Recellularization of whole decellularized lung scaffolds provides a novel approach for generating functional lung tissue ex vivo for subsequent clinical transplantation. To explore the potential utility of stem and progenitor cells in this model, we investigated recellularization of decellularized whole mouse lungs after intratracheal inoculation of bone marrow-derived mesenchymal stromal cells (MSCs). The decellularized lungs maintained structural features of native lungs, including intact vasculature, ability to undergo ventilation, and an extracellular matrix (ECM) scaffold consisting primarily of collagens I and IV, laminin, and fibronectin. However, even in the absence of intact cells or nuclei, a number of cell-associated (non-ECM) proteins were detected using mass spectroscopy, western blots, and immunohistochemistry. MSCs initially homed and engrafted to regions enriched in types I and IV collagen, laminin, and fibronectin, and subsequently proliferated and migrated toward regions enriched in types I and IV collagen and laminin but not provisional matrix (fibronectin). MSCs cultured for up to 1 month in either basal MSC medium or in a small airways growth media (SAGM) localized in both parenchymal and airway regions and demonstrated several different morphologies. However, while MSCs cultured in basal medium increased in number, MSCs cultured in SAGM decreased in number over 1 month. Under both media conditions, the MSCs predominantly expressed genes consistent with mesenchymal and osteoblast phenotype. Despite a transient expression of the lung precursor TTF-1, no other airway or alveolar genes or vascular genes were expressed. These studies highlight the power of whole decellularized lung scaffolds to study functional recellularization with MSCs and other cells.
Members of the metzincin family of metalloproteinases have long been considered merely degradative enzymes for extracellular matrix molecules. Recently, however, there has been growing appreciation for these proteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), as fine modulators of nervous system physiology and pathology. Present all along the phylogenetic tree, in all neural cell types, from the nucleus to the synapse and in the extracellular space, metalloproteinases exhibit a complex spatiotemporal profile of expression in the nervous parenchyma and at the neurovascular interface. The irreversibility of their proteolytic activity on numerous biofactors (e.g., growth factors, cytokines, receptors, DNA repair enzymes, matrix proteins) is ideally suited to sustain structural changes that are involved in physiological or postlesion remodeling of neural networks, learning consolidation or impairment, neurodegenerative and neuroinflammatory processes, or progression of malignant gliomas. The present review provides a state of the art overview of the involvement of the metzincin/TIMP system in these processes and the prospects of new therapeutic strategies based on the control of metalloproteinase activity.The importance of proteolysis in tissue structure/function is reflected not only in the evolutionary conservation of protease genes in all kingdoms (e.g., from archaea and eubacteria to plants and animals) but also the genomic complexity of this protein class. The "degradome," the repertoire of proteases produced by cells, consists of at least 569 human, 629 rat, and 644 mouse proteases or protease-like proteins and homologs, whereas 156 human protease inhibitor genes have been identified (Puente et al., 2003). The proteases are classified into five major catalytic classes, including metalloproteinases and serine, cysteine, threonine, and aspartic proteinases, with the metalloproteinases representing the largest class (Fig. 1 A). The metzincin family of metalloproteinases is so named for the conserved Met residue at the active site and the use of a zinc ion in the enzymatic reaction. This family comprises matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and ADAM proteases with thrombospondin motifs (ADAMTSs). Interest in MMPs began with the identification of an enzyme that contributes to tail resorption during tadpole metamorphosis (collagenase-1, MMP-1) (Gross and Lapiere, 1962) and increased on the discovery that these enzymes not only play a role in normal tissue remodeling but were upregulated in diverse human diseases, including chronic inflammatory disorders and cancer. MMPsMMPs, encoded by 24 human and 23 mouse genes, include secreted and membrane-associated members divided into four main subgroups according to their domain structure, including collagenases, stromelysins, gelatinases, and membrane-type MMPs (MT-MMPs) (Fig.
Rationale: Myocardial infarction (MI) results in remodeling of the myocardium and the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) are critical regulators of ECM integrity via inhibiting matrix metalloproteinases (MMPs). TIMP2 is highly expressed in the heart and is the only TIMP that, in addition to inhibiting MMPs, is required for cell surface activation of pro-MMP2. Hence, it is difficult to predict the function of TIMP2 as protective (MMP-inhibiting) or harmful (MMP-activating) in heart disease. Objective: We examined the role of TIMP2 in the cardiac response to MI. Methods and Results: MI was induced in 11-to 12-week-old male TIMP2؊/؊ and age-matched wild-type mice. Cardiac function was monitored by echocardiography at 1 and 4 weeks post-MI. ECM fibrillar structure was visualized using second harmonic generation and multiphoton imaging of unfixed/unstained hearts. Molecular analyses were performed at 3 days and 1 week post-MI on flash-frozen infarct, periinfarct, and noninfarct tissue. Membrane type 1 (MT1)-MMP levels and activity were measured in membrane protein fractions. TIMP2؊/؊ -MI mice exhibited a 25% greater infarct expansion, markedly exacerbated left ventricular dilation (by 12%) and dysfunction (by 30%), and more severe inflammation compared to wild-type MI mice. Adverse ECM remodeling was detected by reduced density and enhanced disarray of fibrillar collagen in TIMP2 ؊/؊ -MI compared to wild-type MI hearts. TIMP2 deficiency completely abrogated MMP2 activation but markedly increased collagenase activity, particularly MT1-MMP activity post-MI. Key Words: myocardial infarction Ⅲ extracellular matrix Ⅲ remodeling Ⅲ tissue inhibitor of metalloproteinase Ⅲ matrix metalloproteinase C oronary artery disease is the most common cause of heart failure in Western nations. 1 Myocardial infarction (MI) results in adverse remodeling of the myocardium including the extracellular matrix (ECM). 2 ECM is a dynamic structure which undergoes constant turnover, and its intact structure is essential for optimal cardiac structure and function. Tissue inhibitors of metalloproteinases (TIMPs) play a critical role in regulating the activity of the ECM-degrading enzymes matrix metalloproteinases (MMPs). [3][4][5] Four TIMPs have been identified so far (TIMP1 to TIMP4), with TIMP2 and TIMP3 being the highly expressed TIMPs in the heart and TIMP4 showing a tissue-specific expression pattern which includes the heart and the brain. 6 TIMP2 levels have been shown to be altered in patients with different types of heart disease such as pressure overload 7 and atrial fibrillation. 8 In patients with ischemic or idiopathic dilated cardiomyopathy, myocardial protein levels of TIMP2 remained unaltered 9,10 or increased in end-stage dilated cardiomyopathy patients, 11 whereas plasma analyses revealed a rise in TIMP2 levels in late stages of MI. 12 TIMP2 is unique among TIMPs because, in addition to inhibiting a number of MMPs, it mediates MMP activation. TIMP2 is required for cell surface activati...
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