Analyses of fibroblast growth factor signaling in lung fibrosis

Increased fibroblast growth factor 10 (Fgf10) expression in vivo and administration of exogenous FGF7 recombinant protein enhance lung repair due to bleomycin injury by sending survival signals to lung epithelial cells via tyrosine kinase fibroblast growth factor receptor 2b (Fgfr2b). Given the prophylactic effects of FGF7 and therapeutic effects of FGF10 during bleomycin injury in mice, it was hypothesized that activation of the Fgfr2b endogenous pathway is critical for lung repair after bleomycin injury in mice. Furthermore, as new studies for the treatment of Idiopathic Pulmonary Fibrosis (IPF) have begun to target tyrosine kinases, the aim was to 1) assess the levels of FGF10 and FGF7 signaling in end-stage IPF lungs, 2) assess the level of recruitment of the endogenous Fgfr2b pathway after bleomycin lung injury in mice, and 3) assess the effect of FGF10 treatment on IPF fibroblasts in vitro. Compared to donor, non-IPF controls, FGF7 and FGF10 transcripts were increased in end-stage IPF patient lung homogenates. However, receptors as well as downstream targets of FGF7 and 10 were significantly decreased. In contrast, wild type mice undergoing spontaneous repair after bleomycin injury, expressed Fgf10 and downstream targets from 14 days post injury, indicating potential recruitment of this pathway during repair. Using three different genetic mouse lines congenitally deficient in endogenous Fgfr2b signaling, we found that Fgf10 deficient animals incurred the highest trend towards increased fibrosis. Surprisingly, in a fourth mouse line allowing for induction of a soluble, dominant negative Fgfr2b receptor, induced mice did not show increased bleomycin-induced fibrosis compared to non-induced controls. Thus FGFR2b ligands signaling seemed to be recruited in a model of spontaneous repair, and dysregulated in non- repairing IPF patients. Thus the endogenous FGFR2b pathway may be redundant with other repair pathways in mice, as attenuating it did not result in a significant increase in bleomycin- induced fibrosis. IPF fibroblasts responded to FGF10 treatment by decreasing their size, increasing proliferation and increasing expression of lipofibroblast markers. In addition, FGF10 inhibited transforming growth factor beta (TGF-β) stimulated induction of TGF-β signaling downstream target, pSMAD3. Likewise the ability of FGF10 to reduce cell size and inhibit TGF-β signaling in IPF fibroblasts, suggests that it could effectively mediate a contractile to synthetic-like phenotype, which may be an important step towards UIP lesion repair. Taken together, this work highlights and discusses 1) the attenuation of FGFR2b ligand signaling in end-stage IPF, 2) FGFR2b signaling recruitment in wild type mice during bleomycin injury, yet redundant role in injury, and 3) the potential therapeutic effect of FGF10 given the effects of treatment on IPF fibroblasts.Eine in-vivo erhöhte Expression von Fibroblasten-Wachstumsfaktor 10 (FGF10) und exogene Verabreichung von rekombinanten FGF7 Protein fördert über Signalwege des Tyrosinkinase-abhängigen Fibroblastenwachstumsfaktor-Rezeptors r2b (Fgfr2b) an das Lungenepithel die Lungenregeneration nach Bleomycin-induzierter Lungenfibrose. Angesichts der prophylaktischen Wirkungen von FGF7 und therapeutischen Wirkungen von FGF10 für die Bleomycin-induzierte Lungenschädigung im Mausmodell, lautet unsere Hypothese, dass die Aktivierung des endogenen FGFR2b Signalweges in diesem Zusammenhang eine kritische Rolle spielt. Darüber hinaus, wird in neuen klinischen Studien zur Behandlung von idiopathischer pulmonaler Fibrose (IPF) die Blockade von Tyrosin-Kinasen untersucht. Die vorliegende Arbeit hat folgendes zum Ziel: 1) Das Ausmaß der pulmonalen FGF10 und FGF7 Signalwege im Endstadium von IPF zu beurteilen, 2) Das Ausmaß der Aktivierung des endogenem FGFR2b Signalweges nach Bleomycin-induzierte Lungenschädigung bei Mäusen zu evaluieren und 3) Den Effekt der Behandlung von IPF Fibroblasten mit FGF10 in-vitro zu untersuchen. Im Vergleich zum Spender (Kontrollgruppe ohne IPF), waren FGF7 und FGF10 Transkripte in Lungenhomogenaten von IPF Patienten im Endstadium erhöht. Allerdings waren die Expression der Rezeptoren sowie nachgeschaltete Signalwege von FGF7 und 10 signifikant erniedrigt. Im Gegensatz dazu wiesen Wildtyp-Mäuse, die ab Tag 14 nach Bleomycin-induzierte Lungenschädigung eine spontane Lungenregeneration zeigten, eine Aktivierung des FGF10 Signalweges auf. Dies weist darauf hin, dass der FGF 10 Signalweg eine potentielle Rolle während der Lungenregeneration spielen könnte. Unter den drei verwendeten verschiedenen genetischen Mauslinien mit kongenital reduziertem endogenem FGFR2b Signalweg, stellten wir fest, dass die konstitutiv FGF10 defizienten Mäuse den höchsten Trend zur vermehrten Fibrosebildung zeigten. Überraschenderweise, zeigte sich in einer vierten transgenen Mauslinie, in der die Expression eines dominant negativen FGFR2b Rezeptor induziert werden kann, in der Gruppe mit induzierte Blockade des FGFR2B Signalweges im Vergleich zur Kontrollgruppe keine vermehrte Fibrosebildung. Schlußfolgern einerseits wird, dass der FGFR2b Signalweg bei der spontanen Regeneration rekrutiert wird und in nicht-regenerierenden IPF-Patienten im Endstadium fehlreguliert ist. Andererseits, scheint neben dem endogenen FGFR2b Signalweg andere regenerative Signalwege zu existieren, da eine Blockade dieser nicht zur vermehrten Fibrosebildung führte. Obwohl es kann mit anderen Reparaturwege bei Mäusen redundant sein, als mildernde es nicht in einem deutlichen Anstieg der Bleomycin-induzierten Fibrose führen. IPF Fibroblasten reagierten auf die in-vitro FGF10 Behandlung mit eine Reduktion der Zellgröße sowei eine Erhöhung der Proliferation und der Expression von Marker für Lipofibroblasten. Darüber hinaus führte die in-vitro Behandlung mit FGF10 zur Hemmung des TGF-ß (transformierenden Wachstumsfaktor beta ) Signalweges, was an der Reduktion des nachgeschalteten Signalproteins pSMAD3 gezeigt werden konnte. Die Fähigkeit von FGF10, die Zellgröße der IPF Fibroblasten zu reduzieren und den TGF-β Signalweg zu hemmen, deutet darauf hin, dass FGF10 womöglich in der Lage sein könnte, die Veränderungen der Fibroblasten vom kontraktilen zum synthetischen Phänotyp zu fördern. Dies stellt ein wichtiger Schritt in der Regeneration der UIP (Usual Interstitial Pneumonia) Läsionen dar. Zusammenfassend handelt die vorliegende Arbeit von 1) der Abschächung des endogenen FGFR2b Signalweges im Endstadium von IPF Patienten, 2) der Rekrutierung des FGFR2b Signalweges im Rahmen der spontanen Regeneration nach Bleomycin-induzierter Lungenschädigung bei Wildtyp-Mäusen und 3) der potentiellen therapeutisch Wirkung von FGF10 auf der Grundlage der in-vitro Daten an IPF Fibroblasten

Aerobic Exercise Attenuated Bleomycin-Induced Lung Fibrosis in Th2-Dominant Mice

Introduction The aim of this study was to investigate the effect of aerobic exercise (AE) in reducing bleomycin- induced fibrosis in mice of a Th2-dominant immune background (BALB/c). Methods BALB/c mice were distributed into: sedentary, control (CON), Exercise-only (EX), sedentary, bleomycin-treated (BLEO) and bleomycin-treated+exercised (BLEO+EX);(n = 8/group). Following treadmill adaptation, 15 days following a single, oro-tracheal administration of bleomycin (1.5U/kg), AE was performed 5 days/week, 60min/day for 4 weeks at moderate intensity (60% of maximum velocity reached during a physical test) and assessed for pulmonary inflammation and remodeling, and cytokine levels in bronchoalveolar lavage (BAL). Results At 45 days post injury, compared to BLEO, BLEO+EX demonstrated reduced collagen deposition in the airways (p<0.001) and also in the lung parenchyma (p<0.001). In BAL, a decreased number of total leukocytes (p<0.01), eosinophils (p<0.001), lymphocytes (p<0.01), macrophages (p<0.01), and neutrophils (p<0.01), as well as reduced pro-inflammatory cytokines (CXCL-1;p<0.01), (IL-1 beta;p<0.001), (IL-5;p<0.01), (IL-6;p<0.001), (IL-13;p<0.01) and pro-fibrotic growth factor IGF-1 (p<0.001) were observed. Anti-inflammatory cytokine IL-10 was increased (p<0.001). Conclusion AE attenuated bleomycin-induced collagen deposition, inflammation and cytokines accumulation in the lungs of mice with a predominately Th2-background suggesting that therapeutic AE (15-44 days post injury) attenuates the pro-inflammatory, Th2 immune response and fibrosis in the bleomycin model

Aerobic exercise inhibits acute lung injury: from mouse to human evidence Exercise reduced lung injury markers in mouse and in cells

Acute respiratory distress syndrome (ARDS) is defined as hypoxemic respiratory failure with intense pulmonary inflammation, involving hyperactivation of endothelial cells and neutrophils. Given the anti-inflammatory effects of aerobic exercise (AE), this study investigated whether AE performed daily for 5 weeks would inhibit extra-pulmonary LPS-induced ARDS. C57Bl/6 mice were distributed into Control, Exercise, LPS and Exercise+ LPS groups. AE was performed on a treadmill for 5x/week for four weeks before LPS administration. 24hours after the final AE physical test, animals received 100ug of LPS intra-peritoneally. In addition, whole blood cell culture, neutrophils and human endothelial cells were pre-incubated with IL-10, an anti-inflammatory cytokine induced by exercise. AE reduced total protein levels (p<0.01) and neutrophil accumulation in bronchoalveolar lavage (BAL) (p<0.01) and lung parenchyma (p<0.01). AE reduced BAL inflammatory cytokines IL-1 beta, IL-6 and GM-CSF (p<0.001), CXCL1/KC, IL-17, TNF-alpha and IGF-1 (p<0.01). Systemically, AE reduced IL-1 beta, IL-6 and IFN-gamma (p<0.001), CXCL1/KC (p<0.01) and TNF-alpha (p<0.05). AE increased IL-10 levels in serum (p<0.001) and BAL (p<0.001). Furthermore, AE increased superoxide dismutase SOD (p<0.01) and decreased superoxide anion accumulation in the lungs (p<0.01). Lastly, pre-incubation with IL-10 significantly reduced LPS-induced activation of whole blood cells, neutrophils and HUVECs, as observed by reduced production of IL-1 beta, IL-6, IL-8 and TNF-alpha. Our data suggest that AE inhibited LPS-induced lung inflammation by attenuating inflammatory cytokines and oxidative stress markers in mice and human cell culture via enhanced IL-10 production.Sao Paulo Research Foundation (FAPESP) [2012/15165-2]Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) [311335-2015-2]Comissao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) [12804/13-4, 1303/13-9]FAPESP [2013/24076-6, 2014/23196-0, 2012/14604-8, 2012/25435-7, 2012/24880-7]CAPESNove Julho Univ, Sao Paulo, SP, BrazilBrazilian Inst Teaching & Res Pulm & Exercise Imm, Sao Jose Dos Campos, SP, BrazilFed Univ Sao Paulo UNIFESP, Postgrad Program Sci Human Movement & Rehabil, Santos, SP, BrazilUniv Brasil, Sao Paulo, SP, BrazilUniv Sao Paulo, Sch Med, Dept Pathol LIM 59, Sao Paulo, SP, BrazilUniv Fed Lavras UFLA, Sci Dept Hlth, Lavras, MG, BrazilFed Univ Sao Paulo UNIFESP, Campus Sao Paulo, Sao Paulo, SP, BrazilHarbor UCLA Med Ctr, Div Resp & Crit Care Physiol & Med, Los Angeles Biomed Res Inst, Torrance, CA 90509 USAUniv Tubingen, Inst Clin & Expt Transfus Med IKET, Tubingen, GermanyFed Univ Sao Paulo UNIFESP, Postgrad Program Sci Human Movement & Rehabil, Santos, SP, BrazilFed Univ Sao Paulo UNIFESP, Campus Sao Paulo, Sao Paulo, SP, BrazilFAPESP [2012/15165-2]CNPq [311335-2015-2]CAPES [12804/13-4, 1303/13-9]FAPESP [2013/24076-6, 2014/23196-0, 2012/14604-8, 2012/25435-7, 2012/24880-7]Web of Scienc

Characterization of a Novel Fibroblast Growth Factor 10 (Fgf10) Knock-In Mouse Line to Target Mesenchymal Progenitors during Embryonic Development

Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10LacZ), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10Cre-ERT2 knock-in line (Fgf10iCre) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10iCre; Tomatoflox double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10iCre line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10iCre line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner

Characterization of a novel fibroblast growth factor 10 (Fgf10) knock-in mouse line to target mesenchymal progenitors during embryonic development.

Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10(LacZ)), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10(Cre-ERT2) knock-in line (Fgf10(iCre)) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10(iCre); Tomato(flox) double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10(iCre) line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10(iCre) line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner

Aerobic Exercise Attenuated Bleomycin-Induced Lung Fibrosis in Th2-Dominant Mice

Introduction The aim of this study was to investigate the effect of aerobic exercise (AE) in reducing bleomycin- induced fibrosis in mice of a Th2-dominant immune background (BALB/c). Methods BALB/c mice were distributed into: sedentary, control (CON), Exercise-only (EX), sedentary, bleomycin-treated (BLEO) and bleomycin-treated+exercised (BLEO+EX);(n = 8/group). Following treadmill adaptation, 15 days following a single, oro-tracheal administration of bleomycin (1.5U/kg), AE was performed 5 days/week, 60min/day for 4 weeks at moderate intensity (60% of maximum velocity reached during a physical test) and assessed for pulmonary inflammation and remodeling, and cytokine levels in bronchoalveolar lavage (BAL). Results At 45 days post injury, compared to BLEO, BLEO+EX demonstrated reduced collagen deposition in the airways (p<0.001) and also in the lung parenchyma (p<0.001). In BAL, a decreased number of total leukocytes (p<0.01), eosinophils (p<0.001), lymphocytes (p<0.01), macrophages (p<0.01), and neutrophils (p<0.01), as well as reduced pro-inflammatory cytokines (CXCL-1;p<0.01), (IL-1 beta;p<0.001), (IL-5;p<0.01), (IL-6;p<0.001), (IL-13;p<0.01) and pro-fibrotic growth factor IGF-1 (p<0.001) were observed. Anti-inflammatory cytokine IL-10 was increased (p<0.001). Conclusion AE attenuated bleomycin-induced collagen deposition, inflammation and cytokines accumulation in the lungs of mice with a predominately Th2-background suggesting that therapeutic AE (15-44 days post injury) attenuates the pro-inflammatory, Th2 immune response and fibrosis in the bleomycin model

Twisted gastrulation limits apoptosis in the distal region of the mandibular arch in mice

The mandibular arch (BA1) is critical for craniofacial development. The distal region of BA1, which gives rise to most of the mandible, is dependent upon an optimal level of bone morphogenetic protein (BMP) signaling. BMP activity is modulated in the extracellular space by BMP-binding proteins such as Twisted gastrulation (TWSG1). Twsg1(-/-) mice have a spectrum of craniofacial phenotypes, including mandibular defects that range from micrognathia to agnathia. At E9.5, the distal region of the mutant BA1 was prematurely and variably fused with loss of distal markers eHand and Msx1. Expression of proximal markers Fgf8 and Barx1 was expanded across the fused BA1. The expression of Bmp4 and Msx2 was preserved in the distal region, but shifted ventrally. While wild type embryos showed a gradient of BMP signaling with higher activity in the distal region of BA1, this gradient was disrupted and shifted ventrally in the mutants. Thus, loss of TWSG1 results in disruption of the BMP4 gradient at the level of signaling activity as well as mRNA expression. Altered distribution of BMP signaling leads to a shift in gene expression and increase in apoptosis. The extent of apoptosis may account for the variable degree of mandibular defects in Twsg1 mutants

Comparison of the antifibrotic effects of the pan-histone deacetylase-inhibitor panobinostat versus the IPF-drug pirfenidone in fibroblasts from patients with idiopathic pulmonary fibrosis.

BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro. METHODS:Primary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined. RESULTS:Treatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9. CONCLUSIONS:We conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF

Tomato expression in E18.5 <i>Fgf10^iCre/+; Tomato^flox/+</i> embryos.

<p>Recombination was induced at E15.5 by a single IP injection of tamoxifen. Note the absence of Tomato expression in <i>Fgf10^+/+; Tomato^flox/+</i> embryos (<b>A–D</b>). Tomato-positive cells are detected in the ear, skin, limbs and cecum (<b>A’–D’</b>). (<b>A”–D”)</b> Higher magnifications of dotted boxes in <b>A’, B’, C’, D’</b>. <i>n = 3</i>. Tam: tamoxifen.</p