Progression Of Fibrotic Diseases Research Articles

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2.

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.

Atropisomerism Observed in Galactose-Based Monosaccharide Inhibitors of Galectin-3 Comprising 2-Methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione.

Galectin-3 (Gal-3) is a carbohydrate binding protein that has been implicated in the development and progression of fibrotic diseases. Proof-of-principal animal models have demonstrated that inhibition of Gal-3 is a potentially viable pathway for the treatment of fibrosis─with small molecule Gal-3 inhibitors advanced into clinical trials. We hereby report the discovery of novel galactose-based monosaccharide Gal-3 inhibitors comprising 2-methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (compound 20) and 4-phenyl-4H-1,2,4-triazole (compound 15). Notably, hindered rotation caused by steric interaction between the 3-thione and ortho-trifluoromethyl group of compounds 20, 21 induced formation of thermodynamically stable atropisomers. Distinct X-ray cocrystal structures of 20 and 21 were obtained, which clearly demonstrated that the configuration of 21 proscribes a key halogen bonding σ-hole interaction of 3-chloro with carbonyl oxygen of Gly182, thereby leading to significant loss in potency. Ultimately, 20 and 15 were evaluated in mouse pharmacokinetic studies, and both compounds exhibited oral exposures suitable for further in vivo assessment.

Amphiregulin secreted by umbilical cord multipotent stromal cells protects against ferroptosis of macrophages via the activating transcription factor 3-CD36 axis to alleviate endometrial fibrosis.

Endometrium fibrosis is the leading cause of uterine infertility. Macrophages participated in the occurrence and development of endometrial fibrosis. We previously reported that human umbilical cord multipotent stromal cells (hUC-MSCs) exerted their therapeutic effect in a macrophage-dependent manner in endometrial fibrosis. However precise mechanisms by which hUC-MSCs may influence macrophages in endometrial fibrosis remain largely unexplored. Here, we demonstrated that abnormal iron and lipid metabolism occurred in patients with intrauterine adhesions (IUA) and murine models. Ferroptosis has been proven to contribute to the progression of fibrotic diseases. Our results revealed that pharmacological activation of ferroptosis by Erastin aggravated endometrial fibrosis, while inhibition of ferroptosis by Ferrostatin-1 ameliorated endometrial fibrosis in vivo. Moreover, ferroptosis of macrophages was significantly upregulated in endometria of IUA murine models. Of note, transcriptome profiles revealed that CD36 gene expression was significantly increased in patients with IUA and immunofluorescence analysis showed CD36 protein was mainly located in macrophages. Silencing CD36 in macrophages could reverse cell ferroptosis. Dual luciferase reporter assay revealed that CD36 was the direct target of activation transcription factor 3 (ATF3). Furthermore, through establishing coculture system and IUA murine models, we found that hUC-MSCs had a protective role against macrophage ferroptosis and alleviated endometrial fibrosis related to decreased CD36 and ATF3. The effect of hUC-MSCs on macrophage ferroptosis was attributed to the upregulation of amphiregulin (AREG). Our data highlighted that macrophage ferroptosis occurred in endometrial fibrosis via the ATF3-CD36 pathway and hUC-MSCs protected against macrophage ferroptosis to alleviate endometrial fibrosis via secreting AREG. These findings provided a potential target for therapeutic implications of endometrial fibrosis.

O146: Investigating the cell-matrix interplay in fibrotic remodelling using renal 3D in vitro models

Abstract Introduction The dynamic biophysical microenvironment of the renal extracellular matrix is integral in regulating tissue homeostasis. Matrix remodelling is an iterative process, which guides cell behaviour and pathological fibrosis occurs due to aberrant tissue repair. Fibrosis, characterised by the desmoplastic ECM deposition and tissue stiffening, is prominent in CKD and renal carcinomas. We engineered biomimetic 3D constructs to recapitulate cell-matrix interactions in fibrotic disease progression. Methods Soft collagen-I and laminin constructs (0.2%w/v) were engineered with renal cancer cells (786-O or ACHN) or renal fibroblasts (TK188). Plastic compression was used to generate dense collagen constructs, at 6%w/v collagen density. Mechanical properties were analysed using shear rheology. Gene expression of markers defining the healthy/diseased kidney matrix was determined using qPCR. Results Over 21 days, 786-O cancer cells softened the collagen matrix. Matrix degradation was supported by collagen-I downregulation and MMP7 and vimentin upregulation. Whereas metastatic cancer cells (ACHN) stiffened the dense matrix to ∼1.25kPa, with significant gene upregulation of collagen-I, collagen-IV and LOX. Fibrotic fibroblasts soften the dense matrix by ∼1kPa, with α-SMA and MMP9 upregulation. However, matrix stiffness significantly increases with cancer cell co-cultures. Discussion Renal cancer cells remodel their biophysical environment, altering the construct’s material properties. In our models, collagen density and stromal complexity influences cell behaviour, causing alterations in biomechanics and gene expression. These models can generate physiologically relevant stiffness, enabling investigation between key cell types and the physical environment. Drugs targeting MMPs and LOX will be used to determine if we can disrupt the fibrotic remodelling.

Insights into Disease Progression of Translational Preclinical Rat Model of Interstitial Pulmonary Fibrosis through Endpoint Analysis.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose-response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFβ1, IL6, TNFα, IL1β, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFβ/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model.

Inhibition of the ITGB1 gene attenuates crystalline silica-induced pulmonary fibrosis via epithelial-mesenchymal transformation.

Silicosis is a systemic disease caused by long-term exposure to high concentrations of free silica dust particles in the workplace. It is characterized by a persistent inflammatory response, fibroblast proliferation, and excessive collagen deposition, leading to pulmonary interstitial fibrosis. Epithelial interstitial transformation (EMT) can cause epithelial cells to lose their tight junctions, cell polarity, and epithelial properties, thereby enhancing the properties of interstitial cells, which can lead to the progression of fibrosis and the formation of scar tissue. Integrin 1 (ITGB1) is considered an important factor for promoting EMT and tumor invasion in a variety of tumors and also plays an important role in the progression of fibrotic diseases. Therefore, ITGB1 can be used as a potential target for the treatment of silicosis. In this study, we found that silica exposure induced epithelial-mesenchymal transformation in rats and that the expression of integrin ITGB1 was elevated along with the EMT. We used CRISPR/Cas9 technology to construct integrin ITGB1 knockdown cell lines for in vitro experiments. We compared the expression of the EMT key proteins E-cadherin and vimentin in the ITGB1 knockdown cells and wild-type cells simultaneously stimulated by silica and detected the aggregation point distribution of E-cadherin and vimentin in the cells using laser confocal microscopy. Our results showed that ITGB1 knockout inhibited the ITGB1/ILK/Snail signaling pathway and attenuated the EMT occurrence compared to control cells. These results suggested that ITGB1 is associated with silica-induced EMT and may be a potential target for the treatment of silicosis.

Spatial and Temporal Control of 3D Hydrogel Viscoelasticity through Phototuning.

The mechanical properties of the extracellular environment can regulate a variety of cellular functions, such as spreading, migration, proliferation, and even differentiation and phenotypic determination. Much effort has been directed at understanding the effects of the extracellular matrix (ECM) elastic modulus and, more recently, stress relaxation on cellular processes. In physiological contexts such as development, wound healing, and fibrotic disease progression, ECM mechanical properties change substantially over time or space. Dynamically tunable hydrogel platforms have been developed to spatiotemporally modulate a gel's elastic modulus. However, dynamically altering the stress relaxation rate of a hydrogel remains a challenge. Here, we present a strategy to tune hydrogel stress relaxation rates in time or space using a light-triggered tethering of poly(ethylene glycol) to alginate. We show that the stress relaxation rate can be tuned without altering the elastic modulus of the hydrogel. We found that cells are capable of sensing and responding to dynamic stress relaxation rate changes, both morphologically and through differences in proliferation rates. We also exploited the light-based technique to generate spatial patterns of stress relaxation rates in 3D hydrogels. We anticipate that user-directed control of the 3D hydrogel stress relaxation rate will be a powerful tool that enables studies that mimic dynamic ECM contexts or as a means to guide cell fate in space and time for tissue engineering applications.

The Role of Small Airway Disease in Pulmonary Fibrotic Diseases.

Small airway disease (SAD) is a pathological condition that affects the bronchioles and non-cartilaginous airways 2 mm or less in diameter. These airways play a crucial role in respiratory function and are often implicated in various pulmonary disorders. Pulmonary fibrotic diseases are characterized by the thickening and scarring of lung tissue, leading to progressive respiratory failure. We aimed to present the link between SAD and fibrotic lung conditions. The evidence suggests that SAD may act as a precursor or exacerbating factor in the progression of fibrotic diseases. Patients with fibrotic conditions often exhibit signs of small airway dysfunction, which can contribute to worsening respiratory symptoms and decreased lung function. Moreover, individuals with advanced SAD are at a heightened risk of developing fibrotic changes in the lung. The interplay between inflammation, environmental factors, and genetic predisposition further complicates this association. The early detection and management of SAD can potentially mitigate the progression of fibrotic diseases, highlighting the need for comprehensive clinical evaluation and research. This review emphasizes the need to understand the evolving connection between SAD and pulmonary fibrosis, urging further detailed research to clarify the causes and potential treatment between the two entities.

Lysyl oxidase-like 2 processing by factor Xa modulates its activity and substrate preference

Lysyl oxidase-like 2 (LOXL2) has been identified as an essential mediator of extracellular matrix (ECM) remodeling in several disease processes including cardiovascular disease. Thus, there is growing interest in understanding the mechanisms by which LOXL2 is regulated in cells and tissue. While LOXL2 occurs both in full length and processed forms in cells and tissue, the precise identity of the proteases that process LOXL2 and the consequences of processing on LOXL2’s function remain incompletely understood. Here we show that Factor Xa (FXa) is a protease that processes LOXL2 at Arg-338. Processing by FXa does not affect the enzymatic activity of soluble LOXL2. However, in situ in vascular smooth muscle cells, LOXL2 processing by FXa results in decreased cross-linking activity in the ECM and shifts substrate preference of LOXL2 from type IV collagen to type I collagen. Additionally, processing by FXa increases the interactions between LOXL2 and prototypical LOX, suggesting a potential compensatory mechanism to preserve total LOXs activity in the vascular ECM. FXa expression is prevalent in various organ systems and shares similar roles in fibrotic disease progression as LOXL2. Thus, LOXL2 processing by FXa could have significant implications in pathologies where LOXL2 is involved.

The uPA/uPAR System Orchestrates the Inflammatory Response, Vascular Homeostasis, and Immune System in Fibrosis Progression.

Fibrotic diseases, such as systemic sclerosis (SSc), idiopathic pulmonary fibrosis, renal fibrosis and liver cirrhosis are characterized by tissue overgrowth due to excessive extracellular matrix (ECM) deposition. Fibrosis progression is caused by ECM overproduction and the inhibition of ECM degradation due to several events, including inflammation, vascular endothelial dysfunction, and immune abnormalities. Recently, it has been reported that urokinase plasminogen activator (uPA) and its receptor (uPAR), known to be fibrinolytic factors, orchestrate the inflammatory response, vascular homeostasis, and immune homeostasis system. The uPA/uPAR system may show promise as a potential therapeutic target for fibrotic diseases. This review considers the role of the uPA/uPAR system in the progression of fibrotic diseases.

Macrophage Polarization in Tissue Inflammation and Fibrosis

Macrophages are mononuclear phagocytic cells, which have varied phenotypes and perform diverse intricate functions ranging from inflammation to their role in onset and progression of fibrotic diseases

Hydrogel mechanics regulate fibroblast DNA methylation and chromatin condensation.

Cellular mechanotransduction plays a central role in fibroblast activation during fibrotic disease progression, leading to increased tissue stiffness and reduced organ function. While the role of epigenetics in disease mechanotransduction has begun to be appreciated, little is known about how substrate mechanics, particularly the timing of mechanical inputs, regulate epigenetic changes such as DNA methylation and chromatin reorganization during fibroblast activation. In this work, we engineered a hyaluronic acid hydrogel platform with independently tunable stiffness and viscoelasticity to model normal (storage modulus, G' ∼ 0.5 kPa, loss modulus, G'' ∼ 0.05 kPa) to increasingly fibrotic (G' ∼ 2.5 and 8 kPa, G'' ∼ 0.05 kPa) lung mechanics. Human lung fibroblasts exhibited increased spreading and nuclear localization of myocardin-related transcription factor-A (MRTF-A) with increasing substrate stiffness within 1 day, with these trends holding steady for longer cultures. However, fibroblasts displayed time-dependent changes in global DNA methylation and chromatin organization. Fibroblasts initially displayed increased DNA methylation and chromatin decondensation on stiffer hydrogels, but both of these measures decreased with longer culture times. To investigate how culture time affected the responsiveness of fibroblast nuclear remodeling to mechanical signals, we engineered hydrogels amenable to in situ secondary crosslinking, enabling a transition from a compliant substrate mimicking normal tissue to a stiffer substrate resembling fibrotic tissue. When stiffening was initiated after only 1 day of culture, fibroblasts rapidly responded and displayed increased DNA methylation and chromatin decondensation, similar to fibroblasts on static stiffer hydrogels. Conversely, when fibroblasts experienced later stiffening at day 7, they showed no changes in DNA methylation and chromatin condensation, suggesting the induction of a persistent fibroblast phenotype. These results highlight the time-dependent nuclear changes associated with fibroblast activation in response to dynamic mechanical perturbations and may provide mechanisms to target for controlling fibroblast activation.

The Pro-Fibrotic Response to Lens Injury Is Signaled in a PI3K Isoform-Specific Manner.

The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as resident immune cells to produce and organize a fibronectin-EDA rich provisional matrix and transition to collagen I-producing αSMA+ myofibroblasts. While the PI3K effector Akt was alone insufficient to regulate myofibroblast differentiation, our studies revealed a role for Rac, another potential PI3K effector, in this process. Our studies further uncovered a critical role for PI3K p110α in signaling the proliferation of CD44+ leader cells, which is important to the emergence and expansion of myofibroblasts. Thus, these studies identify activation of PI3K p110α as a critical signaling input following wounding to the development and progression of fibrotic disease.

CD26-inhibition correlates with the absence of chronic lung allograft dysfunction and decreases fibroblast activity in vitro

Abstract Objective Chronic lung allograft dysfunction (CLAD) limits the survival after lung transplantation (Tx). CLAD is characterized by progressive fibrosis of small airways and lung parenchyma. No effective therapy is available that reverses or prevents CLAD. CD26 is a molecule with enzymatic activity also playing a key role in the progression of fibrotic diseases. Here, we analyzed the inhibitory effect of CD26 on fibroblast activity in vitro and the role of CD26-inhibition on allograft rejection in lung transplant patients. Methods Profibrogenic mRNA and protein levels were analyzed in vitro on the CD26-expressing fibroblast cell line Wi-38 using RT-qPCR and Western blot. CD26 was inhibited by Vildagliptin. Migration and proliferation activity of activated fibroblasts were analyzed by Incucyte® and Celltiter-Glo®. Characteristics of patients undergoing lung Tx between 2004 and 2021 were reviewed. Lung biopsies were analyzed by immunohistochemistry (IHC) for CD26. Results In vitro, the expression of profibrogenic genes (αSMA, FAPα, IGFBP7, Collagen 3 and Fibronectin) was significantly reduced in activated lung fibroblasts by Vildagliptin treatment. Also, migration and proliferation activity were attenuated by Vildagliptin. In 221 patients analyzed, CLAD was absent in 34 patients treated with the CD26-inhibitor Sitagliptin vs. an incidence of 18% in patients without Sitagliptin intake (p=0.02). Five-year survival in patients on Sitagliptin was significantly improved vs. patients without CD26-inhibitor intake (80% vs. 58%, p=0.006). Likewise, the incidence of acute cellular rejection (ACR) was significantly reduced in patients on Sitagliptin (7% vs. 35%, p=0.01). IHC of patient lung biopsies showed expression of CD26 in perifibrotic areas of CLAD lesions. Additional clinical data from University Hospital Zurich and from University Hospital Padua confirmed the finding that Sitagliptin intake correlated with the absence of acute and chronic allograft rejection. Conclusion CD26-inhibition attenuates key pro-fibrotic mediators and fibroblast activity in vitro. Impressively, patients on CD26-inhibitor did not show any CLAD. Moreover, ACR was significantly reduced. Gliptins which are in routine clinical use for the treatment of type II diabetes therefore seem to have great potential to be repurposed for a novel clinical application against lung allograft rejection.

Function and regulation of GPX4 in the development and progression of fibrotic disease.

Fibrosis is a common feature of fibrotic diseases that poses a serious threat to global health due to high morbidity and mortality in developing countries. There exist some chemical compounds and biomolecules associated with the development of fibrosis, including cytokines, hormones, and enzymes. Among them, glutathione peroxidase 4 (GPX4), as a selenoprotein antioxidant enzyme, is widely found in the embryo, testis, brain, liver, heart, and photoreceptor cells. Moreover, it is shown that GPX4 elicits diverse biological functions by suppressing phospholipid hydroperoxide at the expense of decreased glutathione (GSH), including loss of neurons, autophagy, cell repair, inflammation, ferroptosis, apoptosis, and oxidative stress. Interestingly, these processes are intimately related to the occurrence of fibrotic disease. Recently, GPX4 has been reported to exhibit a decline in fibrotic disease and inhibit fibrosis, suggesting that alterations of GPX4 can change the course or dictate the outcome of fibrotic disease. In this review, we summarize the role and underlying mechanisms of GPX4 in fibrosis diseases such as lung fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and myelofibrosis.

The impact of pulmonary fibrosis on neutrophil aging and trafficking

Abstract Neutrophil aging is the process through which neutrophils released from the bone marrow into circulation acquire specific phenotypic and functional changes over time. After neutrophils age in circulation, they return to the bone marrow or other organs for clearance. Aged neutrophils are highly proinflammatory and can potentially contribute to tissue injury if their return to and clearance within the bone marrow is dysregulated. We have found that the development of pulmonary fibrosis in mice is associated with decreased numbers of aged neutrophils within the bone marrow compared to non-fibrotic mice 21 days after induction of fibrosis, indicating that neutrophils are not returning to the bone marrow for clearance after aging in circulation. Indeed, we observed a corresponding increase in the numbers of aged neutrophils in peripheral blood and bronchioalveolar fluid (BALF) of fibrotic mice compared to non-fibrotic mice, supporting the idea that pulmonary fibrosis prevents neutrophil migration back to the bone marrow after aging. Pulmonary fibrosis is a disease characterized by aberrant wound healing and lung and vascular injury. In accordance with this, we have observed increased levels of extracellular DNA and neutrophil elastase in plasma and BALF of mice 21 days after induction of fibrosis. These products are released in high levels by aged neutrophils during the formation of neutrophil extracellular traps, suggesting that aberrant aged neutrophil behavior may contribute to lung and vascular injury in pulmonary fibrosis. Our study seeks to understand the role of neutrophils in fibrotic disease progression by evaluating how fibrosis may alter the trafficking of aged, highly proinflammatory neutrophils. Supported by grants from NIH (R35 HL144481, F31 HL152509) and the Scleroderma Foundation (Arnold Postlethwaite, M.D. Memorial Pre-Doctoral Summer Fellowship Award)

NUAK1 promotes organ fibrosis via YAP and TGF-β/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-β (TGF-β)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-β-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-β induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-β/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Four-year survival rate in pulmonary sarcoidosis with extensive pulmonary hypertension screening

BackgroundSarcoidosis is a systemic disease of unknown aetiology with significant morbidity and mortality. The PULSAR study prospectively performed cardiac analysis including systematic pulmonary hypertension screening in sarcoidosis patients newly referred to a tertiary sarcoidosis center. In this manuscript we studied the four-year mortality of this population. Methods and main findingsBetween august 2015 and October 2017, 399 patients (58% male, mean age 49.4 years, 90.5% Caucasian) were included and followed for a mean period of 4.3±0.7 years. In total, 10 patients had died at the time of analysis. 1-, 2-, 3- and 4-year survival rate was 100% (n=399), 99.0% (n=399), 98.2% (n=399) and 94.6% (n=276). Most patients died of respiratory failure, other causes were heterogeneous including cardiac, neurological and non-sarcoidosis origin. A low CPI score or modified Walsh score was associated with higher mortality, similar for high PH probability on echocardiography and elevated right ventricular systolic pressure. ConclusionThis study highlights that elevated RVSP and presence of PH on echocardiography and progression of fibrotic disease with subsequent decline in pulmonary function test are important factors for mortality in sarcoidosis patients.

Loureirin B alleviates cardiac fibrosis by suppressing Pin1/TGF-β1 signaling

It is well-established that cardiac fibrosis contributes to cardiac dysfunction and adverse outcomes. However, the underlying mechanisms remain elusive, warranting further studies to develop new therapeutic strategies. It has been suggested that loureirin B can ameliorate the progression of fibrotic diseases. This study investigated the effects of loureirin B on cardiac fibrosis and explored the underlying mechanisms. Transverse aortic constriction (TAC) was performed to induce cardiac fibrosis in mice. Loureirin B (10 mg/kg/day) or saline was continuously delivered via subcutaneous osmotic mini-pumps. Cardiac fibroblasts (CFs) were treated with angiotensin II (Ang II, 100 nM, 24 h) to simulate fibrosis in vitro. Immunochemistry, echocardiography, and Sircol collagen assays were conducted to evaluate the cardioprotective effects. Quantitative real-time polymerase chain reaction, Western blot, and transfection techniques were performed to elucidate the mechanisms. Results showed that loureirin B prevented cardiac fibrosis and improved cardiac function in mice subjected to TAC. Treatment with loureirin B inhibited the elevation of inflammatory factors (interleukin-1β, interleukin-6, and tumor necrosis factor-α), transforming growth factor-β1 (TGF-β1), and Pin1 induced by TAC. Furthermore, loureirin B treatment inhibited the increased fibroblast activation and collagen synthesis induced by Ang II in CFs. In addition, loureirin B inhibited increased expression of TGF-β1 and Pin1 induced by Ang II or TAC. Mechanistically, overexpression of Pin1 induced increased TGF-β1 expression and blocked the anti-fibrotic effects in Ang II-induced CFs treated with loureirin B. Loureirin B ameliorated cardiac fibrosis and dysfunction both in vitro and in vivo probably through the Pin1/TGF-β1 signaling pathway.