Club cell secretory protein improves survival in a murine obliterative bronchiolitis model.

Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy.

Evidence for early fibrosis and increased airway resistance in bone marrow transplant recipient mice deficient in MMP12.

Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality post-bone marrow transplantation (BMT) in humans. In our established murine IPS model in which lethally conditioned recipients are given allogeneic bone marrow and splenocytes, recruitment of host monocytes occurs early post-BMT, followed by donor T cells concomitant with development of severe lung dysfunction. Because matrix metalloproteinase 12 (MMP12) is important for macrophage infiltration and injury in other mouse models of lung disease such as emphysema, lethally conditioned MMP12(-/-) mice were used as allogeneic recipients to determine whether MMP12 plays a similar role in potentiating lung injury in IPS. Surprisingly, MMP12(-/-) mice developed IPS and exhibited an accelerated allogeneic T cell-dependent decrease in compliance compared with wild-type (WT) recipients. MMP12(-/-), but not WT, mice also had allogeneic T cell-dependent elevated lung resistance post-BMT. Recruitment of monocytes and T cells into the lungs was not altered on day 7 post-BMT, but the lungs of MMP12(-/-) recipients had increased collagen deposition, a feature normally not seen in our IPS model. MMP12(-/-) mice had a compensatory increase in MMP2 in the lungs post-BMT, as well as increased ß6-integrin compared with WT recipients, and only in the presence of allogeneic T cells. Levels of total transforming growth factor (TGF)-ß1 protein in the lungs were elevated compared with WT recipients, consistent with the profibrotic function of ß6-integrin as an activator of TGF-ß. These data indicate that host-derived MMP12 may be important in limiting development of IPS by allowing proper remodeling of extracellular matrix and effective repair of BMT-related injury.

Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.

We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2-3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4',6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4',6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells.

Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network.

A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both furin-specific inhibitor and RNA interference technique, we demonstrate that furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple furin recognition sites: (206)RPRR(209), (209)RAKR(212), or (211)KR(212). The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with furin, suggesting that furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a furin-independent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.