Sean E. Gill

Expression analysis of the entire MMP and TIMP gene families during mouse tissue development

Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane‐bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.

The role of TIMPs in regulation of extracellular matrix proteolysis.

Tissue inhibitors of metalloproteinases (TIMPs), which inhibit matrix metalloproteinases (MMPs) as well as the closely related, a disintegrin and metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs), were traditionally thought to control extracellular matrix (ECM) proteolysis through direct inhibition of MMP-dependent ECM proteolysis. This classical role for TIMPs suggests that increased TIMP levels results in ECM accumulation (or fibrosis), whereas loss of TIMPs leads to enhanced matrix proteolysis. Mice lacking TIMP family members have provided support for such a role; however, studies with these TIMP deficient mice have also demonstrated that loss of TIMPs can often be associated with an accumulation of ECM. Collectively, these studies suggest that the divergent roles of TIMPs in matrix accumulation and proteolysis, which together can be referred to as ECM turnover, are dependent on the TIMP, specific tissue, and local tissue environment (i.e. health vs. injury/disease). Ultimately, these combined factors dictate the specific metalloproteinases being regulated by a given TIMP, and it is likely the diversity of metalloproteinases and their physiological substrates that determines whether TIMPs inhibit matrix proteolysis or accumulation. In this review, we discuss the evidence for the dichotomous roles of TIMPs in ECM turnover highlighting some of the common findings between different TIMP family members. Importantly, while we now have a better understanding of the role of TIMPs in regulating ECM turnover, much remains to be determined. Data on the specific metalloproteinases inhibited by different TIMPs in vivo remains limited and must be the focus of future studies.

Pericyte TIMP3 and ADAMTS1 Modulate Vascular Stability after Kidney Injury

Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

Pulmonary Macrophage Subpopulations in the Induction and Resolution of Acute Lung Injury

Macrophages are key orchestrators of the inflammatory and repair responses in the lung, and the diversity of their function is indicated by their polarized states and distinct subpopulations and localization in the lung. Here, we characterized the pulmonary macrophage populations in the interstitial and alveolar compartments during the induction and resolution of acute lung injury induced by Pseudomonas aeruginosa infection. We identified macrophage subpopulations and polarity according to FACS analysis of cell surface protein markers, combined with cell sorting for gene expression using real-time PCR. With these techniques, we validated a novel, alternatively activated (M2) marker (transferrin receptor), and we described three interstitial and alveolar macrophage subpopulations in the lung whose distribution and functional state evolved from the induction to resolution phases of lung injury. Together, these findings indicate the presence and evolution of distinct macrophage subsets in the lung that serve specific niches in regulating the inflammatory response and its resolution. Alterations in the balance and function of these subpopulations could lead to nonresolving acute lung injury.

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.

Proteoglycans: key regulators of pulmonary inflammation and the innate immune response to lung infection.

Exposure to viruses and bacteria results in lung infections and places a significant burden on public health. The innate immune system is an early warning system that recognizes viruses and bacteria, which results in the rapid production of inflammatory mediators such as cytokines and chemokines and the pulmonary recruitment of leukocytes. When leukocytes emigrate from the systemic circulation through the extracellular matrix (ECM) in response to lung infection they encounter proteoglycans, which consist of a core protein and their associated glycosaminoglycans. In this review, we discuss how proteoglycans serve to modify the pulmonary inflammatory response and leukocyte migration through a number of different mechanisms including: (1) The ability of soluble proteoglycans or fragments of glycosaminoglycans to activate Toll‐like receptor (TLRs) signaling pathways; (2) The binding and sequestration of cytokines, chemokines, and growth factors by proteoglycans; (3) the ability of proteoglycans and hyaluronan to facilitate leukocyte adhesion and sequestration; and (4) The interactions between proteoglycans and matrix metalloproteinases (MMP) that alter the function of these proteases. In conclusion, proteoglycans fine‐tune tissue inflammation through a number of different mechanisms. Clarification of the mechanisms whereby proteoglycans modulate the pulmonary inflammatory response will most likely lead to new therapeutic approaches to inflammatory lung disease and lung infection. Anat Rec, 293:968–981, 2010.

Kinetics of chemokine-glycosaminoglycan interactions control neutrophil migration into the airspaces of the lungs.

Chemokine–glycosaminoglycan (GAG) interactions are thought to result in the formation of tissue-bound chemokine gradients. We hypothesized that the binding of chemokines to GAGs would increase neutrophil migration toward CXC chemokines instilled into lungs of mice. To test this hypothesis we compared neutrophil migration toward recombinant human CXCL8 (rhCXCL8) and two mutant forms of CXCL8, which do not bind to heparin immobilized on a sensor chip. Unexpectedly, when instilled into the lungs of mice the CXCL8 mutants recruited more neutrophils than rhCXCL8. The CXCL8 mutants appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro. A comparison of the murine CXC chemokines, KC and MIP-2, revealed that KC was more effective in recruiting neutrophils into the lungs than MIP-2. KC appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro than MIP-2. In kinetic binding studies, KC, MIP-2, and rhCXCL8 bound heparin differently, with KC associating and dissociating more rapidly from immobilized heparin than the other chemokines. These data suggest that the kinetics of chemokine–GAG interactions contributes to chemokine function in tissues. In the lungs, it appears that chemokines, such as CXCL8 or MIP-2, which associate and disassociate slowly from GAGs, form gradients relatively slowly compared with chemokines that either bind GAGs poorly or interact with rapid kinetics. Thus, different types of chemokine gradients may form during an inflammatory response. This suggests a new model, whereby GAGs control the spatiotemporal formation of chemokine gradients and neutrophil migration in tissue.

Tissue Inhibitor of Metalloproteinases 3 Regulates Resolution of Inflammation following Acute Lung Injury

Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits not only matrix metalloproteinases but also a disintegrin and metalloproteinase domain family members and thus contributes to controlling diverse processes mediated by proteolysis. We used Timp3(-/-) mice to assess the role of this inhibitor in acute lung injury. After bleomycin-induced injury, inflammation, as indicated by the influx of neutrophils in bronchoalveolar lavage (BAL), peaked at 7 days post-injury in the wild-type mice and began to wane thereafter; however, in Timp3(-/-) mice, inflammation persisted up to 28 days. Furthermore, although the level of chemokines in BAL and lung homogenate was similar in both genotypes, BAL from Timp3(-/-) mice 7, 14, and 28 days post-injury had increased neutrophil chemotactic activity compared with wild-type BAL. At day 14, a higher percentage of apoptotic neutrophils were present in wild-type mice compared with Timp3(-/-) mice, further suggesting that TIMP3 constrains continued neutrophil influx. In addition, total matrix metalloproteinase activity was increased in lungs from Timp3(-/-) mice, and treatment of mice with a synthetic inhibitor of metalloproteinases rescued the enhanced neutrophilia phenotype. These data demonstrate that TIMP3 regulates neutrophil influx in the lung following injury through its ability to inhibit metalloproteinase activity and indicates that TIMP3 functions to promote the resolution of inflammation in the lung.

Pulmonary microvascular albumin leak is associated with endothelial cell death in murine sepsis-induced lung injury in vivo.

Sepsis is a systemic inflammatory response that targets multiple components of the cardiovascular system including the microvasculature. Microvascular endothelial cells (MVEC) are central to normal microvascular function, including maintenance of the microvascular permeability barrier. Microvascular/MVEC dysfunction during sepsis is associated with barrier dysfunction, resulting in the leak of protein-rich edema fluid into organs, especially the lung. The specific role of MVEC apoptosis in septic microvascular/MVEC dysfunction in vivo remains to be determined. To examine pulmonary MVEC death in vivo under septic conditions, we used a murine cecal ligation/perforation (CLP) model of sepsis and identified non-viable pulmonary cells with propidium iodide (PI) by intravital videomicroscopy (IVVM), and confirmed this by histology. Septic pulmonary microvascular Evans blue (EB)-labeled albumin leak was associated with an increased number of PI-positive cells, which were confirmed to be predominantly MVEC based on specific labeling with three markers, anti-CD31 (PECAM), anti-CD34, and lectin binding. Furthermore, this septic death of pulmonary MVEC was markedly attenuated by cyclophosphamide-mediated depletion of neutrophils (PMN) or use of an anti-CD18 antibody developed for immunohistochemistry but shown to block CD18-dependent signaling. Additionally, septic pulmonary MVEC death was iNOS-dependent as mice lacking iNOS had markedly fewer PI-positive MVEC. Septic PI-positive pulmonary cell death was confirmed to be due to apoptosis by three independent markers: caspase activation by FLIVO, translocation of phosphatidylserine to the cell surface by Annexin V binding, and DNA fragmentation by TUNEL. Collectively, these findings indicate that septic pulmonary MVEC death, putatively apoptosis, is a result of leukocyte activation and iNOS-dependent signaling, and in turn, may contribute to pulmonary microvascular barrier dysfunction and albumin hyper-permeability during sepsis.

Syndecan-4 Regulates Early Neutrophil Migration and Pulmonary Inflammation in Response to Lipopolysaccharide

Proteoglycans (PGs) and their associated glycosaminoglycan side chains are effectors of inflammation, but little is known about changes to the composition of PGs in response to lung infection or injury. The goals of this study were to identify changes to heparan sulfate PGs in a mouse model of gram-negative pneumonia, to identify the Toll-like receptor adaptor molecules responsible for these changes, and to determine the role of the heparan sulfate PG in the innate immune response in the lungs. We treated mice with intratracheal LPS, a component of the cell wall of gram-negative bacteria, to model gram-negative pneumonia. Mice treated with intratracheal LPS had a rapid and selective increase in syndecan-4 mRNA that was regulated through MyD88-dependent mechanisms, whereas expression of several other PGs was not affected. To determine the role of syndecan-4 in the inflammatory response, we exposed mice deficient in syndecan-4 to LPS and found a significant increase in neutrophil numbers and amounts of CXC-chemokines and total protein in bronchoalveolar lavage fluid. In studies performed in vitro, macrophages and epithelial cells treated with LPS had increased expression of syndecan-4. Studies performed using BEAS-2B cells showed that pretreatment with heparin and syndecan-4 decreased the expression of CXCL8 mRNA in response to LPS and TNF-α. These findings indicate that the early inflammatory response to LPS involves marked up-regulation of syndecan-4, which functions to limit the extent of pulmonary inflammation and lung injury.