Non-canonical IKB kinases regulate YAP/TAZ and pathological vascular remodeling behaviors in pulmonary artery smooth muscle cells.

Pulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell-specific approach. Our previous work demonstrated that targeting non-canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non-canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH-associated PASMC remodeling, with longer-lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non-canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.

A redox-shifted fibroblast subpopulation emerges in the fibrotic lung.

Idiopathic pulmonary fibrosis (IPF) is an aggressive and thus far incurable disease, characterized by aberrant fibroblast-mediated extracellular matrix deposition. Our understanding of the disease etiology is incomplete; however, there is consensus that a reduction-oxidation (redox) imbalance plays a role. In this study we use the autofluorescent properties of two redox molecules, NAD(P)H and FAD, to quantify changes in their relative abundance in living lung tissue of mice with experimental lung fibrosis, and in freshly isolated cells from mouse lungs and humans with IPF. Our results identify cell population-specific intracellular redox changes in the lungs in experimental and human fibrosis. We focus particularly on redox changes within collagen producing cells, where we identified a bimodal distribution of NAD(P)H concentrations, establishing NAD(P)H high and NAD(P)H low sub-populations. NAD(P)H high fibroblasts exhibited elevated pro-fibrotic gene expression and decreased collagenolytic protease activity relative to NAD(P)H low fibroblasts. The NAD(P)H high population was present in healthy lungs but expanded with time after bleomycin injury suggesting a potential role in fibrosis progression. We identified a similar increased abundance of NAD(P)H high cells in freshly dissociated lungs of subjects with IPF relative to controls, and similar reductions in collagenolytic activity in this cell population. These data highlight the complexity of redox state changes in experimental and human pulmonary fibrosis and the need for selective approaches to restore redox imbalances in the fibrotic lung.

Using extracellular matrix derived from sugen-chronic hypoxia lung tissue to study pulmonary arterial hypertension.

Pulmonary arterial hypertension has characteristic changes to the mechanical environment, extracellular matrix, and cellular proliferation. In order to develop a culture system to investigate extracellular matrix (ECM) compositional-dependent changes in pulmonary arterial hypertension, we decellularized and characterized protein and lipid profiles from healthy and Sugen-Chronic Hypoxia rat lungs. Significant changes in lipid profiles were observed in intact Sugen-Hypoxia lungs compared with healthy controls. Decellularized lung matrix retained lipids in measurable quantities in both healthy and Sugen-Chronic Hypoxia samples. Proteomics revealed significantly changed proteins associated with pulmonary arterial hypertension in the decellularized Sugen-Chronic Hypoxia lung ECM. We then investigated the potential role of healthy vs. Sugen-Chronic Hypoxia ECM with controlled substrate stiffness to determine if the ECM composition regulated endothelial cell morphology and phenotype. CD117+ rat lung endothelial cell clones were plated on the variable stiffness gels and cellular proliferation, morphology, and gene expression were quantified. Sugen-Chronic Hypoxia ECM on healthy stiffness gels produced significant changes in cellular gene expression levels of Bmp2, Col1α1, Col3α1 and Fn1. The signaling and cell morphology observed at low substrate stiffness suggests early changes to the ECM composition can initiate processes associated with disease progression. These data suggest that Sugen-Chronic Hypoxia ECM can be used to investigate cell-ECM interactions relevant to pulmonary arterial hypertension.

Dopamine Receptor D1 Is Exempt from Transforming Growth Factor ß-Mediated Antifibrotic G Protein-Coupled Receptor Landscape Tampering in Lung Fibroblasts.

Pulmonary fibroblasts are the primary producers of extracellular matrix (ECM) in the lungs, and their pathogenic activation drives scarring and loss of lung function in idiopathic pulmonary fibrosis (IPF). This uncontrolled production of ECM is stimulated by mechanosignaling and transforming growth factor beta 1 (TGF-ß1) signaling that together promote transcriptional programs including Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). G protein-coupled receptors (GPCRs) that couple to G α s have emerged as pharmacological targets to inactivate YAP/TAZ signaling and promote lung fibrosis resolution. Previous studies have shown a loss of expression of "antifibrotic GPCRs"-receptors that couple to G α s, in IPF patient-derived fibroblasts compared with non-IPF samples. Of the 14 G α s GPCRs we found to be expressed in lung fibroblasts, the dopamine receptor D1 (DRD1) was one of only two not repressed by TGF-ß1 signaling, with the ß2-adrenergic receptor being the most repressed. We compared the potency and efficacy of multiple D1 and ß2 receptor agonists +/- TGF-ß1 treatment in vitro for their ability to elevate cAMP, inhibit nuclear localization of YAP/TAZ, regulate expression of profibrotic and antifibrotic genes, and inhibit cellular proliferation and collagen deposition. Consistently, the activity of ß2 receptor agonists was lost, whereas D1 receptor agonists was maintained, after stimulating cultured lung fibroblasts with TGF-ß1. These data further support the therapeutic potential of the dopamine receptor D1 and highlight an orchestrated and pervasive loss of antifibrotic GPCRs mediated by TGF-ß1 signaling. SIGNIFICANCE STATEMENT: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with limited therapies. GPCRs have emerged as a primary target for the development of novel antifibrotic drugs; however, a challenge to this approach is the dramatic changes in GPCR expression in response to profibrotic stimuli. Here, we investigate the impact of TGF-ß1 on the expression of antifibrotic GPCRs and show the D1 dopamine receptor expression is uniquely maintained in response to TGF-ß1, further implicating it as a compelling target to treat IPF.

The matricellular protein CCN3 supports lung endothelial homeostasis and function.

Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.

Dichotomous role of integrin-ß5 in lung endothelial cells.

Pulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis-resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin-ß5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial-to-mesenchymal transition (EnMT). We found that elevated integrin-ß5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117+ rat lung EC clones. These EC clones exhibited elevated expression of integrin-ß5 and its heterodimerization partner integrin-αν and showed accelerated barrier formation. Inhibition of integrin-ανß5 in vitro partially blocked transforming growth factor (TGF)-ß1-induced EnMT gene expression in rat lung control ECs and less in rat lung EC clones and human lung microvascular ECs. Inhibition of integrin-ανß5 promoted endothelial dysfunction as shown by reduced migration in a scratch assay and increased apoptosis in synergism with TGF-ß1. In vivo, blocking of integrin-ανß5 exaggerated PH induced by chronic hypoxia and CD117+ EC clones in rats. In summary, we found a role for integrin-ανß5 in lung endothelial survival and migration, but also a partial contribution to TGF-ß1-induced EnMT gene expression. Our results suggest that integrin-ανß5 is required for physiologic function of ECs and lung vascular homeostasis.

Dysfunctional ERG signaling drives pulmonary vascular aging and persistent fibrosis.

Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury.

Dopamine receptor signaling regulates fibrotic activation of retinal pigmented epithelial cells.

Retinal pigmented epithelial (RPE) cells play an important role in retinal fibrotic diseases such as proliferative vitreoretinopathy (PVR). The purpose of this study was to elucidate the involvement of dopamine receptor signaling in regulating the fibrotic activation of RPE cells. Dopamine receptor expression, the effect of dopamine on fibrotic activity, and dopamine production were measured in the human RPE cell line ARPE-19. The fibrotic activation of RPE cells was evaluated in response to treatments with selective dopamine receptor agonists and antagonists by measuring gene expression, migration, proliferation, and fibronectin deposition. DRD2 and DRD5 are the dominant dopaminergic receptors expressed in ARPE-19 cells and TGF-ß stimulation enhances the autocrine release of dopamine, which we show further exasperates fibrotic activation. Finally, treatment with D2 dopamine receptor antagonists or D5 dopamine receptor agonists inhibits profibrotic gene expression, migration, proliferation, and fibronectin deposition and thus may serve as effective mechanisms for treating retinal fibrosis including PVR.

Combined control of the fibroblast contractile program by YAP and TAZ.

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 ß1 (TGFß1)-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.

GPCR-mediated YAP/TAZ inactivation in fibroblasts via EPAC1/2, RAP2C, and MAP4K7.

Yes-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.

Cellular mitosis predicts vessel stability in a mechanochemical model of sprouting angiogenesis.

Angiogenesis, the formation of new vessels, occurs in both developmental and pathological contexts. Prior research has investigated vessel formation to identify cellular phenotypes and dynamics associated with angiogenic disease. One major family of proteins involved in angiogenesis are the Rho GTPases, which govern function related to cellular elongation, migration, and proliferation. Using a mechanochemical model coupling Rho GTPase activity and cellular and intercellular mechanics, we investigate the role of cellular mitosis on sprouting angiogenesis. Mitosis-GTPase synchronization was not a strong predictor of GTPase and thus vessel signaling instability, whereas the location of mitotic events was predicted to alter GTPase cycling instabilities. Our model predicts that middle stalk cells undergoing mitosis introduce irregular dynamics in GTPase cycling and may provide a source of aberrant angiogenesis. We also find that cellular and junctional tension exhibit spatial heterogeneity through the vessel, and that tension feedback, specifically in stalk cells, tends to increase the maximum forces generated in the vessel.

ZNF416 is a pivotal transcriptional regulator of fibroblast mechanoactivation.

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.

Spontaneous Lung Fibrosis Resolution Reveals Novel Antifibrotic Regulators.

Fibroblast activation is transient in successful wound repair but persistent in fibrotic pathologies. Understanding fibroblast deactivation during successful wound healing may provide new approaches to therapeutically reverse fibroblast activation. To characterize the gene programs that accompany fibroblast activation and reversal during lung fibrosis resolution, we used RNA sequencing analysis of flow sorted Col1α1-GFP-positive and CD45-, CD31-, and CD326-negative cells isolated from the lungs of young mice exposed to bleomycin. We compared fibroblasts isolated from control mice with those isolated at Days 14 and 30 after bleomycin exposure, representing the peak of extracellular matrix deposition and an early stage of fibrosis resolution, respectively. Bleomycin exposure dramatically altered fibroblast gene programs at Day 14. Principal component and differential gene expression analyses demonstrated the predominant reversal of these trends at Day 30. Upstream regulator and pathway analyses of reversing "resolution" genes identified novel candidate antifibrotic genes and pathways. Two genes from these analyses that were decreased in expression at Day 14 and reversed at Day 30, Aldh2 and Nr3c1, were selected for further analysis. Enhancement of endogenous expression of either gene by CRISPR activation in cultured human idiopathic pulmonary fibrosis fibroblasts was sufficient to reduce profibrotic gene expression, fibronectin deposition, and collagen gel compaction, consistent with roles for these genes in fibroblast deactivation. This combination of RNA sequencing analysis of freshly sorted fibroblasts and hypothesis testing in cultured idiopathic pulmonary fibrosis fibroblasts offers a path toward identification of novel regulators of lung fibroblast deactivation, with potential relevance to understanding fibrosis resolution and its failure in human disease.

Dopamine D1 receptor stimulates cathepsin K-dependent degradation and resorption of collagen I in lung fibroblasts.

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.

Porcine Lung-Derived Extracellular Matrix Hydrogel Properties Are Dependent on Pepsin Digestion Time.

Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM) in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.

Electrosprayed extracellular matrix nanoparticles induce a pro-regenerative cell response.

Decellularized extracellular matrix (ECM) is an effective tissue repair scaffold. Additionally, ECM has recently been shown to be protective to the lungs. However, current processing is inadequate for effective delivery of ECM to the lungs. Processing methods, such as milling, produce large variability in particle sizes. The size variation produced is ineffective at treating the lung because only a small range of sizes reach the distal regions of the alveoli. The aim of this work is to formulate decellularized ECM to reach the distal lung while retaining the pro-regenerative effects of ECM. We first digested the protein in acid and then electrosprayed the solution into nanoparticles. The average size of the nanoparticles was 225 (±67) nm, within size requirements to reach the alveoli. After characterizing the particles, we measured cytotoxicity of the nanoparticles. Adding 0.125 mg/ml of nanoparticles to the media increased cellular proliferation in A549 alveolar epithelial cells and caused no cytotoxicity in BEAS-2B cells. We added the formed nanoparticles to macrophages derived from murine bone marrow-derived monocytes. The macrophages exposed to the formed nanoparticles expressed cell surface marker CD206 (mannose receptor C type 1), commonly attributed to a pro-regeneration phenotype. Electrosprayed ECM formed nanoparticles may improve bronchoalveolar deposition while maintaining the pro-regenerative benefits shown by other decellularized ECM materials.

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 °C and can be used immediately following neutralization or stored at 4 °C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

From Here to There, Progenitor Cells and Stem Cells Are Everywhere in Lung Vascular Remodeling.

The field of stem cell biology, cell therapy, and regenerative medicine has expanded almost exponentially, in the last decade. Clinical trials are evaluating the potential therapeutic use of stem cells in many adult and pediatric lung diseases with vascular component, such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), or pulmonary arterial hypertension (PAH). Extensive research activity is exploring the lung resident and circulating progenitor cells and their contribution to vascular complications of chronic lung diseases, and researchers hope to use resident or circulating stem/progenitor cells to treat chronic lung diseases and their vascular complications. It is becoming more and more clear that progress in mechanobiology will help to understand the various influences of physical forces and extracellular matrix composition on the phenotype and features of the progenitor cells and stem cells. The current review provides an overview of current concepts in the field.

Development and characterization of a naturally derived lung extracellular matrix hydrogel.

The complexity and rapid clearance mechanisms of lung tissue make it difficult to develop effective treatments for many chronic pathologies. We are investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. The main objectives of this study include effective decellularization of porcine lung tissue, development of a hydrogel from the porcine ECM, and characterization of the material's composition, mechanical properties, and ability to support cellular growth. Our evaluation of the decellularized tissue indicated successful removal of cellular material and immunogenic remnants in the ECM. The self-assembly of the lung ECM hydrogel was rapid, reaching maximum modulus values within 3 min at 37°C. Rheological characterization showed the lung ECM hydrogel to have a concentration dependent storage modulus between 15 and 60 Pa. The purpose of this study was to evaluate our novel ECM derived hydrogel and measure its ability to support 3D culture of MSCs in vitro and in vivo delivery of MSCs. Our in vitro experiments using human mesenchymal stem cells demonstrated our novel ECM hydrogel's ability to enhance cellular attachment and viability. Our in vivo experiments demonstrated that rat MSC delivery in pre-gel solution significantly increased cell retention in the lung over 24 h in an emphysema rat model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1922-1935, 2016.