Sicard, Delphine

The mechanical properties of biological tissues influence their function and can predict degenerative conditions before gross histological or physiological changes are detectable. This is especially true for structural tissues such as articular cartilage, which has a primarily mechanical function that declines after injury and in the early stages of osteoarthritis. While atomic force microscopy (AFM) has been used to test the elastic modulus of articular cartilage before, there is no agreement or consistency in methodologies reported. For murine articular cartilage, methods differ in two major ways: experimental parameter selection and sample preparation. Experimental parameters that affect AFM results include indentation force and cantilever stiffness; these are dependent on the tip, sample, and instrument used. The aim of this project was to optimize these experimental parameters to measure murine articular cartilage elastic modulus by AFM micro-indentation. We first investigated the effects of experimental parameters on a control material, polydimethylsiloxane gel (PDMS), which has an elastic modulus on the same order of magnitude as articular cartilage. Experimental parameters were narrowed on this control material, and then finalized on wildtype C57BL/6J murine articular cartilage samples that were prepared with a novel technique that allows for cryosectioning of epiphyseal segments of articular cartilage and long bones without decalcification. This technique facilitates precise localization of AFM measurements on the murine articular cartilage matrix and eliminates the need to separate cartilage from underlying bone tissues, which can be challenging in murine bones because of their small size. Together, the new sample preparation method and optimized experimental parameters provide a reliable standard operating procedure to measure microscale variations in the elastic modulus of murine articular cartilage.

The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

The transcriptional co‐activator TAZ plays a critical role in lung alveolarization (Mitani et al. Am J Respir Crit Care Med. 2009). We recently observed enhanced TAZ expression in the nuclei of fibroblasts present in lung tissue from patients with idiopathic pulmonary fibrosis, suggesting that TAZ is reactivated during fibrosis. To test the role of TAZ in driving a fibroproliferative response, we developed scaffold‐free cultures in which fibroblasts were grown in 3D hanging drop spheroids. This spheroid model removes the influence of the artificial 2D cell culture environment on cell growth. NIH3T3 fibroblasts were stably transduced with doxycycline (dox)‐inducible constructs expressing nuclear‐localizing mutant TAZ4SA or empty vector (courtesy of X. Varelas, Boston University). Spheroids were initiated by seeding 500 cells in hanging drops, and were subsequently fed growth media supplemented with 0, 1, 10, or 100 ng/mL dox every other day up to 15 days. Spheroid growth was tracked by measuring 2D projected area under light microscopy. Control NIH3T3 fibroblast spheroids remained stable in culture, but neither increased nor decreased in size significantly. In contrast, TAZ4SA‐transduced spheroids grew over time, and by day 15 spheroids treated with 100 ng/ml dox had a 2D projected area ~2.2‐fold larger than those grown in the absence of dox (p=0.0054). Intermediate growth levels were observed in 1 and 10 ng/ml doxycycline treated spheroids. Our results demonstrate the utility of the spheroid culture model for studying fibroproliferation, and document a prominent role for TAZ in promoting fibroblast spheroid growth. *Supported by NIH grants RO1 HL092961 and NIH R21 HL113796.

ABSTRACT Matrix resorption is essential to the clearance of the extracellular matrix (ECM) after normal wound healing. A disruption in these processes constitutes a main component of fibrotic diseases, characterized by excess deposition and diminished clearance of fibrillar ECM proteins, such as collagen type I. The mechanisms and stimuli regulating ECM resorption in the lung remain poorly understood. Recently, agonism of dopamine receptor D1 (DRD1), which is predominantly expressed on fibroblasts in the lung, has been shown to accelerate tissue repair and clearance of ECM following bleomycin injury in mice. Therefore, we investigated whether DRD1 receptor signaling promotes the degradation of collagen type I by lung fibroblasts. For cultured fibroblasts, we found that DRD1 agonism enhances extracellular cleavage, internalization and lysosomal degradation of collagen I mediated by cathepsin K, which results in reduced stiffness of cell-derived matrices, as measured by atomic force microscopy. In vivo agonism of DRD1 similarly enhanced fibrillar collagen degradation by fibroblasts, as assessed by tissue labeling with a collagen-hybridizing peptide. Together, these results implicate DRD1 agonism in fibroblast-mediated collagen clearance, suggesting an important role for this mechanism in fibrosis resolution. This article has an associated First Person interview with the first author of the paper.

Extracellular matrix stiffness controls DNA damage response.

Abstract Introduction Impaired plasminogen activation (PA) is causally related to the development of lung fibrosis. Prior studies demonstrate that enhanced PA in the lung limits the severity of scarring following injury and in vitro studies indicate that PA promotes matrix degradation and fibroblast apoptosis. These findings led us to hypothesize that increased PA in an in vivo model would enhance the resolution of established lung fibrosis in conjunction with increased myofibroblast apoptosis. Methods Transgenic C57BL/6 mice with doxycycline inducible lung-specific urokinase plasminogen activator (uPA) expression or littermate controls were treated (day 0) with bleomycin or saline. Doxycycline was initiated on days 1, 9, 14, or 21. Lung fibrosis, stiffness, apoptosis, epithelial barrier integrity, and inflammation were assessed. Results Protection from fibrosis with uPA upregulation from day 1 through day 28 was associated with reduced parenchymal stiffness as determined by atomic force microscopy. Initiation of uPA expression beginning in the late inflammatory or the early fibrotic phase reduced stiffness and fibrosis at day 28. Induction of uPA activity in mice with established fibrosis decreased lung collagen and lung stiffness while increasing myofibroblast apoptosis. Upregulation of uPA did not alter lung inflammation but was associated with improved epithelial cell homeostasis. Conclusion Restoring intrapulmonary PA activity diminishes lung fibrogenesis and enhances the resolution of established lung fibrosis. This PA-mediated resolution is associated with increased myofibroblast apoptosis and improved epithelial cell homeostasis. These studies support the potential capacity of the lung to resolve existing scar in murine models.

Pharmacologic stimulation of a G protein–coupled receptor preferentially expressed on fibroblasts reverses experimental lung and liver fibrosis.

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is thought to present both molecular and mechanical barriers to neuronal regeneration. The scar consists of a meshwork of reactive glia and deposited, cross-linked, extracellular matrix (ECM) that has long been assumed to present a mechanically “stiff” blockade. However, remarkably little quantitative information is available about the rheological properties of chronically injured spinal tissue. In this study we utilize atomic force microscopy microindentation to provide quantitative evidence of chronic mechanical stiffening after SCI. Using the results of this tissue characterization, we assessed the sensitivity of both mouse and human astrocytes in vitro and determined that they are exquisitely mechanosensitive within the relevant range of substrate stiffness observed in the injured/uninjured spinal cord. We then utilized a novel immune modifying nanoparticle (IMP) treatment as a tool to reveal fibrotic scarring as one of the key drivers of mechanical stiffening after SCI in vivo . We also demonstrate that glial scar-forming astrocytes form a highly aligned, anisotropic network of glial fibers after SCI, and that IMP treatment mitigates this pathological alignment. Taken together, our results identify chronic mechanical stiffening as a critically important aspect of the complex lesion milieu after SCI that must be considered when assessing and developing potential clinical interventions for SCI.

Abstract Reciprocal epithelial–mesenchymal interactions are pivotal in lung development, homeostasis, injury, and repair. Organoids have been used to investigate such interactions, but with a major focus on epithelial responses to mesenchyme and less attention to epithelial effects on mesenchyme. In the present study, we used nascent organoids composed of human and mouse lung epithelial and mesenchymal cells to demonstrate that healthy lung epithelium dramatically represses transcriptional, contractile, and matrix synthetic functions of lung fibroblasts. Repression of fibroblast activation requires signaling via the bone morphogenetic protein (BMP) pathway. BMP signaling is diminished after epithelial injury in vitro and in vivo, and exogenous BMP4 restores fibroblast repression in injured organoids. In contrast, inhibition of BMP signaling in healthy organoids is sufficient to derepress fibroblast matrix synthetic function. Our results reveal potent repression of fibroblast activation by healthy lung epithelium and a novel mechanism by which epithelial loss or injury is intrinsically coupled to mesenchymal activation via loss of repressive BMP signaling.

Lung function is inherently mechanical in nature and depends on the capacity to conduct air and blood to and from the gas exchange regions. Variations in the elastic properties of the human lung across anatomical compartments and with aging are likely important determinants of lung function but remain relatively poorly characterized. Here we applied atomic force microscopy microindentation to characterize human lung tissue from subjects ranging in age from 11 to 60 yr old. We observed striking anatomical variations in elastic modulus, with the airways (200- to 350-µm diameter) the stiffest and the parenchymal regions the most compliant. Vessels (diameter < 100 µm) represented an intermediate mechanical environment and displayed diameter-dependent trends in elastic modulus. Binning our samples into younger (11–30 yr old) and older (41–60 yr old) groups, we observed significant age-related increases in stiffness in parenchymal and vessel compartments, with the most pronounced changes in the vessels. To investigate cellular mechanisms that might contribute to vascular stiffening with aging, we studied primary human pulmonary artery smooth muscle cells from subjects ranging in age from 11 to 60 yr old. While we observed no change in the mechanical properties of the cells themselves, we did observe trends toward increases in traction forces and extracellular matrix deposition with aging. These results demonstrate age-related changes in tissue mechanical properties that likely contribute to impaired lung function with aging and underscore the potential to identify mechanisms that contribute to mechanical tissue remodeling through the study of human cells and tissues from across the aging spectrum.