ABSTRACT
Pulmonary fibrosis (PF) is a clinically severe and commonly fatal complication of Systemic Sclerosis (SSc). Our group has previously reported profibrotic roles for Insulin-like Growth Factor II (IGF-II) and Lysyl Oxidase (LOX) in SSc-PF. We sought to identify downstream regulatory mediators of IGF-II. In the present work, we show that SSc lung tissues have higher baseline levels of the total (N-glycosylated/unglycosylated) LOX-Propeptide (LOX-PP) than control lung tissues. LOX-PP-mediated changes were consistent with the extracellular matrix (ECM) deregulation implicated in SSc-PF progression. Furthermore, Tolloid-like 1 (TLL1) and Bone Morphogenetic Protein 1 (BMP1), enzymes that can cleave ProLOX to release LOX-PP, were increased in SSc lung fibrosis and the bleomycin (BLM)-induced murine lung fibrosis model, respectively. In addition, IGF-II regulated the levels of ProLOX, active LOX, LOX-PP, BMP1, and isoforms of TLL1. The Class E Basic Helix-Loop-Helix protein 40 (BHLHE40) transcription factor localized to the nucleus in response to IGF-II. BHLHE40 silencing downregulated TLL1 isoforms and LOX-PP, and restored features of ECM deregulation triggered by IGF-II. Our findings indicate that IGF-II, BHLHE40, and LOX-PP may serve as targets of therapeutic intervention to halt SSc-PF progression.
Subject(s)
Bone Morphogenetic Protein 1 , Insulin-Like Growth Factor II , Protein-Lysine 6-Oxidase , Pulmonary Fibrosis , Animals , Protein-Lysine 6-Oxidase/metabolism , Protein-Lysine 6-Oxidase/genetics , Insulin-Like Growth Factor II/metabolism , Insulin-Like Growth Factor II/genetics , Mice , Humans , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/genetics , Bone Morphogenetic Protein 1/metabolism , Bone Morphogenetic Protein 1/genetics , Scleroderma, Systemic/pathology , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Extracellular Matrix/metabolism , Lung/metabolism , Lung/pathology , Bleomycin/pharmacology , Disease Models, Animal , Female , MaleABSTRACT
OBJECTIVES: Systemic sclerosis (SSc) has the highest mortality rate among the rheumatic diseases, with lung fibrosis leading as the cause of death. A characteristic of severe SSc-related lung fibrosis is its progressive nature. Although most research has focused on the pathology of the fibrosis, the mechanism mediating the fibrotic spread remains unclear. We hypothesized that extracellular vesicle (EV) communication drives the propagation of SSc lung fibrosis. METHODS: EVs were isolated from normal (NL) or SSc-derived human lungs and primary lung fibroblasts (pLFs). EVs were also isolated from human fibrotic lungs and pLFs induced experimentally with transforming growth factor-ß (TGFß). Fibrotic potency of EVs was assessed using functional assays in vitro and in vivo. Transmission electron microscopy, nanoparticle tracking analysis, real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunofluorescence were used to analyze EVs, their cargo, extracellular matrix (ECM) fractions, and conditioned media. RESULTS: SSc lungs and pLFs released significantly more EVs than NL lungs, and their EVs showed increased fibrotic content and activity. TGFß-stimulated NL lung cores and pLFs increased packaging of fibrotic proteins, including fibronectin, collagens, and TGFß, into released EVs. The EVs induced a fibrotic phenotype in recipient pLFs and in vivo in mouse lungs. Furthermore, EVs interacted with and contributed to the ECM. Finally, suppressing EV release in vivo reduced severity of murine lung fibrosis. CONCLUSIONS: Our findings highlight EV communication as a novel mechanism for propagation of SSc lung fibrosis. Identifying therapies that reduce EV release, activity, and/or fibrotic cargo in SSc patient lungs may be a viable therapeutic strategy to improve fibrosis.
Subject(s)
Extracellular Vesicles , Pulmonary Fibrosis , Scleroderma, Systemic , Humans , Animals , Mice , Pulmonary Fibrosis/pathology , Signal Transduction , Scleroderma, Systemic/pathology , Fibrosis , Lung/pathology , Transforming Growth Factor beta/metabolism , Extracellular Vesicles/pathology , Fibroblasts/metabolismABSTRACT
Systemic sclerosis (SSc), also known as scleroderma, is an autoimmune disorder that affects the connective tissues and has the highest mortality rate among the rheumatic diseases. One of the hallmarks of SSc is fibrosis, which may develop systemically, affecting the skin and virtually any visceral organ in the body. Fibrosis of the lungs leads to interstitial lung disease (ILD), which is currently the leading cause of death in SSc. The identification of effective treatments to stop or reverse lung fibrosis has been the main challenge in reducing SSc mortality and improving patient outcomes and quality of life. Thus, understanding the molecular mechanisms, altered pathways, and their potential interactions in SSc lung fibrosis is key to developing potential therapies. In this review, we discuss the diverse molecular mechanisms involved in SSc-related lung fibrosis to provide insights into the altered homeostasis state inherent to this fatal disease complication.
Subject(s)
Lung Diseases, Interstitial , Pulmonary Fibrosis , Scleroderma, Systemic , Humans , Pulmonary Fibrosis/pathology , Quality of Life , Scleroderma, Systemic/complications , Scleroderma, Systemic/genetics , Lung Diseases, Interstitial/complications , Lung/pathology , FibrosisABSTRACT
OBJECTIVES: Lung fibrosis is the leading cause of death in SSc, with no cure currently available. Antifibrotic Endostatin (ES) production does not reach therapeutic levels in SSc patients, suggesting a deficit in its release from Collagen XVIII by the main cleavage enzyme, Cathepsin L (CTSL). Thus, elucidating a potential deficit in CTSL expression and activity unravels an underlying molecular cause for SSc-driven lung fibrosis. METHODS: Fibrosis was induced experimentally using TGF-ß in vitro, in primary human lung fibroblasts (pLFs), and ex vivo, in human lung tissues. ES and CTSL expression was quantified using ELISA, RT-qPCR, immunoblotting or immunofluorescence. Recombinant NC1-FLAG peptide was used to assess CTSL cleavage activity. CTSL expression was also compared between SSc vs normal (NL)-derived pLFs and lung tissues. RESULTS: ES levels were significantly reduced in media conditioned by TGF-ß-induced pLFs. TGF-ß-stimulated pLFs significantly reduced expression and secretion of CTSL into the extracellular matrix (ECM). CTSL was also sequestered in its inactive form into extracellular vesicles, further reducing its availability in the ECM. Media conditioned by TGF-ß-induced pLFs showed reduced cleavage of NC1-Flag and reduced release of the antifibrotic ES fragment. SSc-derived pLFs and lung tissues expressed significantly lower levels of CTSL compared with NL. CONCLUSIONS: Our findings identify CTSL as a protein protective against lung fibrosis via its activation of antifibrotic ES, and whose expression in SSc pLFs and lung tissues is suppressed. Identifying strategies to boost CTSL endogenous levels in SSc patients could serve as a viable therapeutic strategy.
Subject(s)
Pulmonary Fibrosis , Scleroderma, Systemic , Humans , Cathepsin L/metabolism , Cells, Cultured , Fibroblasts/metabolism , Fibrosis , Pulmonary Fibrosis/pathology , Scleroderma, Systemic/pathology , Skin/pathology , Transforming Growth Factor beta/metabolismABSTRACT
One of the challenges plastic surgeons face is the unpredictability of postoperative scarring. The variability of wound healing and subsequent scar formation across patients makes it virtually impossible to predict if a patient's surgery will result in excessive fibrosis and scarring, possibly amounting to keloids or hypertrophic scars. There is a need to find predictive molecular indicators of patients or skin location with high risk of excessive scarring. We hypothesized that baseline expression levels of fibrotic genes in the skin can serve as a potential indicator of excessive scarring. Methods: An ex vivo model of skin fibrosis was used with abdominal and breast skin tissue from 45 patients undergoing breast reduction and/or abdominoplasty. Fibrosis was induced in skin explants in organ culture with transforming growth factor-ß (TFGß). Fibrotic gene response was assessed via quantitative real-time polymerase chain reaction and correlated with skin location, age, and baseline levels of fibrotic genes. Results: The increase in TFGß-induced fibronectin1 (FN1) gene expression in skin explants was significantly higher than for Collagen 1A1, alpha smooth muscle actin, and connective tissue growth factor. Also, FN1 expression positively correlated with donor age. Moreover, lower expression of the fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin correlated with a more pronounced fibrotic response, represented by higher induction levels of these genes. Conclusions: Skin sites exhibit different baseline levels of profibrotic genes. Further, low baseline expression levels of fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin, in donor skin may indicate a potential for excessive scarring of the skin.
ABSTRACT
Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21Cip1 and p27Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21Cip1 and p27Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.
Subject(s)
Cell Cycle/drug effects , Cell Survival/drug effects , Hydroxamic Acids/therapeutic use , Rhabdomyosarcoma/drug therapy , Animals , Apoptosis , Cell Line, Tumor , Child , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Hydroxamic Acids/pharmacology , Male , MiceABSTRACT
With the increasing levels of atmospheric ozone depletion, there has been much concern about the causal effects of high levels of ultraviolet radiation reaching the Earth's surface on skin cancer. This has led to growing interest in identifying new active ingredients for use in commercial sunscreens. In our study, the chemical compound 2-benzoyl-3-phenylquinoxaline 1,4-dioxide (BPQ) prepared by the Beirut reaction was tested for its ability to protect a human keratinocyte cell line (HaCaT) against ultraviolet B radiation (280-315 nm). We show that BPQ exhibited strong absorbance in the UVB range, with an overall absorption spectrum very similar to that of Padimate-O, a well-known active ingredient used in commercial sunscreens. HaCaT cells, which were irradiated with UVB in the presence of multiple doses of BPQ, exhibited, in a dose-dependent fashion, a significantly higher viability and lower oxidative stress levels than cells irradiated in the absence of drug. Our results show that BPQ is a potential photoprotective drug that holds great promise for use as an active ingredient in commercial sunscreens.