oligonucleotide microarray analyses, and differential expression analyses were performed for comparison of various data sets. A significant majority of differentially expressed genes were upregulated with aging in both strains. Aging D2 lungs uniquely exhibited upregulation in stress-response genes including xenobiotic detoxification cascades. In contrast, aging B6 lungs showed downregulation of heat shock-response genes. Age-dependent downregulation of genes common to both B6 and D2 strains included several collagen genes (e.g., Col1a1 and Col3a1). There was a greater elastin gene (Eln) expression in D2 mice at 2 mo, and Eln was uniquely downregulated with age in this strain. The matrix metalloproteinase 14 gene (Mmp14), critical to alveolar structural integrity, was also downregulated with aging in D2 mice only. Several polymorphisms in the regulatory and untranslated regions of Mmp14 were identified between strains, suggesting that variation in Mmp14 gene regulation contributes to accelerated aging of lungs in D2 mice. In summary, lungs of B6 and D2 mice age with variable rates at the gene expression level, and these quantifiable genomic differences provide a template for understanding the variability in age-dependent changes in lung structure and function.     

Loss of PECAM-1 function impairs alveolarization

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.