Abstract: In conclusion, our studies address two important topics in lung biology: the lineage relationships of epithelial cells in the distal gas exchange region of the lung and the cellular origins of pulmonary fibrosis. A deeper understanding of epithelial progenitors will help to identify therapies to reverse the cell loss that occurs in a number of lung diseases and not just fibrosis. For fibrosis, defining the relative contributions of each of the different stromal cell types, including pericytes, to the fibrotic process will be important in future studies. In particular, we need to understand whether contributions are direct (through synthesis of ECM) or indirect (through production of growth factors and cytokines). This knowledge has the potential to lead to novel therapeutic strategies for the treatment of pulmonary fibrosis. It has been suggested that 30–50% of the myofibroblasts in the bleomycin model originate from epithelial cells, including type 2 alveolar epithelial cells (AEC2), through a process known as epithelial to mesenchymal transition. According to this model, epithelial cells lose their polarity and assume a fibroblast phenotype, including the expression of stromal markers and secretion of ECM. We addressed this popular hypothesis by generating a knock-in allele to induce the heritable expression of a fluorescent lineage tag in AEC2 cells. In the distal lung, these cells are a source of surfactant proteins, which are critical for proper lung function, and they may also act as a population of stem cells capable of differentiating into type 1 alveolar epithelial cells (AEC1s) ( 4 ). The lineage tag also enabled us to purify AEC2s and their descendants to confirm the morphological findings by examining the expression of different marker genes. Using confocal microscopy and our panel of stromal markers, we saw no evidence that lineage-labeled AEC2 gave rise to any kind of stromal cell. Rather, they differentiate into AEC1 cells, supporting classical models for the lineage relationship of these two cell types. Significantly, the slow conversion of AEC2 to AEC1 cells seen in control lungs is greatly enhanced in response to bleomycin-induced lung injury. Currently, we do not understand the mechanism underlying this change in behavior, but additional analysis of gene expression in AEC2 cells will address this question. We also used our Secretoglobin1a1-CreER knock-in allele to follow the response of epithelial cells of the airways (bronchioles) and a subset of alveolar epithelial cells to bleomycin. Unlike our previous findings with other injury models ( 5 ), we found striking changes in the proliferation and differentiation of these cells after bleomycin injury. However, again, we found no evidence that this labeled population generates fibroblasts. With this foundation in place, we used genetic lineage tracing in the mouse to determine the origin of the fibroblasts within the bleomycin-induced lesions. First, we tested the hypothesis that pericytes are a source of myofibroblasts. We used two different mouse strains carrying transgenes, Ng2-CreER and FoxJ1-CreER, to induce the expression of a fluorescent protein that is heritable and specifically expressed in pericyte-like cells within the alveolar wall. This florescent marker allowed us to follow the fate of this cell type in response to bleomycin. We found that these cells proliferated and expanded within fibroblast foci. Surprisingly, most of the lineage-labeled cells did not express high levels of aSMA. We began by staining tissue sections with antibodies against a panel of commonly used markers for stromal cells and viewing them by confocal microscopy to characterize these cell populations before bleomycin treatment and at different times after it. The markers included α-smooth muscle actin (aSMA and Acta2), which is generally considered a hallmark of myofibroblasts. We also examined other markers including S100a4 (Fsp1), desmin, vimentin, Pdgfra, Pdgfrb, and the surface glycoprotein Ng2 (Cspg4). Some of these markers mark pericytes, a population of cells implicated as a source of myofibroblasts in other fibrotic processes ( 2 , 3 ). These studies revealed a surprising diversity of cells in the fibrotic lesions, with aSMA-positive cells showing the greatest abundance relatively early after the administration of bleomycin. A survey of fresh biopsy samples from patients with IPF suggested that a similar diversity exists in human fibrotic lesions. One factor complicating the study of IPF is that surprisingly little is known about the identities, behaviors, and lineage relationships of the different cell types that make up the gas exchange region of the lung where the disease occurs. This region has a complex 3D structure in which a vast network of capillaries and nonepithelial stromal cells surrounds the millions of thin-walled, air-filled epithelial sacs known as alveoli ( Fig. P1 ; ref. 1 ). To fully understand the process of fibrosis, we focused on high-resolution microscopy of intact normal and fibrotic lung tissue rather than study of cells grown in culture. Idiopathic pulmonary fibrosis (IPF) is a debilitating disease in which the delicate gas exchange region of the lung is gradually replaced by accumulations of ECM and myofibroblasts. These cells are commonly thought to produce components of the ECM, including collagen. Conflicting ideas about the cellular origin of the fibrotic lesions may slow progress to the development of effective therapies for IPF. To address this problem, we combined the power of cell lineage tracing in mice with a model of pulmonary fibrosis induced by bleomycin, an anticancer agent that often results in pulmonary fibrosis as a side effect. We provide evidence that the behaviors of pericytes, a cell type associated with blood vessels, and epithelial cells are modulated in response to bleomycin-induced lung injury. However, neither cell type is a major source of myofibroblasts. These findings should help to focus future efforts at developing therapies for the treatment of IPF.

Abstract: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-fixed heterograft tissue, such as bovine pericardium (BP), are widely used for treating heart valve disease, a group of disorders that affects millions. Structural valve degeneration (SVD) of BHV due to both calcification and the accumulation of advanced glycation end products (AGE) with associated serum proteins limits durability. We hypothesized that BP modified with poly-2-methyl-2-oxazoline (POZ) to inhibit protein entry would demonstrate reduced accumulation of AGE and serum proteins, mitigating SVD. In vitro studies of POZ-modified BP demonstrated reduced accumulation of serum albumin and AGE. BP-POZ in vitro maintained collagen microarchitecture per two-photon microscopy despite AGE incubation, and in cell culture studies was associated with no change in tumor necrosis factor-α after exposure to AGE and activated macrophages. Comparing POZ and polyethylene glycol (PEG)–modified BP in vitro, BP-POZ was minimally affected by oxidative conditions, whereas BP-PEG was susceptible to oxidative deterioration. In juvenile rat subdermal implants, BP-POZ demonstrated reduced AGE formation and serum albumin infiltration, while calcification was not inhibited. However, BP-POZ rat subdermal implants with ethanol pretreatment demonstrated inhibition of both AGE accumulation and calcification. Ex vivo laminar flow studies with human blood demonstrated BP-POZ enhanced thromboresistance with reduced white blood cell accumulation. We conclude that SVD associated with AGE and serum protein accumulation can be mitigated through POZ functionalization that both enhances biocompatibility and facilitates ethanol pretreatment inhibition of BP calcification.

Abstract: One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA , through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.