Genetic deficiency of the transcription factor NFAT1 confers protection against fibrogenic responses independent of immune influx.

Idiopathic pulmonary fibrosis (IPF) is marked by unremitting matrix deposition and architectural distortion. Multiple profibrotic pathways contribute to the persistent activation of mesenchymal cells (MCs) in fibrosis, highlighting the need to identify and target common signaling pathways. The transcription factor nuclear factor of activated T cells 1 (NFAT1) lies downstream of second messenger calcium signaling and has been recently shown to regulate key profibrotic mediator autotaxin (ATX) in lung MCs. Herein, we investigate the role of NFAT1 in regulating fibroproliferative responses during the development of lung fibrosis. Nfat1-/--deficient mice subjected to bleomycin injury demonstrated improved survival and protection from lung fibrosis and collagen deposition as compared with bleomycin-injured wild-type (WT) mice. Chimera mice, generated by reconstituting bone marrow cells from WT or Nfat1-/- mice into irradiated WT mice (WT→WT and Nfat1-/-→WT), demonstrated no difference in bleomycin-induced fibrosis, suggesting immune influx-independent fibroprotection in Nfat1-/- mice. Examination of lung tissue and flow sorted lineageneg/platelet-derived growth factor receptor alpha (PDGFRα)pos MCs demonstrated decreased MC numbers, proliferation [↓ cyclin D1 and 5-ethynyl-2'-deoxyuridine (EdU) incorporation], myofibroblast differentiation [↓ α-smooth muscle actin (α-SMA)], and survival (↓ Birc5) in Nfat1-/- mice. Nfat1 deficiency abrogated ATX expression in response to bleomycin in vivo and MCs derived from Nfat1-/- mice demonstrated decreased ATX expression and migration in vitro. Human IPF MCs demonstrated constitutive NFAT1 activation, and regulation of ATX in these cells by NFAT1 was confirmed using pharmacological and genetic inhibition. Our findings identify NFAT1 as a critical mediator of profibrotic processes, contributing to dysregulated lung remodeling and suggest its targeting in MCs as a potential therapeutic strategy in IPF.NEW & NOTEWORTHY Idiopathic pulmonary fibrosis (IPF) is a fatal disease with hallmarks of fibroblastic foci and exuberant matrix deposition, unknown etiology, and ineffective therapies. Several profibrotic/proinflammatory pathways are implicated in accelerating tissue remodeling toward a honeycombed end-stage disease. NFAT1 is a transcriptional factor activated in IPF tissues. Nfat1-deficient mice subjected to chronic injury are protected against fibrosis independent of immune influxes, with suppression of profibrotic mesenchymal phenotypes including proliferation, differentiation, resistance to apoptosis, and autotaxin-related migration.

Rev-erbα agonists suppresses TGFß1-induced fibroblast-to-myofibroblast transition and pro-fibrotic phenotype in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by excessive scarring of the lungs that can lead to respiratory failure and death. Lungs of patients with IPF demonstrate excessive deposition of extracellular matrix (ECM) and an increased presence of pro-fibrotic mediators such as transforming growth factor-beta 1 (TGFß1), which is a major driver of fibroblast-to-myofibroblast transition (FMT). Current literature supports that circadian clock dysfunction plays an essential role in the pathophysiology of various chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and IPF. The circadian clock transcription factor Rev-erbα is encoded by Nr1d1 that regulates daily rhythms of gene expression linked to immunity, inflammation, and metabolism. However, investigations into the potential roles of Rev-erbα in TGFß-induced FMT and ECM accumulation are limited. In this study, we utilized several novel small molecule Rev-erbα agonists (GSK41122, SR9009, and SR9011) and a Rev-erbα antagonist (SR8278) to determine the roles of Rev-erbα in regulating TGFß1-induced FMT and pro-fibrotic phenotypes in human lung fibroblasts. WI-38 cells were either pre-treated/co-treated with or without Rev-erbα agonist/antagonist along with TGFß1. After 48 h, the following parameters were evaluated: secretion of COL1A1 (Slot-Blot analysis) and IL-6 (ELISA) into condition media, expressions of α-smooth muscle actin (αSMA: immunostaining and confocal microscopy), and pro-fibrotic proteins (αSMA and COL1A1 by immunoblotting), as well as gene expression of pro-fibrotic targets (qRT-PCR: Acta2, Fn1, and Col1a1). Results revealed that Rev-erbα agonists inhibited TGFß1-induced FMT (αSMA and COL1A1), and ECM production (reduced gene expression of Acta2, Fn1, and Col1a1), and decreased pro-inflammatory cytokine IL-6 release. The Rev-erbα antagonist promoted TGFß1-induced pro-fibrotic phenotypes. These findings support the potential of novel circadian clock-based therapeutics, such as Rev-erbα agonist, for the treatment and management of fibrotic lung diseases and disorders.

Fatal COVID-19 and Non-COVID-19 Acute Respiratory Distress Syndrome Is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State without Fibrosis.

Acute respiratory distress syndrome (ARDS) due to coronavirus disease 2019 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; recovery requires epithelial regeneration. During physiological regeneration in mice, type 2 AECs (AEC2s) proliferate, exit the cell cycle, transiently assume a transitional state, then differentiate into type 1 AECs (AEC1s); in humans, persistence of the transitional state is associated with pulmonary fibrosis. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiological regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or single-cell RNA sequencing revealed that transitional cells in mouse models of physiological regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.

Dedicator of Cytokinesis 2 (DOCK2) Deficiency Attenuates Lung Injury Associated with Chronic High-Fat and High-Fructose Diet-Induced Obesity.

Obesity is a major risk factor for lung disease development. However, little is known about the impact of chronic high-fat and high-fructose (HFHF) diet-induced obesity on lung inflammation and subsequent pulmonary fibrosis. Herein we hypothesized that dedicator of cytokinesis 2 (DOCK2) promotes a proinflammatory phenotype of lung fibroblasts (LFs) to elicit lung injury and fibrosis in chronic HFHF diet-induced obesity. An HFHF diet for 20 weeks induced lung inflammation and profibrotic changes in wild-type C57BL/6 mice. CD68 and monocyte chemoattractant protein-1 (MCP-1) expression were notably increased in the lungs of wild-type mice fed an HFHF diet. An HFHF diet further increased lung DOCK2 expression that co-localized with fibroblast-specific protein 1, suggesting a role of DOCK2 in regulating proinflammatory phenotype of LFs. Importantly, DOCK2 knockout protected mice from lung inflammation and fibrosis induced by a HFHF diet. In primary human LFs, tumor necrosis factor-α (TNF-α) and IL-1ß induced DOCK2 expression concurrent with MCP-1, IL-6, and matrix metallopeptidase 2. DOCK2 knockdown suppressed TNF-α-induced expression of these molecules and activation of phosphatidylinositol 3-kinase/AKT and NF-κB signaling pathways, suggesting a mechanism of DOCK2-mediated proinflammatory and profibrotic changes in human LFs. Taken together, these findings reveal a previously unrecognized role of DOCK2 in regulating proinflammatory phenotype of LFs, potentiation of lung inflammation, and pulmonary fibrosis in chronic HFHF diet-caused obesity.

Upregulation of dihydropyrimidinase-like 3 (DPYSL3) protein predicts poor prognosis in urothelial carcinoma.

BACKGROUND: Dihydropyrimidinase-like 3 (DPYSL3) is a cytosolic phosphoprotein expressed in the nervous system and is crucial for neurogenesis. A previous study showed that increased DPYSL3 expression promotes tumour aggressiveness in pancreatic ductal adenocarcinoma, gastric cancer, and colon cancer. However, the role of DPYSL3 in affecting the biological behaviour of urothelial carcinoma (UC) is not yet understood. METHODS: A UC transcriptomic dataset from the Gene Expression Omnibus and the Urothelial Bladder Cancer (BLCA) dataset from The Cancer Genome Atlas were used for the in silico study. We collected 340 upper urinary tract urothelial carcinoma (UTUC) and 295 urinary bladder urothelial carcinoma (UBUC) samples for the immunohistochemical study. Fresh tumour tissue from 50 patients was used to examine the DPYSL3 mRNA level. In addition, urothelial cell lines with and without DPYSL3 knockdown were used for the functional study. RESULTS: The in silico study revealed that DPYSL3 correlated with advanced tumour stage and metastasis development while functioning primarily in the nucleobase-containing compound metabolic process (GO:0006139). DPYSL3 mRNA expression is significantly upregulated in advanced UC. Furthermore, overexpression of the DPYSL3 protein is significantly associated with the aggressive behaviour of UTUC and UBUC. DPYSL3 expression independently predicts disease-specific survival (DSS) and metastatic-free survival (MFS) in patients with UC. In non-muscle-invasive UBUC, DPYSL3 expression predicts local recurrence-free survival. UC cell lines with DPYSL3 knockdown exhibited decreased proliferation, migration, invasion, and human umbilical vein endothelial cells (HUVECs) tube formation but increased apoptosis and G1 arrest. Gene ontology enrichment analysis revealed that the enriched processes related to DPYSL3 overexpression in UC were tissue morphogenesis, cell mesenchyme migration, smooth muscle regulation, metabolic processes, and RNA processing. In vivo study revealed DPYSL3 knockdown in UC tumours significantly suppressed the growth of tumours and decreased MYC and GLUT1 protein expression. CONCLUSIONS: DPYSL3 promotes the aggressiveness of UC cells by changing their biological behaviours and is likely associated with cytoskeletal and metabolic process modifications. Furthermore, DPYSL3 protein overexpression in UC was associated with aggressive clinicopathological characteristics and independently predicted poor clinical outcomes. Therefore, DPYSL3 can be used as a novel therapeutic target for UC.

Low expression of ZSCAN4 predicts unfavorable outcome in urothelial carcinoma of upper urinary tract and urinary bladder.

BACKGROUND: With the advance in genome-wide analyses, genetic alternations have been found to play an important role in carcinogenesis and aggressiveness of UC. Through bioinformatic analysis of gene expression profiles of urinary bladder urothelial carcinoma (UBUC) from publicly available GEO dataset (GSE31684), Zinc finger and SCAN domain containing 4 (ZSCAN4) was identified as a significant downregulated gene in muscle-invasive bladder cancer when compared with non-muscle-invasive bladder cancer. METHODS: The expression of ZSCAN4 was evaluated by immunohistochemistry in 340 upper urinary tract urothelial carcinomas (UTUCs) and 295 UBUCs. The expression profiles of ZSCAN4 and potential signaling pathways were analyzed bioinformatically. RESULTS: In UTUC, low expression of ZSCAN4 was significantly associated with advanced primary pT stage (P = 0.011), increased nodal metastasis (P = 0.002) and increased vascular invasion (P = 0.019). In UBUC, low expression of ZSCAN4 was significantly correlated with advanced primary pT stage (P < 0.001), increased nodal metastasis (P = 0.001), high histological grade (P = 0.003) and increased vascular invasion (P = 0.003). In survival analysis, low expression of ZSCAN4 acted as an independent negative prognostic factor for disease-specific survival and metastasis-free survival both in UTUC and UBUC. Gene ontology analysis showed that ZSCAN4 mRNA and its co-downregulated genes are associated with the mitotic cell cycle. CONCLUSIONS: Low expression of ZSCAN4 predicted worse outcome in urothelial carcinoma and might have potential regulatory role in cell mitosis.

Impact of pathological response on oncological outcomes in patients with upper tract urothelial cancer receiving neo-adjuvant chemotherapy.

PURPOSE: The purpose of this study is to evaluate the rates of pathological complete response (ypT0N0/X) and pathological response (ypT1N0/X or less) in patients with upper tract urothelial cancer who were treated with neo-adjuvant chemotherapy and to examine their impact on oncological outcomes. METHODS: This study is a multi-institutional retrospective analysis of patients with high-risk upper tract urothelial cancer who underwent neoadjuvant chemotherapy and radical nephroureterectomy between 2002 and 2021. Logistic regression analyses were used to investigate all clinical parameters for response after neoadjuvant chemotherapy. Cox proportional hazard models were performed to assess the effect of the response on the oncological outcomes. RESULTS: A total of 84 patients with UTUC who received neo-adjuvant chemotherapy were identified. Among them, 44 (52.4%) patients received cisplatin-based chemotherapy, and 22 (26.2%) patients had a carboplatin-based regimen. The pathological complete response rate was 11.6% (n = 10), and the pathological response rate was 42.9% (n = 36). Multifocal tumors or tumors larger than 3 cm significantly reduced the odds of pathological response. In the multivariable Cox proportional hazard model, pathological response was independently associated with better overall survival (HR 0.38, p = 0.024), cancer-specific survival (HR 0.24, p = 0.033), and recurrence-free survival (HR 0.17, p = 0.001), but it was not associated with bladder recurrence-free survival (HR 0.84, p = 0.69). CONCLUSION: Pathological response after neo-adjuvant chemotherapy and radical nephroureterectomy is strongly associated with patient survival and recurrence, and it might be a good surrogate for evaluating the efficacy of neo-adjuvant chemotherapy in the future.

High MT2A Expression Predicts Worse Prognosis in Patients with Urothelial Carcinoma.

Introduction: Dysregulation of metal ion homeostasis is associated with urothelial carcinogenesis. From a published urinary bladder urothelial carcinoma (UBUC) transcriptome, we identified metallothionein 2A (MT2A) as the most significantly upregulated gene implicated in cancer progression among metal ion binding-related genes. Therefore, we analyzed the association between MT2A expression and clinical significance in our well-characterized cohort of patients with upper tract urothelial carcinoma (UTUC) and UBUC. Methods: We retrospectively reviewed the clinicopathological characteristics of 295 and 340 patients with UBUC and UTUC, respectively. MT2A expression was assessed using real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. We further correlated MT2A expression with clinicopathological factors, disease-specific survival (DSS) and metastasis-free survival (MFS) using the Pearson's χ2 test, Kaplan-Meier analysis, and multivariate Cox proportional hazards model. Results: High MT2A expression was significantly associated with aggressive pathological features including high tumor stage, lymph node metastasis, high tumor grade, vascular invasion, and perineural invasion. In the Kaplan-Meier analysis, high MT2A expression was significantly correlated with poor DSS (p < 0.0001) and MFS (p < 0.0001); in the multivariate analysis, it was an independent predictor of CSS (p < 0.001) and MFS (p = 0.001). Gene coexpression analysis demonstrated that MT2A overexpression promotes UC progression through complement activation. Conclusion: High MT2A expression correlated with aggressive UC features and was an independent predictor of cancer metastasis and patient survival, suggesting its role in risk stratification and decision-making in patients with UTUC and UBUC.

Comparison of mini endoscopic combined intrarenal surgery and multitract minimally invasive percutaneous nephrolithotomy specifically for kidney staghorn stones: a single-centre experience.

BACKGROUND: Staghorn stones require surgical treatment to prevent serious complications. Multitract percutaneous nephrolithotomy (PNL) causes great renal parenchymal injury and blood loss. One-stage endoscopic combined intrarenal surgery (ECIRS) entails the combined use of antegrade nephroscope and retrograde flexible ureteroscope to clear the staghorn stone, which may overcome the limitations of multitract PNL. We aimed to compare the perioperative outcomes of mini ECIRS and multitract minimally invasive PNL in staghorn stone management. METHODS: This was a retrospective single-center study of patients with staghorn stones who underwent ECIRS (n = 17) or multitract minimally invasive PNL (n = 17) between January 2018 and September 2021. RESULTS: There was a significant between-group difference with respect to Guy's stone score. Stone size, stone burden (ECIRS group, 21.41 cm3; multitract minimally invasive PNL group, 20.88 cm3 [P = 0.94]), and degree of hydronephrosis were comparable in the two groups. There was no significant between-group difference with respect to one-step or final stone-free rates. The mean operative time was also not significantly different between the groups (ECIRS group, 140 min; multitract minimally invasive PNL group, 183 min [P = 0.63]). ECIRS was associated with significantly lesser postoperative pain (visual analog scale; ECIRS group: 0; multitract minimally invasive PNL group: 2.7 [P < 0.001]). Hemoglobin loss, postoperative blood transfusion rate, complications, and length of hospital stay were comparable in the two groups. CONCLUSION: Both mini ECIRS and multitract minimally invasive PNL were effective and safe for the management of renal staghorn stones with comparable operation time and stone-free rate, and complications. ECIRS was associated with less severe postoperative pain.

Clioquinol Attenuates Pulmonary Fibrosis through Inactivation of Fibroblasts via Iron Chelation.

Strict control of iron homeostasis is critical for the maintenance of normal lung function. Iron accumulates in the lungs of patients with idiopathic pulmonary fibrosis (PF), but the characteristics of iron metabolism in the pathogenesis of PF and related targeting therapeutics are not well studied. In this study, we investigated the cellular and molecular characteristics of iron metabolism in fibrotic lungs and further explored the efficacy of clioquinol (CQ) for the treatment of PF as well as its functional mechanism. Iron aggregates accumulated in the lungs of patients with idiopathic PF, and FTL (ferritin light chain) transcripts were increased in their pulmonary fibroblasts. In the bleomycin (BLM)-induced PF (BLM-PF) mouse model, pulmonary iron accumulation is a very early and concomitant event of PF. Labile iron pool levels in both fibroblasts and macrophages from the BLM-PF model were elevated, and iron metabolism was dysregulated. CQ attenuated PF induced by BLM and FITC, and iron-saturated CQ did not alleviate BLM-PF. Furthermore, CQ inhibited the activation of fibroblasts, including proliferation, fibrotic differentiation, proinflammatory cytokine secretion, and migration. In conclusion, our study demonstrated that CQ, acting as an iron chelator, attenuates experimental PF through inactivation of fibroblasts, providing support for targeting iron metabolism as a basis for PF treatment.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

Angiotensin-converting enzyme 2 expression in COPD and IPF fibroblasts: the forgotten cell in COVID-19.

The COVID-19 pandemic is associated with severe pneumonia and acute respiratory distress syndrome leading to death in susceptible individuals. For those who recover, post-COVID-19 complications may include development of pulmonary fibrosis. Factors contributing to disease severity or development of complications are not known. Using computational analysis with experimental data, we report that idiopathic pulmonary fibrosis (IPF)- and chronic obstructive pulmonary disease (COPD)-derived lung fibroblasts express higher levels of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 entry and part of the renin-angiotensin system that is antifibrotic and anti-inflammatory. In preclinical models, we found that chronic exposure to cigarette smoke, a risk factor for both COPD and IPF and potentially for SARS-CoV-2 infection, significantly increased pulmonary ACE2 protein expression. Further studies are needed to understand the functional implications of ACE2 on lung fibroblasts, a cell type that thus far has received relatively little attention in the context of COVID-19.

High glycosyltransferase 8 domain containing two protein levels contribute to poor prognosis in urothelial carcinoma.

OBJECTIVE: To examine the expression levels of the glycosyltransferase 8 domain containing protein 2 and its clinical implications in urothelial carcinoma patients. METHODS: Data mining, immunohistochemistry together with H-score calculation was carried out to evaluate the glycosyltransferase 8 domain containing protein 2 levels on tissue specimens from urothelial carcinoma patients, retrospectively. Correlations between glycosyltransferase 8 domain containing protein 2 H-score and imperative clinicopathological factors were measured. The indication of glycosyltransferase 8 domain containing protein 2 level on disease-specific and metastasis-free survivals were next analyzed. RESULTS: In upper tract urothelial carcinomas (n = 340) and bladder urothelial carcinomas (n = 295), 170 (50%) and 148 (50%) patients, respectively, were identified to have high glycosyltransferase 8 domain containing protein 2 expression. The glycosyltransferase 8 domain containing protein 2 levels were correlated to several clinicopathological characteristics and patient survival. Upregulation of the glycosyltransferase 8 domain containing protein 2 was correlated to primary tumor (P < 0.001), nodal metastasis (P < 0.001), histological grade (P < 0.001), vascular invasion (P < 0.001), perineural invasion (P < 0.05) and mitotic rate (P < 0.001). High glycosyltransferase 8 domain containing protein 2 levels independently predicted poor disease-specific survival (P = 0.049) and metastasis-free survival (P = 0.008) in upper tract urothelial carcinoma and urinary bladder urothelial carcinoma, respectively. Gene Ontology enrichment analysis additionally showed that multiple biological processes were enriched including "ECM organization" (Gene Ontology:0030198), "extracellular structure organization" (Gene Ontology:0043062), "biological adhesion" (Gene Ontology:0022610), "cell adhesion" (Gene Ontology:0007155), "collagen fibril organization" (Gene Ontology:0030199) and "vasculature development" (Gene Ontology:0001944). CONCLUSIONS: The present findings suggest that upregulation of the glycosyltransferase 8 domain containing protein 2 is an independent and disadvantageous prognosticator in urothelial carcinoma. High glycosyltransferase 8 domain containing protein 2 level might play a crucial role in progression of urothelial carcinoma.

The Role of KCNMB1 and BK Channels in Myofibroblast Differentiation and Pulmonary Fibrosis.

The differentiation of fibroblasts into myofibroblasts is critical for the development of fibrotic disorders, including idiopathic pulmonary fibrosis (IPF). Previously, we demonstrated that fibroblasts from patients with IPF exhibit changes in DNA methylation across the genome that contribute to a profibrotic phenotype. One of the top differentially methylated genes identified in our previous study was KCNMB1, which codes for the ß subunit of the large-conductance potassium (BK, also known as MaxiK or KCa1.1) channel. Here, we examined how the expression of KCNMB1 differed between IPF fibroblasts and normal cells, and how BK channels affected myofibroblast differentiation. Fibroblasts from patients with IPF exhibited increased expression of KCNMB1, which corresponded to increased DNA methylation within the gene body. Patch-clamp experiments demonstrated that IPF fibroblasts had increased BK channel activity. Knockdown of KCNMB1 attenuated the ability of fibroblasts to contract collagen gels, and this was associated with a loss of α-smooth muscle actin (SMA) expression. Pharmacologic activation of BK channels stimulated α-SMA expression, whereas BK channel inhibitors blocked the upregulation of α-SMA. The ability of BK channels to enhance α-SMA expression was dependent on intracellular calcium, as activation of BK channels resulted in increased levels of intracellular calcium and the effects of BK agonists were abolished when calcium was removed. Together, our findings demonstrate that epigenetic upregulation of KCNMB1 contributes to increased BK channel activity in IPF fibroblasts, and identify a newfound role for BK channels in myofibroblast differentiation.

Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease characterized by excessive scarring and fibroblast activation. We previously showed that fibroblasts from patients with IPF are hypermethylated at the CDKN2B gene locus, resulting in decreased CDKN2B expression. Here, we examine how diminished CDKN2B expression in normal and IPF fibroblasts affect fibroblast function, and how loss of CDKN2B contributes to IPF pathogenesis. We first confirmed that protein expression of CDKN2B was diminished in IPF lungs in situ. Loss of CDKN2B was especially notable in regions of increased myofibroblasts and fibroblastic foci. The degree of CDKN2B hypermethylation was particularly elevated in patients with radiographic honeycombing, a marker of more advanced fibrosis, and increased DNA methylation correlated with decreased expression. Although CDKN2B is traditionally considered a cell cycle inhibitor, loss of CDKN2B did not result in an increase in fibroblast proliferation, but instead was associated with an increase in myofibroblast differentiation. An increase in myofibroblast differentiation was not observed when CDKN2A was silenced. Loss of CDKN2B was associated with an increase in the transcription factors serum response factor and myocardin-related transcription factor A, and overexpression of CDKN2B in IPF fibroblasts inhibited myofibroblast differentiation. Finally, decreased CDKN2B expression was noted in fibroblasts from a murine model of fibrosis, and Cdkn2b-/- mice developed greater histologic fibrosis after bleomycin injury. These findings identify a novel function for CDKN2B that differs from its conventional designation as a cell cycle inhibitor and demonstrate the importance of this protein in pulmonary fibrosis.

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli.

BACKGROUND: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-ß1 and substrate stiffness affect fibroblast Fas expression are not well understood. METHODS: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-ß1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. RESULTS: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-ß1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-ß1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-ß1. CONCLUSIONS: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

Successful reuse of a transplanted kidney 9 years after initial transplantation: 4-year follow-up.

BACKGROUND: Kidney transplantation is the preferred renal replacement therapy for patients with end-stage renal disease, but the waiting list for kidneys continues to grow because of a shortage of donor organs. The reuse of transplanted kidneys would seem to be a good approach to expand the pool of available organs. Here, we describe the reuse of a kidney 9 years after the initial transplantation. At 4-year follow-up, the second recipient is showing good renal function. CASE PRESENTATION: In 2005, a kidney was transplanted from a 40-year-old man, who suffered brain death due to an intracranial hemorrhage, into a 45-year-old man. Nine years later, the recipient suffered a ruptured cerebral aneurysm, resulting in brain death. The kidney was re-transplanted into a 40-year-old man with diabetic nephropathy who had received hemodialysis for 5 years. During 4 years of follow-up, the graft has functioned well. CONCLUSIONS: This case demonstrates the successful regrafting of a transplanted kidney. We believe this is the longest period for reuse of kidney after initial transplantation. The outcome suggests that a well-functioning transplanted kidney can be reused years after transplantation.

Epigenetic Regulation of Caveolin-1 Gene Expression in Lung Fibroblasts.

Fibrotic disorders are associated with tissue accumulation of fibroblasts. We recently showed that caveolin (Cav)-1 gene suppression by a profibrotic cytokine, transforming growth factor (TGF)-ß1, contributes to fibroblast proliferation and apoptosis resistance. Cav-1 has been shown to be constitutively suppressed in idiopathic pulmonary fibrosis (IPF), but mechanisms for this suppression are incompletely understood. We hypothesized that epigenetic processes contribute to Cav-1 down-regulation in IPF lung fibroblasts, and after fibrogenic stimuli. Cav-1 expression levels, DNA methylation status, and histone modifications associated with the Cav-1 promoter were examined by PCR, Western blots, pyrosequencing, or chromatin immunoprecipitation assays in IPF lung fibroblasts, normal fibroblasts after TGF-ß1 stimulation, or in murine lung fibroblasts after bleomycin injury. Methylation-specific PCR demonstrated methylated and unmethylated Cav-1 DNA copies in all groups. Despite significant changes in Cav-1 expression, no changes in DNA methylation were observed in CpG islands or CpG island shores of the Cav-1 promoter by pyrosequencing of lung fibroblasts from IPF lungs, in response to TGF-ß1, or after bleomycin-induced murine lung injury, when compared with respective controls. In contrast, the association of Cav-1 promoter with the active histone modification mark, H3 lysine 4 trimethylation, correlated with Cav-1 down-regulation in activated/fibrotic lung fibroblasts. Our data indicate that Cav-1 gene silencing in lung fibroblasts is actively regulated by epigenetic mechanisms that involve histone modifications, in particular H3 lysine 4 trimethylation, whereas DNA methylation does not appear to be a primary mechanism. These findings support therapeutic strategies that target histone modifications to restore Cav-1 expression in fibroblasts participating in pathogenic tissue remodeling.

Transforming Growth Factor-ß1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts.

DNA methylation is a fundamental epigenetic mark that plays a critical role in differentiation and is mediated by the actions of DNA methyltransferases (DNMTs). TGF-ß1 is one of the most potent inducers of fibroblast differentiation, and although many of its actions on fibroblasts are well described, the ability of TGF-ß1 to modulate DNA methylation in mesenchymal cells is less clear. Here, we examine the ability of TGF-ß1 to modulate the expression of various DNMTs in primary lung fibroblasts (CCL210). TGF-ß1 increased the protein expression, but not RNA levels, of both DNMT1 and DNMT3a. The increases in DNMT1 and DNMT3a were dependent on TGF-ß1 activation of focal adhesion kinase and PI3K/Akt. Activation of mammalian target of rapamycin complex 1 by Akt resulted in increased protein translation of DNMT3a. In contrast, the increase in DNMT1 by TGF-ß1 was not dependent on new protein synthesis and instead was due to decreased protein degradation. TGF-ß1 treatment led to the phosphorylation and inactivation of glycogen synthase kinase-3ß, which resulted in inhibition of DNMT1 ubiquitination and proteosomal degradation. The phosphorylation and inactivation of glycogen synthase kinase-3ß was dependent on mammalian target of rapamycin complex 1. These results demonstrate that TGF-ß1 increases expression of DNMT1 and DNMT3a through different post-transcriptional mechanisms. Because DNA methylation is critical to many processes including development and differentiation, for which TGF-ß1 is known to be crucial, the ability of TGF-ß1 to increase expression of both DNMT1 and DNMT3a demonstrates a novel means by which TGF-ß1 may regulate DNA methylation in these cells.