Adult Lung Fibroblasts Research Articles

Development of a small cell lung cancer organoid model to study cellular interactions and survival after chemotherapy.

Introduction: Small-cell-lung-cancer (SCLC) has the worst prognosis of all lung cancers because of a high incidence of relapse after therapy. While lung cancer is the second most common malignancy in the US, only about 10% of cases of lung cancer are SCLC, therefore, it is categorized as a rare and recalcitrant disease. Therapeutic discovery for SCLC has been challenging and the existing pre-clinical models often fail to recapitulate actual tumor pathophysiology. To address this, we developed a bioengineered 3-dimensional (3D) SCLC co-culture organoid model as a phenotypic tool to study SCLC tumor kinetics and SCLC-fibroblast interactions after chemotherapy. Method: We used functionalized alginate microbeads as a scaffold to mimic lung alveolar architecture and co-cultured SCLC cell lines with primary adult lung fibroblasts (ALF). We found that SCLCs in the model proliferated extensively, invaded the microbead scaffold and formed tumors within just 7days. We compared the bioengineered tumors with patient tumors and found them to recapitulate the pathology and immunophenotyping of the patient tumors. When treated with standard chemotherapy drugs, etoposide and cisplatin, we observed that some of the cells survived the chemotherapy and reformed the tumor in the organoid model. Result and Discussion: Co-culture of the SCLC cells with ALFs revealed that the fibroblasts play a key role in inducing faster and more robust SCLC cell regrowth in the model. This is likely due to a paracrine effect, as conditioned media from the same fibroblasts could also support this accelerated regrowth. This model can be used to study cell-cell interactions and the response to chemotherapy in SCLC and is also scalable and amenable to high throughput phenotypic or targeted drug screening to find new therapeutics for SCLC.

Effects of bone morphogenetic protein 4 on TGF-β1-induced cell proliferation, apoptosis, activation and differentiation in mouse lung fibroblasts via ERK/p38 MAPK signaling pathway.

Fibroblasts, in particular myofibroblasts, are the critical effector cells in idiopathic pulmonary fibrosis (IPF), a deadly lung disease characterized by abnormal lung remodeling and the formation of “fibroblastic foci”. Aberrant activation of TGF-β1 is frequently encountered and promotes fibroblast proliferation, activation, and differentiation in pulmonary fibrosis. Hence, the inhibition of TGF-β1-induced lung fibroblast activation holds promise as a therapeutic strategy for IPF. The present study aimed to investigate the potential effect and underlying mechanisms of bone morphogenetic protein 4 (BMP4) on TGF-β1-induced proliferation, apoptosis, activation and myofibroblast differentiation of adult lung fibroblasts. Here, we demonstrated that BMP4 expression was significantly decreased in TGF-β1-stimulated mouse primary lung fibroblasts (PLFs). BMP4 inhibited proliferation and apoptosis resistance of TGF-β1-stimulated mouse PLFs. BMP4 suppressed TGF-β1-induced fibroblast activation and differentiation in mouse PLFs. We also found that BMP4 inhibited TGF-β1-induced ERK and p38 MAPK phosphorylation. Our findings indicate that BMP4 exerts its anti-fibrotic effects by regulating fibroblast proliferation, apoptosis, activation and differentiation via the inhibition of the ERK/p38 MAPK signaling pathway, and thus has a potential for the treatment of pulmonary fibrosis.

Loss of p19Arf promotes fibroblast survival during leucine deprivation

ABSTRACTFibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated, however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here, we show that prolonged leucine deprivation results in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation. This article has an associated First Person interview with the first author of the paper.

Pregnancy-associated plasma protein-A (PAPP-A) is a key component of an interactive cellular mechanism promoting pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with few effective treatment options. We found a highly significant correlation between pregnancy-associated plasma protein (PAPP)-A expression in IPF lung tissue and disease severity as measured by various pulmonary and physical function tests. PAPP-A is a metalloproteinase that enhances local insulin-like growth factor (IGF) activity. We used primary cultures of normal adult human lung fibroblasts (NHLF) to test the hypothesis that PAPP-A plays an important role in the development of pulmonary fibrosis.Treatment of NHLF with pro-fibrotic transforming growth factor (TGF)-β stimulated marked increases in IGF-I mRNA expression (>20-fold) and measurable IGF-I levels in 72-hconditioned medium (CM). TGF-β treatment also increased PAPP-A levels in CM fourfold (p = 0.004) and proteolytic activity ~2-fold. There was an indirect effect of TGF-β to stimulate signaling through the PI3K/Akt pathway, which was significantly inhibited by both IGF-I-inactivating and PAPP-A inhibitory antibodies.Induction of senescence in NHLF increased PAPP-A levels in CM 10-fold (p = 0.006) with attendant increased proteolytic activity. Thus, PAPP-A is a novel component of the senescent lung fibroblast secretome. In addition, NHLF secreted extracellular vehicles (EVs) with surface-bound active PAPP-A that were increased fivefold with senescence.Regulation of PAPP-A and IGF signaling by TGF-β and cell senescence suggests an interactive cellular mechanism underlying the resistance to apoptosis and the progression of fibrosis in IPF. Furthermore, PAPP-A-associated EVs may be a means of pro-fibrotic, pro-senescent communication with other cells in the lung and, thus, a potential therapeutic target for IPF.

Regulator of Cell Cycle Protein (RGCC/RGC-32) Protects against Pulmonary Fibrosis.

Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either RGCC (regulator of cell cycle) or RGC-32 (response gene to complement 32 protein). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with transforming growth factor (TGF)-β in cell cultures elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-β on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased invivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-β stimulation, induced notable transcriptomic changes that negated the effects of TGF-β, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-β-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.

Activation of COX‐2/PGE 2 Pathway Is Not Involved in Dedifferentiation of Cardiac Myofibroblasts Induced by Phorbol 12‐myristate 13‐acetate

The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGF-β1 is one of the essential molecules that promotes transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. In our previous studies, Phorbol 12-Myristate 13-Acetate (PMA) induces reversal of myofibroblast differentiation via protein kinase C (PKC)-independent mechanism. Prostaglandin E2 (PGE2) has been shown to reserve differentiation of myofibroblasts of fetal and adult lung fibroblasts. The role of PGE2 in cardiac myofibroblast dedifferentiation remains unknown. Human cardiac fibroblasts were cultured in fibroblast medium (FM)-2. TGF-β1 (2ng/mL) was added to FM-2 for 48 hours to convert fibroblasts into myofibroblasts. PMA (50 ng/mL) or PGE2 (500 nM) was added into cultured cells for 48 hours, respectively. Expression of α-smooth muscle action (SMA), a biomarker of myofibroblasts, and fibroblast specific protein 1 (FSP-1), the biomarker of fibroblasts, were detected by using western blotting and immunofluorescence. To explore the involvement of cyclooxygenase 2 (COX-2)/PGE2 pathway in PMA-induced reversal of cardiac myofibroblast differentiation, NS-398, the selective COX-2 inhibitor, and PF-04418948, a selective PGE2 receptor antagonist, were applied. Endogenous levels of PGE2 in cardiac myofibroblasts were detected by using Elisa assay kit. TGF-β1 promoted conversion of cardiac fibroblasts to myofibroblasts as evidenced by increased expression of α-SMA and reduced expression of FSP-1. Treatment with PMA dose-dependently attenuated expression of de novo myofibroblasts. Both NS-398 and PF-04418948 exerted no effects on PMA-induced reversal of cardiac myofibroblasts. Addition of PGE2 into culture medium had no effect on expression of α-SMA from myofibroblasts. PMA dose-dependently enhanced formation of PGE2 levels in cardiac myofibroblasts. In conclusion, PMA-induced reversal of cardiac myofibroblast is independent of activation of COX-2 and PGE2 pathway. The mechanism in PMA-induced reversal of cardiac myofibroblasts remains to be further explored.

Deubiquitinase USP13 promotes extracellular matrix expression by stabilizing Smad4 in lung fibroblast cells

Smad4 plays a central role in the regulation of extracellular matrix (ECM) protein expression and cell differentiation; however, the molecular regulation of Smad4 protein stability by a deubiquitinase has not been reported. In the current study, we reveal that a deubiquitinase USP13 stabilizes Smad4, ultimately modulating ECM protein expression in lung fibroblast cells. USP13 was increased in primary adult lung fibroblasts isolated from bleomycin-challenged mice and transforming growth factor (TGF)-β1-treated primary mouse lung fibroblasts. In a bleomycin-induced murine model of lung fibrosis, USP13-deficientmiceshowed reduced ECM levels such as fibronectin (FN) and collagen compared with wild-type mice. The reductions in both protein levels and mRNA expression of ECM were observed in the isolated lung fibroblasts from USP13-deficient mice, suggesting that downregulation of USP13 reduces ECM levels through inhibiting its transcription. To investigate the molecular mechanisms by which USP13 modulates ECM expression, we focused on the role of USP13 on Smad4 expression. Overexpression of USP13 increased FN and Smad4 protein levels in lung fibroblasts, while downregulation ofUSP13 reduced Smad4 protein levels, without altering Smad4 mRNA expression, suggesting that USP13 regulates Smad4 protein stability. Knockdown of USP13 decreased Smad4 half-life and promoted Smad4 ubiquitination. Both Smad4 and USP13 were co-localized in the cytoplasm in treated cell, and co-translocated into the nucleus in response to TGF-β1.The results indicate that USP13 promotes ECM expression by stabilizing Smad4 in lung fibroblasts and plays a role in the maintenance of the extracellular matrix in lungs.

Long Noncoding RNA FENDRR Exhibits Antifibrotic Activity in Pulmonary Fibrosis.

Abnormal activation of lung fibroblasts contributes to the initiation and progression of idiopathic pulmonary fibrosis (IPF). The objective of the present study was to investigate the role of fetal-lethal noncoding developmental regulatory RNA (FENDRR) in the activation of lung fibroblasts. Dysregulated long noncoding RNAs in IPF lungs were identified by next-generation sequencing analysis from the two online datasets. FENDRR expression in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis was determined by quantitative real-time PCR. IRP1 (iron-responsive element-binding protein 1), a protein partner of FENDRR, was identified by RNA pulldown-coupled mass spectrometric analysis and confirmed by RNA immunoprecipitation. The interaction region between FENDRR and IRP1 was determined by cross-linking immunoprecipitation. The in vivo role of FENDRR in pulmonary fibrosis was studied using adenovirus-mediated gene transfer in mice. The expression of FENDRR was downregulated in fibrotic human and mouse lungs as well as in primary lung fibroblasts isolated from bleomycin-treated mice. TGF-β1 (transforming growth factor-β1)-SMAD3 signaling inhibited FENDRR expression in lung fibroblasts. FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts and bound IRP1, suggesting its role in iron metabolism. FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation by reducing iron concentration and acting as a competing endogenous RNA of the profibrotic microRNA-214. Adenovirus-mediated FENDRR gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that FENDRR is an antifibrotic long noncoding RNA and a potential therapeutic target for pulmonary fibrosis.

Transcriptional regulation of the IL-13R\u03b12 gene in human lung fibroblasts

Interleukin (IL)−13 is a type 2 cytokine with important roles in allergic diseases, asthma, and tissue fibrosis. Its receptor (R) α1 is primarily responsible for the biological actions of this cytokine, while Rα2 possesses a decoy function which can block IL-13 signaling. Although the expression of Rα2 is known to be subject to modulation, information about its transcriptional regulation is limited. In this study, we sought to expand the understanding of transcriptional control of Rα2 in lung fibroblasts. We confirmed previous reports that IL-13 elicited modest induction of Rα2 in normal adult human lung fibroblasts, but found that prostaglandin E2 (PGE2) and fibroblast growth factor 2 (FGF-2) –mediators known to influence fibroblast activation in tissue fibrosis but not previously investigated in this regard – led to a much greater magnitude of Rα2 induction. Although both PGE2 (via protein kinase A) and FGF-2 (via protein kinase B, also known as AKT) depended on activation of cAMP-responsive element-binding protein (CREB) for induction of Rα2 expression, they nevertheless demonstrated synergy in doing so, likely attributable to their differential utilization of distinct transcriptional start sites on the Rα2 promoter. Our data identify CREB activation via PGE2 and FGF-2 as a previously unrecognized molecular controller of Rα2 gene induction and provide potential new insights into strategies for therapeutic manipulation of this endogenous brake on IL-13 signaling.

Reduced microRNA-503 expression augments lung fibroblast VEGF production in chronic obstructive pulmonary disease.

Alterations in microRNA (miRNA) expression may contribute to COPD pathogenesis. In COPD, lung fibroblast repair functions are altered in multiple ways, including extracellular mediator release. Our prior study revealed miR-503 expression is decreased in COPD lung fibroblasts, although the exact role played by miR-503 is undetermined. The current study examined a role of miR-503 in cytokine, growth factor and fibronectin production by lung fibroblasts from patients with and without COPD. Primary adult lung fibroblasts were isolated from patients with or without COPD. MiR-503 expression and interleukin (IL)-6, -8, PGE2, HGF, KGF, VEGF and fibronectin release were examined with or without inflammatory cytokines, IL-1β and tumor necrosis factor (TNF)-α. MiR-503 expression was decreased in COPD lung fibroblasts. The expression of miR-503 was positively correlated with %FVC, %FEV1, and %DLco as well as IL-6, -8, PGE2, HGF, KGF, and VEGF in the absence or presence of IL-1ß/TNF-α. In addition, IL-8 and VEGF release from COPD lung fibroblasts were increased compared to those from control. Exogenous miR-503 inhibited VEGF release from primary adult and fetal lung fibroblasts but not IL-8 release. As expected, COPD fibroblasts proliferated more slowly than control fibroblasts. MiR-503 did not affect proliferation of either control or COPD lung fibroblasts. MiR-503 inhibition of VEGF protein production and mRNA was mediated by direct binding to the 3’ untranslated region of VEGF mRNA. Endogenous miR-503 was differently regulated by exogenous stimulants associated with COPD pathogenesis, including IL-1ß/TNF-α, TGF-ß1 and PGE2. Endogenous miR-503 inhibition augmented VEGF release by IL-1ß/TNF-α and TGF-ß1 but not by PGE2, demonstrating selectivity of miR-503 regulation of VEGF. In conclusions, reduced miR-503 augments VEGF release from lung fibroblasts from patients with COPD. Since VEGF contributes to disturbed vasculature in COPD, altered miR-503 production might play a role in modulating fibroblast-mediated vascular homeostasis in COPD.

Human airway organoid engineering as a step toward lung regeneration and disease modeling.

Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-β1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues.

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice.

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated β-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders.

CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells.

Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid‐regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal–epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/− mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal–epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)‐induced human fetal lung fibroblast line (MRC5). LPS‐induced upregulation of the proinflammatory cytokines IL‐8 and CCL2 was exacerbated in MRC5‐CRISPLD2 knockdown cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL‐8, IL‐6, CCL2. LPS‐stimulated expression of proinflammatory mediators by human lung epithelial HAEo‐ cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF‐CRISPLD2 knockdown suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood.

Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors

Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.

The IκB kinase inhibitor ACHP strongly attenuates TGFβ1-induced myofibroblast formation and collagen synthesis.

Excessive accumulation of a collagen-rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGFβ1) is a strong inducer of myofibroblast formation and subsequent collagen production. Currently, there are no remedies for the treatment of fibrosis. Activation of the nuclear factor kappa B (NF-κB) pathway by phosphorylating IκB with the enzyme IκB kinase (IKK) plays a major role in the induction of fibrosis. ACHP {2-Amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3 pyridinecarbonitrile}, a selective inhibitor of IKK, prohibits the activation of the NF-κB pathway. It is not known whether ACHP has potential anti-fibrotic properties. Using adult human dermal and lung fibroblasts we have investigated whether ACHP has the ability to inhibit the TGFβ1-induced transition of fibroblasts into myofibroblasts and its excessive synthesis of ECM. The presence of ACHP strongly suppressed the induction of the myofibroblast markers alpha-smooth muscle actin (αSMA) and SM22α, as well as the deposition of the ECM components collagen type I and fibronectin. Furthermore, post-treatment with ACHP partly reversed the expression of αSMA and collagen type I production. Finally, ACHP suppressed the expression of the three collagen-modifying enzymes lysyl hydroxylase (PLOD1, PLOD2 and PLOD3) in dermal fibroblasts, but did not do so in lung fibroblasts. We conclude that the IKK inhibitor ACHP has potent antifibrotic properties, and that the NF-κB pathway plays an important role in myofibroblast biology.

Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

The stimulating effect of interleukin-33 on proliferation of the primary human lung fibroblasts

Interleukin (IL)-33 is a multifunctional cytokine that belongs to the IL-1 cytokine family and expressed by multiple organs and cell types. Recent studies have showed that IL-33 plays an etiological role in several fibrotic disorders and may be involved in the pathogenesis of chronic respiratory diseases. It has been reported that IL-33–induced cutaneous fibrosis is associated with the increased fibroblast proliferation and altered expression of extracellular matrix-modifying genes. However, the role of IL-33 in regulating of functions of lung fibroblasts remains unclear. In the present study we examined the effect of IL-33 on proliferation of human lung fibroblasts. Five primary lines of normal adult human lung fibroblasts were cultured for 3-7 days in the presence of increasing concentrations of IL-33. We have observed that normal human lung fibroblasts responded in a dose-dependent manner to treatment with recombinant human IL-33 by increasing proliferation rates 1.5- to 2.3 fold compared to the non-stimulated control. The maximum effect of IL-33 on fibroblast proliferation was observed in the cytokine concentrations range from 2 ng/ml to 100 ng/ml. These results suggest that IL-33 may play an important role in the regulation of the human lung fibroblast proliferation. Human lung fibroblasts activated by IL-33 may act as effector cells not only in the pathogenesis of lung diseases, but also in lung remodeling processes.

Epithelial-mesenchymal co-culture model for studying alveolar morphogenesis

Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.

Fibroblasts that resist cigarette smoke-induced senescence acquire profibrotic phenotypes.

This study assessed the effect of extended exposure to cigarette smoke extract (CSE) on tissue repair functions in lung fibroblasts. Human fetal (HFL-1) and adult lung fibroblasts were exposed to CSE for 14 days. Senescence-associated β-galactosidase (SA β-gal) expression, cell proliferation, and tissue repair functions including chemotaxis and gel contraction were assessed. HFL-1 proliferation was inhibited by CSE and nearly half of the CSE-exposed cells were SA β-gal positive after 14 days exposure, whereas 33% of adult lung fibroblasts were SA β-gal positive in response to 10% CSE exposure. The SA β-gal-positive cells did not proliferate as indicated by bromodeoxyuridine incorporation. In contrast, cells negative for SA β-gal after CSE exposure proliferated faster than cells never exposed to CSE. These nonsenescent cells migrated more and contracted collagen gels more than control cells. CSE exposure stimulated TGF-β1 production, and both inhibition of TGF-β receptor kinase and TGF-β1 siRNA blocked CSE modulation of fibroblast function. Extended exposure to CSE might induce two different fibroblast phenotypes, a senescent and a profibrotic phenotype. The fibroblasts that resist CSE-induced cellular senescence may contribute to the pathogenesis of idiopathic pulmonary fibrosis and could contribute to fibrotic lesions in chronic obstructive pulmonary disease acting through a TGF-β1-mediated pathway. In contrast, the senescent cells may contribute to the pathogenesis of emphysema.

Histamine induces human lung fibroblast-mediated collagen gel contraction via histamine H1 receptor

ABSTRACTBackground: Airway remodeling is implicated in irreversible airflow limitation of refractory asthma, which includes increased smooth muscle mass and subepithelial fibrosis. Activated fibroblasts acquire contractile phenotype to participate in tissue contraction and structural alteration of extracellular matrices. Histamine is a potent mediator of allergic inflammation, substantially involved in asthmatic pathophysiology. Objective: We hypothesized that histamine might play a role in airway remodeling, and investigated its effect on fibroblast-mediated collagen gel contraction. Methods: Fibroblast-mediated collagen gel contraction was studied. Histamine's regulation of collagen gel contraction was characterized by using specific histamine-receptor antagonists, an IP3 receptor antagonist and a PKC inhibitor. Results: Histamine induced contraction of collagen gels embedded with human lung fibroblasts, in a time-dependent manner, and at the concentration more than 10−6 M, both in four primary cultured adult lung fibroblasts and three fetal lung fibroblast cell lines. This effect was attenuated by H1 receptor antagonist, whereas those for H2 to H4 receptors failed to show an inhibitory effect. Furthermore, IP3 receptor-mediated Ca2+ mobilization was implicated in histamine's action on collagen gel contraction. Conclusions: Our results suggest that histamine is involved in airway remodeling through its action on lung fibroblasts, and antihistamine drugs, especially H1 receptor antagonists, might be potentially beneficial for a subset of asthmatic patients.