[64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via µPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

[68 Ga]Ga-FAPI-46 PET for non-invasive detection of pulmonary fibrosis disease activity.

PURPOSE: The lack of effective molecular biomarkers to monitor idiopathic pulmonary fibrosis (IPF) activity or treatment response remains an unmet clinical need. Herein, we determined the utility of fibroblast activation protein inhibitor for positron emission tomography (FAPI PET) imaging in a mouse model of pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intratracheal administration of bleomycin (1 U/kg) while intratracheal saline was administered to control mice. Subgroups from each cohort (n = 3-5) underwent dynamic 1 h PET/CT after intravenously injecting FAPI-46 radiolabeled with gallium-68 ([68 Ga]Ga-FAPI-46) at 7 days and 14 days following disease induction. Animals were sacrificed following imaging for ex vivo gamma counting and histologic correlation. [68 Ga]Ga-FAPI-46 uptake was quantified and reported as percent injected activity per cc (%IA/cc) or percent injected activity (%IA). Lung CT density in Hounsfield units (HU) was also correlated with histologic examinations of lung fibrosis. RESULTS: CT only detected differences in the fibrotic response at 14 days post-bleomycin administration. [68 Ga]Ga-FAPI-46 lung uptake was significantly higher in the bleomycin group than in control subjects at 7 days and 14 days. Significantly (P = 0.0012) increased [68 Ga]Ga-FAPI-46 lung uptake in the bleomycin groups at 14 days (1.01 ± 0.12%IA/cc) vs. 7 days (0.33 ± 0.09%IA/cc) at 60 min post-injection of the tracer was observed. These findings were consistent with an increase in both fibrinogenesis and FAP expression as seen in histology. CONCLUSION: CT was unable to assess disease activity in a murine model of IPF. Conversely, FAPI PET detected both the presence and activity of lung fibrogenesis, making it a promising tool for assessing early disease activity and evaluating the efficacy of therapeutic interventions in lung fibrosis patients.

Expression of serum response factor in the lung mesenchyme is essential for development of pulmonary fibrosis.

Extracellular matrix deposition characterizes idiopathic pulmonary fibrosis (IPF) and is orchestrated by myofibroblasts. The lung mesenchyme is an essential source of myofibroblasts in pulmonary fibrosis. Although the transcription factor serum response factor (SRF) has shown to be critical in the process of myofibroblast differentiation, its role in development of pulmonary fibrosis has not been determined in vivo. In this study, we observed that SRF expression localized to mesenchymal compartments, areas of dense fibrosis, and fibroblastic foci in human (IPF and normal) and bleomycin-treated mouse lungs. To determine the role of mesenchymal SRF in pulmonary fibrosis, we utilized a doxycycline-inducible, Tbx4 lung enhancer (Tbx4LE)-driven Cre-recombinase to disrupt SRF expression in the lung mesenchyme in vivo. Doxycycline-treated Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f (and controls) were treated with a single intratracheal dose of bleomycin to induce pulmonary fibrosis and examined for lung mesenchymal expansion, pulmonary fibrosis, and inflammatory response. Bleomycin-treated Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f mice showed decreased numbers of Tbx4LE-positive lung mesenchymal cells (LMCs) and collagen accumulation (via hydroxyproline assay) compared with controls. This effect was associated with SRF-null LMCs losing their proliferative and myofibroblast differentiation potential compared with SRF-positive controls. Together, these data demonstrate that SRF plays a critical role in LMC myofibroblast expansion during bleomycin-induced pulmonary fibrosis. This sets the stage for pharmacological strategies that specifically target SRF in the lung mesenchyme as a potential means of treating pulmonary fibrosis.

Analysis of fibroblast migration dynamics in idiopathic pulmonary fibrosis using image-based scaffolds of the lung extracellular matrix.

Idiopathic pulmonary fibrosis (IPF) is characterized by a profound remodeling of the collagen in the extracellular matrix (ECM), where the fibers become both denser and more highly aligned. However, it is unknown how this reconfiguration of the collagen matrix affects disease progression. Here, we investigate the role of specific alterations in collagen fiber organization on cell migration dynamics by using biomimetic image-based collagen scaffolds representing normal and fibrotic lung, where the designs are derived directly from high-resolution second harmonic generation microscopy images. The scaffolds are fabricated by multiphoton-excited (MPE) polymerization, where the process is akin to three-dimensional printing, except that it is performed at much greater resolution (∼0.5 microns) and with collagen and collagen analogs. These scaffolds were seeded with early passaged primary human normal and IPF fibroblasts to enable the decoupling of the effect of cell-intrinsic characteristics (normal vs. IPF) versus ECM structure (normal vs. IPF) on migration dynamics. We found that the highly aligned IPF collagen structure promoted enhanced cell elongation and F-actin alignment along with increased cell migration speed and straightness relative to the normal tissues. Collectively, the data are consistent with an enhanced contact guidance mechanism on the aligned IPF matrix. Although cell intrinsic effects were observed, the aligned collagen matrix morphology had a larger effect on these metrics. Importantly, these biomimetic models of the lung cannot be synthesized by conventional fabrication methods. We suggest that the MPE image-based fabrication method will enable additional hypothesis-based testing studies of cell-matrix interactions in the context of tissue fibrosis.

Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response.

Pdgfra-expressing (Pdgfra+) cells have been implicated as progenitors in many mesenchymal tissues. To determine lineage potential, we generated PdgfrartTA knockin mice using CRISPR/Cas9. During lung maturation, counter to a prior study reporting that Pdgfra+ cells give rise equally to myofibroblasts and lipofibroblasts, lineage tracing using PdgfrartTA;tetO-cre mice indicated that ~95% of the lineaged cells are myofibroblasts. Genetic ablation of Pdgfra+ cells using PdgfrartTA-driven diphtheria toxin (DTA) led to alveolar simplification, demonstrating that these cells are essential for building the gas exchange surface area. In the adult bleomycin model of lung fibrosis, lineaged cells increased to contribute to pathological myofibroblasts. In contrast, in a neonatal hyperoxia model of bronchopulmonary dysplasia (BPD), lineaged cells decreased and do not substantially contribute to pathological myofibroblasts. Our findings revealed complexity in the behavior of the Pdgfra-lineaged cells as exemplified by their distinct contributions to myofibroblasts in normal maturation, BPD and adult fibrosis.

The Novel mTOR Complex 1/2 Inhibitor P529 Inhibits Human Lung Myofibroblast Differentiation.

Idiopathic pulmonary fibrosis is a progressive and deadly disorder with very few therapeutic options. Palomid 529 (8-(1-hydroxyethyl)-2-methoxy-3-(4-methoxybenzyloxy)-benzo[c]chromen-6-one; P529) is a novel dual inhibitor of mechanistic target of rapamycin complex 1/2 (mTORC1/2). In these studies, we investigated the effect of P529 on TGF-ß-dependent signaling and myofibroblast differentiation. TGF-ß-induced phosphorylation of the mTORC1 targets, p70 S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), were both dose dependently inhibited by P529 in human lung fibroblasts with maximal inhibition occurring between 10 and 20 µM. mTORC2-mediated phosphorylation of Akt at the S473 site was partially inhibited with a similar dose dependency, as was TGF-ß-induced myofibroblast differentiation. Protein levels of TGF-ß-induced fibronectin and collagen were similarly decreased by P529. At this dose, there was also inhibition of mRNA transcript levels for Col1 and α-SMA, suggesting inhibition of transcriptional activation. However, there was no effect of P529 on canonical TGF-ß-induced Smad signaling, as assessed by receptor-associated Smad2/3 phosphorylation, Smad2/3/4 translocation, or Smad-driven gene expression, as assessed by Smad-binding element driven luciferase. Conversely, activation of mTORC1/2 signaling was dependent on TGF-ß type I receptor (ALK5) signaling and on Smad2/3 expression. P529 treatment disrupted TGF-ß-induced actin stress fiber formation during myofibroblast differentiation, the deposition of new extracellular fibronectin matrix, and linear wound closure by fibroblasts. Likewise, mTOR knockdown inhibited TGF-ß-induced myofibroblast differentiation. In conclusion, P529 inhibits TGF-ß-induced myofibroblast differentiation, actin stress fiber formation, and matrix protein expression and deposition. Inhibition of mTORC1/2 by P529 may be a promising approach to inhibit in vivo fibrosis. J. Cell. Biochem. 118: 2241-2249, 2017. © 2017 Wiley Periodicals, Inc.

Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation.

Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF). Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore, endogenous and exogenous semaphorin-7A may have opposite effects on the fibroblast phenotype.

Tensin 1 Is Essential for Myofibroblast Differentiation and Extracellular Matrix Formation.

Myofibroblasts, the primary effector cells that mediate matrix remodeling during pulmonary fibrosis, rapidly assemble an extracellular fibronectin matrix. Tensin (TNS) 1 is a key component of specialized cellular adhesions (fibrillar adhesions) that bind to extracellular fibronectin fibrils. We hypothesized that TNS1 may play a role in modulating myofibroblast-mediated matrix formation. We found that TNS1 expression is increased in fibroblastic foci from lungs with idiopathic pulmonary fibrosis. Transforming growth factor (TGF)-ß profoundly up-regulates TNS1 expression with kinetics that parallel the expression of the myofibroblast marker, smooth muscle α-actin. TGF-ß-induced TNS1 expression is dependent on signaling through the TGF-ß receptor 1 and is Rho coiled-coiled kinase/actin/megakaryoblastic leukemia-1/serum response factor dependent. Small interfering RNA-mediated knockdown of TNS1 disrupted TGF-ß-induced myofibroblast differentiation, without affecting TGF-ß/Smad signaling. In contrast, loss of TNS1 resulted in disruption of focal adhesion kinase phosphorylation, focal adhesion formation, and actin stress fiber development. Finally, TNS1 was essential for the formation of fibrillar adhesions and the assembly of nascent fibronectin and collagen matrix in myofibroblasts. In summary, our data show that TNS1 is a novel megakaryoblastic leukemia-1-dependent gene that is induced during pulmonary fibrosis. TNS1 plays an essential role in TGF-ß-induced myofibroblast differentiation and myofibroblast-mediated formation of extracellular fibronectin and collagen matrix. Targeted disruption of TNS1 and associated signaling may provide an avenue to inhibit tissue fibrosis.

In Vivo Tracking of Human Neural Progenitor Cells in the Rat Brain Using Magnetic Resonance Imaging Is Not Enhanced by Ferritin Expression.

Rapid growth in the field of stem cell research has generated a lot of interest in their therapeutic use, especially in the treatment of neurodegenerative diseases. Specifically, human neural progenitor cells (hNPCs), unique in their capability to differentiate into cells of the neural lineage, have been widely investigated due to their ability to survive, thrive, and migrate toward injured tissues. Still, one of the major roadblocks for clinical applicability arises from the inability to monitor these cells following transplantation. Molecular imaging techniques, such as magnetic resonance imaging (MRI), have been explored to assess hNPC transplant location, migration, and survival. Here we investigated whether inducing hNPCs to overexpress ferritin (hNPCs(Fer)), an iron storage protein, is sufficient to track these cells long term in the rat striatum using MRI. We found that increased hypointensity on MRI images could establish hNPC(Fer) location. Unexpectedly, however, wild-type hNPC transplants were detected in a similar manner, which is likely due to increased iron accumulation following transplantation-induced damage. Hence, we labeled hNPCs with superparamagnetic iron oxide (SPIO) nanoparticles to further increase iron content in an attempt to enhance cell contrast in MRI. SPIO-labeling of hNPCs (hNPCs-SPIO) achieved increased hypointensity, with significantly greater area of decreased T2* compared to hNPC(Fer) (p < 0.0001) and all other controls used. However, none of the techniques could be used to determine graft rejection in vivo, which is imperative for understanding cell behavior following transplantation. We conclude that in order for cell survival to be monitored in preclinical and clinical settings, another molecular imaging technique must be employed, including perhaps multimodal imaging, which would utilize MRI along with another imaging modality.

Megakaryoblastic leukemia-1 is required for the development of bleomycin-induced pulmonary fibrosis.

BACKGROUND: Fibrosing disorders of the lung, such as idiopathic pulmonary fibrosis, are characterized by progressive extracellular matrix accumulation that is driven by myofibroblasts. The transcription factor megakaryoblastic leukemia-1 (MKL1) mediates myofibroblast differentiation in response to several profibrotic stimuli, but the role it plays in mediating pulmonary fibrosis has not been fully elucidated. In this study, we utilized mice that had a germline deletion of MKL1 (MKL1 (-,-)) to determine the role that MKL1 plays in the development of bleomycin-induced pulmonary fibrosis. METHODS: Bleomycin or normal saline were intratracheally delivered to 9 to 12 week old female MKL1 (+,+) and MKL1 (-,-) mice. Mice were assessed for weight loss and survival to 28 days. Inflammatory responses were assessed through bronchoalveolar lavage at days 3 and 7 post-treatment. The development of pulmonary fibrosis was characterized using hydroxyproline assay and histological staining. MKL1 (+,+) and MKL1 (-,-) mouse lung fibroblasts were isolated to compare morphologic, gene expression and functional differences. RESULTS: MKL1 (-,-) mice demonstrated increased survival, attenuated weight loss, and decreased collagen accumulation compared to wild-type animals 28-days after intratracheal instillation of bleomycin. Histological analysis demonstrated decreased trichrome, smooth muscle α-actin, and fibronectin staining in MKL1(-,-) mice compared to MKL1 (+,+) controls. Differential cell counts from bronchoalveolar lavage demonstrated that there was attenuated neutrophilia 3 days after bleomycin administration, but no difference at day 7. Isolated mouse lung fibroblasts from MKL1 (-,-) mice had decreased contractility and deposited less fibronectin matrix compared to wild-type controls, suggesting a defect in key remodeling functions. CONCLUSIONS: Altogether, these data demonstrate that MKL1 plays a significant role in mediating the fibrotic response to bleomycin injury. Loss of MKL1 attenuated early neutrophil influx, as well as myofibroblast-mediated remodeling. Targeting MKL1 activity may therefore be a useful strategy in treating pulmonary fibrosis.

Myofibroblasts exhibit enhanced fibronectin assembly that is intrinsic to their contractile phenotype.

Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

In vivo tracking of human neural progenitor cells in the rat brain using bioluminescence imaging.

BACKGROUND: Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity. NEW METHOD: In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC(Luc2)) in the rat striatum long-term. RESULTS: We show that hNPC(Luc2) are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC(Luc2) contralateral migration. COMPARISON WITH EXISTING METHODS: Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood-brain barrier remains intact. CONCLUSIONS: These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders.

Neonatal immune-tolerance in mice does not prevent xenograft rejection.

Assessing the efficacy of human stem cell transplantation in rodent models is complicated by the significant immune rejection that occurs. Two recent reports have shown conflicting results using neonatal tolerance to xenografts in rats. Here we extend this approach to mice and assess whether neonatal tolerance can prevent the rapid rejection of xenografts. In three strains of neonatal immune-intact mice, using two different brain transplant regimes and three independent stem cell types, we conclusively show that there is rapid rejection of the implanted cells. We also address specific challenges associated with the generation of humanized mouse models of disease.

Synergistic effects of GDNF and VEGF on lifespan and disease progression in a familial ALS rat model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.