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Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFβ1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFβ1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

Rationale: TGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined., Objective: To investigate myofibroblast differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis., Methods: Lung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β1, TGF-β signaling (Smad2 phosphorylation), myofibroblast differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain)., Results: TGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both myofibroblast differentiation and tissue fibrosis are significantly augmented (p<0.005)., Conclusions: These studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.

Thy-1 is a glycosylphosphytidylinositol-linked cell-surface glycoprotein present on a subset of lung fibroblasts, which plays an important role in postnatal alveolarization. In the present study, we define the role of Thy-1 in pulmonary lipofibroblast differentiation and in the regulation of lipid homeostasis via peroxisome proliferator-activated receptor-γ (PPARγ). Thy-1 was associated with interstitial cells containing lipid droplets in vivo. The transfection of Thy-1 into Thy-1 (-) fibroblasts increased triglyceride content, fatty-acid uptake, and the expression of the lipofibroblast marker adipocyte differentiation-related protein. Thy-1 (+) fibroblasts exhibited 2.4-fold higher PPARγ activity, and the inhibition or activation of PPARγ reduced and increased triglyceride content, respectively. Thy-1 (-) fibroblasts were not responsive to either of the PPARγ agonists ciglitazone or prostaglandin J(2), supporting the importance of Thy-1 in signaling via PPARγ. Thy-1 (+) fibroblasts expressed significantly higher concentrations of fatty-acid transporter protein-3 mRNA, and demonstrated higher rates of fatty-acid uptake and increased triglyceride content. The inhibition of fatty-acid transporter protein function reduced Thy-1 (+) fibroblast lipid content. The expression of Thy-1 in C57BL/6 lung fibroblasts increased during the neonatal period, coinciding with the onset of alveolarization. Thy-1 promoted lipofibroblast differentiation via the expression of PPARγ, stimulated lipid accumulation via fatty-acid esterification, and enhanced the fatty-acid uptake mediated by fatty-acid transporter proteins. Thy-1 is important in the regulation of lipofibroblast differentiation in the developing lung.

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin alpha(v)beta(5)-dependent latent transforming growth factor (TGF)-beta1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin alpha(v)beta(5), both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin alpha(v)beta(5) interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin alpha(v)beta(5)-dependent latent TGF-beta1 activation and TGF-beta1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-alpha(v)beta(5) interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin alpha(v)beta(5) interactions inhibit contraction-induced latent TGF-beta1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-beta1 with integrin alpha(v)beta(5). Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.

Thy-1 is a glycosylphosphatidyl-inositol-linked cell surface glycoprotein whose exact biological role remains unclear. Differential expression of Thy-1 affects fibroblast proliferation and fibrogenic signaling. In idiopathic pulmonary fibrosis, the proliferating myofibroblasts within the fibroblastic foci are Thy-1(-), whereas normal lung fibroblasts are predominantly Thy-1(+). In this study, we used rat lung fibroblasts sorted for Thy-1 expression to examine myofibroblastic differentiation in response to fibrogenic stimuli. We examined the effects of transforming growth factor-beta, endothelin-1, and connective tissue growth factor on the expression of myofibroblast proteins and myogenic regulatory factors by real-time RT-PCR and immunoblotting. Thy-1(-) cells have significantly higher myofibroblast and myogenic regulatory factor gene and protein expression compared with Thy-1(+) cells, confirmed by immunofluorescence. We also used floating collagen matrix contraction assays to assess the functional differentiation of the fibroblasts. At baseline and after stimulation with transforming growth factor-beta and endothelin-1, Thy-1(-) cells caused significantly greater collagen contraction than did Thy-1(+) cells, supporting the hypothesis that Thy-1(-) cells are more fully differentiated myofibroblasts. Because apoptosis has been implicated in the regression of myofibroblasts, we examined the percentage of apoptotic cells in the contracted collagen matrices at baseline and after stimulation with fibrogenic agents. A significantly greater proportion of Thy-1(+) cells underwent apoptosis in all conditions compared with Thy-1(-) fibroblasts. Transfection of Thy-1 into Thy-1(-) cells inhibits collagen matrix contraction and reduces cell survival. Our data indicate that Thy-1 regulates myogenic gene expression, myofibroblastic differentiation, and survival in lung fibroblasts.