Espinosa, Ana M. Diaz

Abstract Rationale Pulmonary Fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), primarily collagen I, leading to progressive lung scarring and loss of function. Many anti-fibrotic compounds have been found to attenuate collagen deposition. However, identifying compounds that stimulate collagen I degradation, a necessary step in fibrosis clearance, is less common. To identify such compounds, we developed in vitro and ex vivo assays to quantify extracellular collagen I and collagen I degradation products. Methods Primary lung fibroblasts (passage 1-3, N = 5) from patients with idiopathic pulmonary fibrosis (IPF) were cultured in DMEM/F12 with 2% FBS, 2 ng/mL TGF-β, and 50 µg/mL ascorbic acid for 72 hours to generate collagen-rich cell-derived ECM. Cells were then treated with test compounds for 24 hours in the same media. Nuclear labeling with Hoechst 33342 was used to quantify cell numbers by automated microscopy. Decellularization was then performed with a solution containing 20 mM ammonium hydroxide and 0.5% Triton X-100. Cell-derived ECM was then fixed, stained with anti-Collagen I antibody, and quantified by automated microscopy. Collagen I degradation was further assessed by measuring type I collagen C-telopeptide (ICTP) via ELISA in cell culture media. To investigate the role of matrix metalloproteinase-13 (MMP-13) in collagen degradation, we used T26-C, a selective MMP-13 inhibitor, or transfected cells with MMP-13 siRNA. Ex vivo validation of collagen-degrading activity was conducted by measuring type I collagen N-terminal telopeptide (NTX-I) in the culture media of human precision-cut lung slices (PCLS, N = 3) generated from upper and lower lobes of IPF tissue and stimulated with select compounds. Results Thirty-four candidate compounds (metabolic, epigenetic, and cAMP modulators) were tested for their ability to reduce collagen I levels in cell-derived ECM. Six hits and two positive controls were then validated using ICTP ELISAs. In total five compounds were found to stimulate collagen degradation: EGCG (1 μM), Nintedanib (0.25 μM), SB431542 (1 μM), and two dopamine receptor agonists, A68930 (10 μM) and Dihydrexidine (10 µM). MMP-13 inhibition or knockdown prevented collagen degradation by all five compounds. In human IPF PCLS, collagen degradation was confirmed in response to EGCG, Nintedanib and SB431542 but not Dihydrexidine, with much greater responses seen in PCLS from upper lobes than lower lobes. Conclusion Collagen degradation can be stimulated in cultured fibroblasts and IPF PCLS. These systems will assist in uncovering mechanisms and regulators of collagen degradation and may pave the way to better fibrosis-resolving therapies. This abstract is funded by: NIH U01 HL152967

Regenerative medicine is transforming how we restore tissue function, leveraging advances in cell and molecular biology, biomaterials, and engineered microenvironments. While there have been notable advances and rapid progress over the past few decades, ongoing challenges persist in the technical development and effective translation of these advancements to clinical care. This perspective highlights clinically promising examples and critically assesses present challenges in translating tissue regenerative medicine therapies from the bench to the clinic. We further examine the evolving landscape of regenerative medicine by describing strategies to optimize the cellular microenvironment, the impact of patient demographics, and the use of artificial intelligence to shape the future of this field.

Recent studies highlight transcriptionally distinct fibroblast subpopulations in human lungs. We observed the loss of these native transcriptional programs as fresh isolated cells are maintained in traditional culture conditions. Identifying the signals defining native fibroblast identities will be pivotal to creating culture models that preserve unique subpopulations. The screening system developed here will allow the investigation of a broad selection of cues, leading to better culture models for studying human lung fibroblast function and plasticity.

Abstract Rationale: Application of single cell RNAseq (scRNAseq) in idiopathic pulmonary fibrosis (IPF) and multiple murine models has revealed two unique disease associated fibroblast subsets, the Cthrc1+ fibrotic population, and a population expressing inflammatory markers whose function is unclear. We, and others, have demonstrated that inflammatory fibroblasts are derived from homeostatic fibroblasts and can differentiate to Cthrc1+ fibroblasts. There is also data in mice suggesting that TGFβ inhibition prevents the emergence of Cthrc1+ fibrotic fibroblasts without impacting the inflammatory fibroblast population. Using our inducible genetic murine model of PF, reductionist organoid systems, and scRNAseq from IPF we present evidence and mechanism for inflammatory fibroblasts as a potential regenerative population in the alveolar niche. Methods: Tamoxifen administration to SftpcI73T conditional allelic knock-in mice, induces expression of mutant protein and promotes an inflammatory cascade followed by fibrogenesis. ScRNA-seq and population RNA-seq of bleomycin and SftpcI73T murine models were compared with publicly available data sets from IPF to identify population specific surface marker expression and potential epithelial ligand-receptor pairs. Flow cytometry approaches developed to isolate fibroblast populations were applied for in-vitro organoid modeling and validation of signaling pathways. In-vivo murine intervention in TGFβ signaling was further applied to confirm functional biology of inflammatory fibroblasts. Results: Informatic analysis of human and mouse inflammatory fibroblasts identifies expression of multiple BMP antagonists that have previously been associated with epithelial proliferation. Furthermore, human inflammatory fibroblasts also express key epithelial growth factors including FGF2 and FGF7. Murine organoid modeling confirms that inflammatory fibroblasts support alveolar organoid formation through the inhibition of BMP signaling. As was previously demonstrated in bleomycin, the inhibition of TGFβ signaling in SftpcI73T does not alter the inflammatory fibroblast population, rather this population is sustained, and alveolar epithelial proliferation increases. Additionally, we did not find any evidence for increase inflammation in the mouse lung due to inflammatory fibroblasts preservation. Finally, human data suggests that two populations of inflammatory fibroblasts are present in the lung with only one subset found to express the BMP antagonists and growth factors required to promote alveolar epithelial proliferation. Conclusions: Our data identifies that inflammatory fibroblasts in the alveolar niche inhibit BMP signaling and allow for epithelial expansion. We also determine differences between murine and human inflammatory fibroblasts suggesting that human inflammatory fibroblasts have a greater potential to drive epithelial expansion. These data shed light on the functional role of inflammatory fibroblasts, further refining our understanding of fibroblast heterogeneity in IPF.

Cellular senescence is emerging as a driver of idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease with limited effective therapies. The senescence-associated secretory phenotype (SASP), involving the release of inflammatory cytokines and profibrotic growth factors by senescent cells, is thought to be a product of multiple cell types in IPF, including lung fibroblasts. NF-κB is a master regulator of the SASP, and its activity depends on the phosphorylation of p65/RelA. The purpose of this study was to assess the role of Pim-1 kinase as a driver of NF-κB-induced production of inflammatory cytokines from low-passage IPF fibroblast cultures displaying markers of senescence. Our results demonstrate that Pim-1 kinase phosphorylates p65/RelA, activating NF-κB activity and enhancing IL-6 production, which in turn amplifies the expression of PIM1, generating a positive feedback loop. In addition, targeting Pim-1 kinase with a small molecule inhibitor dramatically inhibited the expression of a broad array of cytokines and chemokines in IPF-derived fibroblasts. Furthermore, we provide evidence that Pim-1 overexpression in low-passage human lung fibroblasts is sufficient to drive premature senescence, in vitro. These findings highlight the therapeutic potential of targeting Pim-1 kinase to reprogram the secretome of senescent fibroblasts and halt IPF progression.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor β1 (TGFβ1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2, CNN1, and TAGLN. We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.

AbstractYes‐associated protein (YAP) and PDZ‐binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs‐coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2‐mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis.

Matrix stiffness is a central regulator of fibroblast function. However, the transcriptional mechanisms linking matrix stiffness to changes in fibroblast phenotype are incompletely understood. Here, we evaluated the effect of matrix stiffness on genome-wide chromatin accessibility in freshly isolated lung fibroblasts using ATAC-seq. We found higher matrix stiffness profoundly increased global chromatin accessibility relative to lower matrix stiffness, and these alterations were in close genomic proximity to known profibrotic gene programs. Motif analysis of these regulated genomic loci identified ZNF416 as a putative mediator of fibroblast stiffness responses. Genome occupancy analysis using ChIP-seq confirmed that ZNF416 occupies a broad range of genes implicated in fibroblast activation and tissue fibrosis, with relatively little overlap in genomic occupancy with other mechanoresponsive and profibrotic transcriptional regulators. Using loss- and gain-of-function studies, we demonstrated that ZNF416 plays a critical role in fibroblast proliferation, extracellular matrix synthesis, and contractile function. Together, these observations identify ZNF416 as novel mechano-activated transcriptional regulator of fibroblast biology.

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.