Fibrosis-related Markers Research Articles

Proteomic Screening Identifies Megakaryocyte Derived PF4/Cxcl4 As a Critical Driver of Myelofibrosis

Background: Despite advances in understanding the genomic landscape of Myeloproliferative Neoplasms (MPNs), the precise pathological mechanisms driving fibrotic progression in MPNs remain incompletely understood. Previous research has shown that Megakaryocytes (Mks) play a role in MPN pathology by promoting inflammation and extracellular matrix (ECM) deposition by activated stromal cells. However, the specific factors derived from Mks that contribute to the inflammatory milieu and myelofibrosis progression are not well defined. Aims: To investigate the events that drive bone marrow (BM) fibrosis due to abnormal megakaryocyte-stromal crosstalk, we employed mass spectrometry-based proteomics in a myelofibrosis-like mouse model. Our goal was to identify differentially expressed proteins involving Mks, platelets and BM cells in fibrotic versus non-fibrotic conditions. Methods: We induced myelofibrosis in mice through repeated injections of supra-pharmacological doses of the thrombopoietin-mimetic Romiplostim (TPO high). Subsequently, we isolated Mks, BM cells and platelets from TPO high and control mice (injected with saline) and analyzed protein extracts using LC-MS/MS assay. Cytokine levels in plasma and BM cell-free fluids of treated mice were assessed using cytokine arrays. To evaluate fibrosis-related markers, we analyzed the expression of myofibroblasts markers (a-SMA, Vimentin) and ECM synthesis (Fibronectin, Type III Collagen) in mouse embryonic fibroblasts (mEFs) and human BM mesenchymal stromal cells after stimulation with recombinant PF4 using western blot. mEFs were co-cultured with BM-derived Mks in direct and indirect co-culture systems in the presence of increasing TPO concentrations. In some experiments, Mks were transfected with siRNA specifically targeting CXCL4 gene or control siRNA before co-culture with mEFs. Additionally, we quantified plasma PF4/Cxcl4 levels using ELISA assay in a large cohort of MPN patients and normal controls. We also sorted CD41/CD61 Mks from BM aspirates of MPN patients and evaluated CXCL4 mRNA levels through qPCR. Results: We observed that signaling pathways related to cytoskeletal reorganization, cell adhesion and inflammation, were commonly activated in Mks, platelets and BM cells of TPO high mice compared to control mice. The MSP-RON signaling pathway and the Unfolded Protein Response were specifically activated in Mks and platelets, respectively. Among the differentially expressed proteins,the chemokine PF4/Cxcl4 was up regulated exclusively in the proteasomes of the TPO high mice. We confirmed Increased levels of PF4/Cxcl4 in plasma and BM cell-free fluids of fibrotic mice using cytokine arrays. In vitro stimulation of BM-derived Mks with high TPO concentrations resulted in increased synthesis and secretion of PF4/Cxcl4. Recombinant PF4/Cxcl4 was rapidly internalized through surface glycosaminoglycans by mEFs and human BM mesenchymal stromal cells, leading to myofibroblast differentiation. Importantly, we demonstrated that these mechanisms were interconnected during Mk-mEF cross-talk in co-culture systems with high TPO concentrations. Genetic down-regulation of CXCL4 in Mks, prior to co-culture in TPO-saturated conditions, mitigated the profibrotic phenotype of mEFs in terms of ECM synthesis and myofibroblast markers expression. Finally, we found higher plasma levels of circulating PF4/Cxcl4 and increased expression in BM-sorted Mks in patients with MPNs. Conclusions: Our findings identify a crucial role of Mk derived PF4 in the fibrosis progression of MPNs, providing further support for the potential therapeutic strategy of neutralizing PF4.

TDO2 deficiency attenuates the hepatic lipid deposition and liver fibrosis in mice with diet-induced non-alcoholic fatty liver disease

PurposeNon-alcoholic fatty liver disease (NAFLD) represents an increasingly prevalent set of liver diseases. Tryptophan 2,3-dioxygenase 2 (TDO2) is the major enzyme of tryptophan catabolism and is abnormally expressed in liver cancer, but the function of TDO2 in NAFLD remains unclear. The current study was designed to probe into the effect and mechanism of TDO2 on NAFLD. MethodsC57BL/6 mice and TDO2-knockout (KO) mice were fed with a high-fat diet for 16 weeks to construct the NAFLD model in vivo; primary hepatocytes isolated from TDO2-KO mice were exposed to palmitate (PA) to establish the NAFLD model in vitro. The expression of TDO2 was determined using Western blot. The function and mechanism of TDO2 were evaluated by enzyme-linked immunosorbent assay, hematoxylin-eosin staining, Oil Red O staining, immunohistochemical assay, and Western blot. ResultsThe expression of TDO2 in the liver tissue of NAFLD mice was more than three times that in the control group. Functionally, TDO2 knockout reduced hepatic lipid deposition and liver fibrosis in NAFLD mice in vivo and primary hepatocytes induced by 200 μM PA in vitro. Mechanistically, the loss of TDO2 restrained hepatic lipid deposition and expression levels of fibrosis-related markers in PA-treated primary hepatocytes, and these trends were partially reversed by 10 ng/ml receptor activator of the nuclear factor kappa-B ligand (RANKL, an activator of the NF-κB pathway). ConclusionKnocking out TDO2 repressed hepatic lipid deposition and liver fibrosis in mice with NAFLD, and reduced hepatic lipid deposition and expressions of fibrosis-related markers in PA-treated primary hepatocytes by inactivating the NF-κB pathway.

Knockdown of lncRNA MALAT1 attenuates renal interstitial fibrosis through miR-124-3p/ITGB1 axis

Renal interstitial fibrosis (RIF) considered the primary irreversible cause of chronic kidney disease. Recently, accumulating studies demonstrated that lncRNAs play an important role in the pathogenesis of RIF. However, the underlying exact mechanism of lncRNA MALAT1 in RIF remains barely known. Here, the aim of our study was to investigate the dysregulate expression of lncRNA MALAT1 in TGF-β1 treated HK2/NRK-49F cells and unilateral ureteral obstruction (UUO) mice model, defining its effects on HK2/NRK-49F cells and UUO mice fibrosis process through the miR-124-3p/ITGB1 signaling axis. It was found that lncRNA MALAT1 and ITGB1 was significantly overexpression, while miR-124-3p was downregulated in HK2/NRK-49F cells induced by TGF-β1 and in UUO mice model. Moreover, knockdown of lncRNA MALAT1 remarkably downregulated the proteins level of fibrosis-related markers, ITGB1, and upregulated the expression of epithelial marker E-cadherin. Consistently, mechanistic studies showed that miR-124-3p can directly binds to lncRNA MALAT1 and ITGB1. And the protect effect of Len-sh-MALAT1 on fibrosis related protein levels could be partially reversed by co-transfected with inhibitor-miR-124-3p. Moreover, the expression trend of LncRNA MALAT1/miR-124-3p/ITGB1 in renal tissues of patients with obstructive nephropathy (ON) was consistent with the results of cell and animal experiments. Taken together, these results indicated that lncRNA MALAT1 could promote RIF process in vitro and in vivo via the miR-124-3p/ITGB1 signaling pathway. These findings suggest a new regulatory pathway involving lncRNA MALAT1, which probably serves as a potential therapeutic target for RIF.

Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids.

Non-alcoholic steatohepatitis (NASH) is known to progress to cirrhosis and hepatocellular carcinoma in some patients. Although NASH is associated with abnormal mitochondrial function related to lipid metabolism, mechanisms for the development and effective treatments are still unclear. Therefore, new approaches to elucidate the pathophysiology are needed. In the previous study, we generated liver organoids from different stages of NASH model mice that could recapitulate the part of NASH pathology. In the present study, we investigated the relationship between mitochondrial function and NASH disease by comparing NASH liver organoids (NLO) and control liver organoids (CLO). Compared with CLO, mitochondrial and organoid morphology was abnormal in NLO, with increased expression of mitochondrial mitogen protein, DRP1, and mitochondria-derived reactive oxygen species (ROS) production. Treatment of NLO with a DPR1 inhibitor, Mdivi-1 resulted in the improvement of morphology and the decreased expression of fibrosis-related markers, Col1a1 and Acta2. In addition, treatment of NASH model mice with Mdivi-1 showed a decrease in fatty liver. Mdivi-1 treatment also prevented fibrosis and ROS production in the liver. These results indicate that NLO undergoes enhanced metabolism and abnormal mitochondrial morphology compared with CLO. It was also suggested that Mdivi-1 may be useful as a therapeutic agent to ameliorate NASH pathology.

Application of recombinant TGF-β1 inhibitory peptide to alleviate isoproterenol-induced cardiac fibrosis.

Cardiac fibrosis is a remodeling process of the cardiac interstitium, characterized by abnormal metabolism of the extracellular matrix, excessive accumulation of collagen fibers, and scar tissue hyperplasia. Persistent activation and transdifferentiation into myofibroblasts of cardiac fibroblasts promote the progression of fibrosis. Transforming growth factor-β1 (TGF-β1) is a pivotal factor in cardiac fibrosis. Latency-associated peptide (LAP) is essential for activating TGF-β1 and its binding to the receptor. Thus, interference with TGF-β1 and the signaling pathways using LAP may attenuate cardiac fibrosis. Recombinant full-length and truncated LAP were previously constructed, expressed, and purified. Their effects on cardiac fibrosis were investigated in isoproterenol (ISO)-induced cardiac fibroblasts (CFs) and C57BL/6 mice. The study showed that LAP and tLAP inhibited ISO-induced CF activation, inflammation, and fibrosis, improved cardiac function, and alleviated myocardial injury in ISO-induced mice. LAP and tLAP alleviated the histopathological alterations and inhibited the elevated expression of inflammatory and fibrosis-related markers in cardiac tissue. In addition, LAP and tLAP decreased the ISO-induced elevated expression of TGF-β, αvβ3, αvβ5, p-Smad2, and p-Smad3. The study indicated that LAP and tLAP attenuated ISO-induced cardiac fibrosis via suppressing TGF-β/Smad pathway. This study may provide a potential approach to alleviate cardiac fibrosis. KEY POINTS: • LAP and tLAP inhibited ISO-induced CF activation, inflammation, and fibrosis. • LAP and tLAP improved cardiac function and alleviated myocardial injury, inflammation, and fibrosis in ISO-induced mice. • LAP and tLAP attenuated cardiac fibrosis via suppressing TGF-β/Smad pathway.

Resveratrol plays an anti-fibrotic and anti-autophagy role by stimulating miR-192-5p expression in urethral fibrosis.

Resveratrol (RSV) exerts anti-fibrotic effects on various fibrotic diseases. Whereas the biological role of RSV on urethral fibrosis remains to be elucidated. This study aimed to determine the mechanisms by which RSV affects urethral fibrosis and autophagy. Sprague‒Dawley rats and primary fibroblasts were treated with transforming growth factor-β1 (TGFβ1) to generate in vivo and in vitro fibrosis models. Then, those were treated with RSV, and autophagy and fibrosis-related indicators were tested. Firstly, we found that RSV reversed the upregulation of indicators related to TGFβ1-induced fibrosis (TGFβ1, α-smooth muscle actin, collagen type I, and collagen type III), autophagy (TFEB and LC3), and TGFβR1/Smad4 pathway, as well as the downregulation of p62 and miR-192-5p expression both in vivo and in vitro. Overexpression of miR-192-5p suppressed the upregulation of fibrosis-related markers expression, as well as TFEB and LC3 expression, induced by TGFβ1, while the expression trend of p62 was the opposite. Inhibiting miR-192-5p reversed the effects of RSV on the model group cells. It was also shown that RSV combined with sh-Smad4 inhibited autophagy more effectively than RSV alone. These results suggest that RSV inhibits urinary fibrosis and autophagy via the miR-192-5p/TGFβR1/Smad4 pathway. RAV may be a potential drug for alleviating urethral fibrosis.

Exercise-induced myocardial hypertrophy preconditioning promotes fibroblast senescence and improves myocardial fibrosis through Nrf2 signaling pathway

ABSTRACT This study aims to investigate how exercise-induced myocardial hypertrophy preconditioning affects cardiac fibroblasts in the context of myocardial fibrosis, a chronic disease that can cause cardiac arrhythmia and heart failure. Heart failure was induced in male C57BL/6 mice via Transverse aortic constriction, and some mice were given swimming exercise before surgery to test the effects of exercise-induced myocardial hypertrophy preconditioning on myocardial fibrosis. Myocardial tissue was evaluated for fibrosis, senescent cells, and apoptotic cells. Myocardial fibroblasts from rats were cultured and treated with norepinephrine to induce fibrosis which were then treated with si-Nrf2 and analyzed for markers of fibrosis, senescence, apoptosis, and cell proliferation. Exercise-induced myocardial hypertrophy preconditioning reduced myocardial fibrosis in mice, as shown by decreased mRNA expression levels of fibrosis-related indicators and increased cell senescence. In vitro data indicated that norepinephrine (NE) treatment increased fibrosis-related markers and reduced apoptotic and senescent cells, and this effect was reversed by pre-conditioning in PRE+NE group. Preconditioning activated Nrf2 and downstream signaling genes, promoting premature senescence in cardiac fibroblasts and tissues isolated from preconditioned mice. Moreover, Nrf2 knockdown reversed proapoptotic effects, restored cell proliferation, reduced senescence-related protein expression, and increased oxidative stress markers and fibrosis-related genes, indicating Nrf2‘s crucial role in regulating oxidative stress response of cardiac fibroblasts. Exercise-induced myocardial hypertrophy preconditioning improves myocardial fibrosis which is Nrf2-dependent, indicating the protective effect of hypertrophy preconditioning. These findings may contribute to the development of therapeutic interventions to prevent or treat myocardial fibrosis.

SPARC-YAP/TAZ inhibition prevents the fibroblasts-myofibroblasts transformation

BackgroundFibrotic scar is a severe side effect of trabeculectomy, resulting in unsatisfactory outcomes for glaucoma surgery. Accumulating evidence showed human Tenon's fibroblasts (HTFs) play an important role in fibrosis formation. We previously reported that the aqueous level of secreted protein acidic and rich in cysteine (SPARC) was higher in the patients with primary angle closure glaucoma, which was associated with the failure of trabeculectomy. In this study, the potential effect and mechanism of SPARC in promoting fibrosis were explored by using HTFs. MethodsHTFs were employed in this study and examined under a phase-contrast microscope. Cell viability was determined by CCK-8. The expressions of SPARC-YAP/TAZ signaling and the fibrosis-related markers were examined with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence, subcellular fractionation was conducted to further determined the variation of YAP and phosphorylated YAP. The differential gene expressions were analyzed with RNA sequencing (RNAseq), followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. ResultsExogenous SPARC induced HTFs-myofibroblast transformation, as evidenced by the increased expression of α-SMA, collagen I and fibronectin in both protein and mRNA levels. SPARC knockdown decreased the expressions of the above genes in TGF-β2-treated HTFs. KEGG analysis showed that the Hippo signaling pathway was mostly enriched. SPARC treatment increased the expressions of YAP, TAZ, CTGF and CYR61 as well as enhanced YAP translocation from cytoplasm to nucleus, and decreased the phosphorylation of YAP and LAST1/2, which was reversed by SPARC knockdown. Knockdown of YAP1 decreased the fibrosis-related markers, such as α-SMA, collagen I and Fibronectin, in SPARC-treated HTFs. ConclusionsSPARC induced HTFs-myofibroblast transformation via activating YAP/TAZ signaling. Targeting SPARC-YAP/TAZ axis in HTFs might provide a novel strategy for inhibiting fibrosis formation after trabeculectomy.

Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible interstitial pneumonia caused by the excessive production and deposition of extracellular matrix components, including type I collagen. Activated fibroblasts, called α-SMA (α-smooth muscle actin)-expressing myofibroblasts, are the major source of type I collagen in pulmonary fibrosis (PF), but the mechanisms underlying disease progression have not been fully elucidated. Here, we obtained lung fibroblasts from patients with IPF from both nonfibrotic and fibrotic areas as determined by a lung computed tomography scan and compared gene expression between these areas by DNA microarray. We found that ANGPTL4 (angiopoietin-like 4) was highly expressed only in fibroblasts from the fibrotic area. ANGPTL4 was selectively expressed in the fibroblastic area of IPF lungs, where the myofibroblast marker α-SMA was also expressed. ANGPTL4 also regulates the gene expression of fibrosis-related markers, cell migration, and proliferation. In addition, ANGPTL4 expression in a murine model of PF induced by treatment with bleomycin was significantly induced in the lungs from the acute to the chronic phase. Single-cell transcriptome analysis during the course of bleomycin-induced PF revealed that Angptl4 was predominantly expressed in the activated fibroblasts and myofibroblasts. Moreover, the administration of recombinant ANGPTL4 to the bleomycin-induced fibrosis model significantly increased collagen deposition and exacerbated the PF. In contrast, the pathogenesis of PF in Angptl4-deficient mice was improved. These results indicate that ANGPTL4 is critical for the progression of PF and might be an early diagnostic marker and therapeutic target for IPF.

Smurf1 Facilitates Oxidative Stress and Fibrosis of Ligamentum Flavum by Promoting Nrf2 Ubiquitination and Degradation.

Lumbar spinal stenosis (LSS), which can lead to irreversible neurologic damage and functional disability, is characterized by hypertrophy and fibrosis in the ligamentum flavum (LF). However, the underlying mechanism is still unclear. In the current study, the effect of Smurf1, a kind of E3 ubiquitin ligase, in promoting the fibrosis and oxidative stress of LF was investigated, and its underlying mechanism was explored. The expression of oxidative stress and fibrosis-related markers was assessed in the tissue of lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). Next, the expression of the top 10 E3 ubiquitin ligases, obtained from Gene Expression Omnibus (GEO) dataset GSE113212, was assessed in LDH and LSS, and confirmed that Smurf1 expression was markedly upregulated in the LSS group. Furthermore, Smurf1 overexpression promotes the fibrosis and oxidative stress of LF cells. Subsequently, NRF2, an important transcription factor for oxidative stress and fibrosis, was predicted to be a target of Smurf1. Mechanistically, Smurf1 directly interacts with Nrf2 and accelerates Nrf2 ubiquitination and degradation. In conclusion, the current study suggests that Smurf1 facilitated the fibrosis and oxidative stress of LF and induced the development of LSS by promoting Nrf2 ubiquitination and degradation.

Tcf21 Alleviates Pancreatic Fibrosis by Regulating the Epithelial-Mesenchymal Transformation of Pancreatic Stellate Cells.

The activation of pancreatic stellate cells (PSCs) plays a key role in the occurrence and development of chronic pancreatitis (CP) and pancreatic fibrosis, which is related to the process of epithelial-mesenchymal transition (EMT). This study was designed to investigate the effect and mechanism of Tcf21 (one of tumor suppressor genes) on pancreatic inflammation and fibrosis in vivo and in vitro. C57BL/6 male mice were intraperitoneally injected with caerulein for 6weeks to establish CP animal model. Fixed pancreatic tissue paraffin-embedded sections were used for immunohistochemistry staining of Tcf21, fibrosis-related markers (α-SMA), interstitial markers (Vimentin) and epithelial markers (E-cadherin). Western blotting and qRT-PCR assay were performed to analyze the change of expression of the above markers after stimulation of TGF-β1 or overexpressed Tcf21 lentivirus transfection in human pancreatic stellate cells (HPSCs). The pancreatic expression of α-SMA and Vimentin of CP mice significantly increased, while the expression of Tcf21 and E-cadherin significantly decreased. TGF-β1 could promote activation and EMT process of HPSCs, and inhibited the expression of Tcf21. Overexpression of Tcf21 could significantly down-regulate the expression of α-SMA, Fibronectin and Vimentin, and up-regulated the expression of ZO-1 of HPSCs. Cell Counting Kit-8 assay and scratch wound-healing assay results showed that overexpression of Tcf21 could significantly inhibit the cell migration and proliferation of HPSCs. Overexpression of Tcf21 could significantly alleviate the activation, proliferation, migration of PSCs by regulating the EMT process. Tcf21 had a potential prospect of a new target for CP therapy.

Sustainably released nanoparticle-based rhynchophylline limits pulmonary fibrosis by inhibiting the TEK-PI3K/AKT signaling pathway.

Pulmonary fibrosis (PF) is a rapidly progressing and irreversible disease, and the currently available types of clinical drugs are limited and inefficient. In our previous study, we observed that Rhynchophylline (Rhy) hindered tendon adhesion and stimulated the healing of injured tendon structures. Considering the similar mechanisms between adhesion formation and PF, we explored the roles of Rhy in PF. The cytotoxicity of Rhy was tested by a Cell Counting Kit-8 (CCK-8) assay. The degree of PF was evaluated by Western blot (WB), Masson and hematoxylin-eosin (HE) staining, and hydroxyproline quantification. The Rhy-loaded nanoparticles were prepared through an emulsification sonication technique and characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The release of the Rhy-loaded nanoparticles was tested using the absorbance value of the supernatant. Transcriptome sequencing was performed to determine the downstream target and pathway of Rhy, which was then verified by WB. In vitro, Rhy decreased Transforming Growth Factor Beta 1 (TGF-β1)-induced abnormal overexpression of fibronectin (FN), collagen I (Col I), α-smooth muscle actin (α-SMA) in a dose-dependent manner in human lung fibroblast (HFL1) cells. In vivo, we confirmed (through Masson staining) that the intraperitoneal injection of Rhy reduced collagen deposition and the fibrotic area in a dose-dependent manner. Our results indicated that the Rhy-loaded nanoparticles intratracheal spray intuitively narrowed collagen deposition, shrank collagen deposition and the fibrotic area (Masson and HE staining), and reduced the expression of fibrosis-related markers (WB). Meanwhile, the lung index value and hydroxyproline content were markedly lower than the bleomycin (BLM)-treated group. By transcriptional sequencing analysis, we identified Receptor Tyrosine Kinase (TEK)-Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/AKT) as the downstream target and pathway of Rhy. It was also observed that Rhy could reverse the TGF-β1-induced TEK and phosphorylated AKT (p-AKT) elevated expression. Our findings indicate that Rhy constrained PF progression by inhibiting TEK-PI3K/AKT signaling pathway. Hence, this sustainable release system of Rhy is a highly effective therapy to limit PF and should be developed.

Time-sequential fibroblast-to-myofibroblast transition in elastin-variable 3D hydrogel environments by collagen networks

BackgroundFibrosis plays an important role in both normal physiological and pathological phenomena as fibroblasts differentiate to myofibroblasts. The activation of fibroblasts is determined through interactions with the surrounding extracellular matrix (ECM). However, how this fibroblast-to-myofibroblast transition (FMT) is regulated and affected by elastin concentration in a three-dimensional (3D) microenvironment has not been investigated.MethodsWe developed an insoluble elastin-gradient 3D hydrogel system for long-lasting cell culture and studied the molecular mechanisms of the FMT in embedded cells by nanoflow LC–MS/MS analysis along with validation through real-time PCR and immunofluorescence staining.ResultsBy optimizing pH and temperature, four 3D hydrogels containing fibroblasts were successfully fabricated having elastin concentrations of 0, 20, 50, and 80% in collagen. At the low elastin level (20%), fibroblast proliferation was significantly increased compared to others, and in particular, the FMT was clearly observed in this condition. Moreover, through mass spectrometry of the hydrogel environment, it was confirmed that differentiation proceeded in two stages. In the early stage, calcium-dependent proteins including calmodulin and S100A4 were highly associated. On the other hand, in the late stage after several passages of cells, distinct markers of myofibroblasts were presented such as morphological changes, increased production of ECM, and increased α-SMA expression. We also demonstrated that the low level of elastin concentration induced some cancer-associated fibroblast (CAF) markers, including PDGFR-β, and fibrosis-related disease markers, including THY-1.ConclusionUsing our developed 3D elastin-gradient hydrogel system, we evaluated the effect of different elastin concentrations on the FMT. The FMT was induced even at a low concentration of elastin with increasing CAF level via calcium signaling. With this system, we were able to analyze varying protein expressions in the overall FMT process over several cellular passages. Our results suggest that the elastin-gradient system employing nonlinear optics imaging provides a good platform to study activated fibroblasts interacting with the microenvironment, where the ECM plays a pivotal role.Graphical

Interleukin-11 Is Elevated in Patients with Atrial Fibrillation, Correlates with Serum Fibrosis Markers, and Represents a Therapeutic Target for Atrial Fibrosis

Introduction: Inflammatory cytokines are closely associated with developing cardiac fibrosis. This research aimed to explore the significant role of IL-11 in atrial fibrosis progression and potential therapeutic targets. Methods: 207 AF patients and 160 healthy subjects were included in the case-control study. Blood samples were analyzed for the level of IL-11 by enzyme-linked immunosorbent assay (ELISA). Angiotensin II (Ang II)-treated fibrosis mouse models were generated, and expression of IL-11 mRNA and protein was detected by RT-qPCR and Western blot. IL-11 antagonist was used to evaluating atrial fibrosis-related markers. Results: The persistent atrial fibrillation patients (n = 76) had significantly larger left atrial size, higher serum levels of hypertrophic protein BNP, proinflammatory cytokine high-sensitivity C-reactive protein (hs-CRP), and interleukin-6 (IL-6) compared to paroxysmal atrial fibrillation patients (n = 131), and healthy subjects (all p < 0.05). Pearson correlation analysis revealed significant positive correlation between serum IL-11 and cardiac fibrosis markers BNP (r = 0.394, p < 0.001), CTX-I (r = 0.418, p < 0.001), PICP (r = 0.306, p < 0.001), PIIINP (r = 0.335, p < 0.001), and TGF-β1 (r = 0.273, p < 0.001). In the fibrosis mouse model, Ang II infusion significantly upregulated IL-11 mRNA and protein expression in the left atrium of mice (p < 0.05), as well as staining intensity of Masson trichrome, the intensity of α-SMA, and it increased mRNA expression of collagen I and III in atrial tissue. IL-11 antagonist treatment significantly attenuated Masson trichrome, number of α-SMA-positive myofibroblasts in atrial tissue. Also, it significantly reduced the p-ERK1/2 in atrial tissue of mice infused with Ang II (p < 0.05). Conclusions: IL-11 is upregulated in the serum of AF patients, and IL-11 inhibitor significantly inhibited Ang II-induced atrial fibrosis, a key pathological feature of AF. Therefore, IL-11 could be a potential therapeutic target for AF.

Ochratoxin A induces endoplasmic reticulum stress and fibrosis in the kidney via the HIF-1α/miR-155-5p link.

Ochratoxin A (OTA) is a ubiquitous fungal toxin found in agricultural products and foods that is toxic to both humans and animals. OTA mainly affects kidney, but the mechanisms underlying OTA-induced nephrotoxicity remain not fully understood. MicroRNA (miRNA) is involved in key cellular processes. The toxic mechanism and regulatory effects of miRNAs on OTA toxicity in kidney, and particularly the role of HIFα-1/miR-155-5p on OTA-caused ER stress and fibrosis, were investigated in this study. OTA induced hypoxia-like conditions such as ER stress and fibrosis in HK-2 cells and renal tissues via modulating HIF-1α, which was followed by regulation of ER stress-related proteins (GRP78 and ATF-4), as well as fibrosis-related markers (fibronectin, α-SMA, and E-cadherin). Notably, a total of 62 miRNAs showed significant differential expression in kidney of OTA-treated mice. Under OTA exposure, HIF-1α enhanced miR-155-5p expression, causing ER stress and fibrosis in HK-2 cells. HIF-1α knockdown decreased OTA-induced miR-155-5p expression as well as ER stress and fibrotic responses, whereas miR-155-5p overexpression restored this. Our data suggest that OTA enhances ER stress and fibrosis in the kidney through upregulating the HIF-1α/miR-155-5p link.

Upregulation of leucine-rich alpha-2 glycoprotein: A key regulator of inflammation and joint fibrosis in patients with severe knee osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of the joints mainly affecting older individuals. Since the etiology behind the progression of OA is not well understood, several associated consequences, such as synovial joint stiffness and its progression due to joint fibrosis, are still poorly understood. Although a lot of developments have been achieved in the diagnosis and management of OA, synovial fibrosis remains one of the major challenging consequences. The present study was therefore focused on understanding the mechanism of synovial fibrosis, which may further contribute to improving symptomatic treatments, leading to overall improvements in the treatment outcomes of patients with OA. We used advanced proteomic techniques including isobaric tag for relative and absolute quantitation and sequential window acquisition of all theoretical mass spectra for the identification of differentially expressed proteins in the plasma samples of patients with OA. An in silico study was carried out to evaluate the association of the identified proteins with their biological processes related to fibrosis and remodeling of the extracellular matrix (ECM). The most significantly upregulated protein was then validated by Western blot and enzyme-linked immunosorbent assay. The target protein was then further investigated for its role in inflammation and joint fibrosis using an in vitro study model. Leucine-rich alpha-2 glycoprotein (LRG1) was found to be the most highly differentially expressed upregulated (9.4-fold) protein in the plasma samples of patients with OA compared to healthy controls. The knockdown of LRG1 followed by in vitro studies revealed that this protein promotes the secretion of the ECM in synovial cells and actively plays a role in wound healing and cell migration. The knockdown of LRG1 further confirmed the reduction of the inflammatory- and fibrosis-related markers in primary cells. LRG1 was identified as a highly significant upregulated protein in the plasma samples of patients with OA. It was found to be associated with increased fibrosis and cell migration, leading to enhanced inflammation and joint stiffness in OA pathogenesis.

E2F1 regulates miR-215-5p to aggravate paraquat-induced pulmonary fibrosis via repressing BMPR2 expression.

Pulmonary fibrosis is considered to be an irreversible lung injury, which can be caused by paraquat (PQ) poisoning. MiRNAs have been demonstrated crucial roles in pulmonary fibrosis caused by numerous approaches including PQ induction. The purpose of this study was to investigate the role and the underlying mechanism of miR-215 in PQ-induced pulmonary fibrosis. The cell and animal models of pulmonary fibrosis were established through PQ intervention. Cell viability was performed to test by MTT assay. Immunofluorescence assay was used to detect COL1A1 expression and its location. The relationships among E2F1, miR-215-5p, and BMPR2 were validated by dual luciferase reporter gene assay, chromatin immunoprecipitation and RNA-binding protein immunoprecipitation. Lung morphology was evaluated by hematoxylin and eosin staining. MiR-215-5p was upregulated in PQ-induced pulmonary fibrosis in vitro and in vivo. MiR-215-5p silencing relieved PQ-induced pulmonary fibrosis progression by enhancing cell viability and reducing the expression of fibrosis-related markers (COL1A1, COL3A1, and α-SMA). Mechanistically, miR-215-5p directly targeted BMRP2. BMPR2 knockdown abolished the suppressive effects of miR-215-5p knockdown on PQ-induced pulmonary fibrosis. In addition, E2F1 interacted with miR-215-5p promoter and positively regulated miR-215-5p expression. E2F1 downregulation reduced miR-215-5p level and promoted BMPR2 level via regulating TGF-β/Smad3 pathway, and then suppressed PQ-induced pulmonary fibrosis, whereas these effects were compromised by miR-215-5p sufficiency. MiR-215-5p was activated by E2F1 to repress BMPR2 expression and activate TGF-β/Smad3 pathway, which aggravated PQ-induced pulmonary fibrosis progression. Targeting the E2F1/miR-215-5p/BMPR2 axis might be a new approach to alleviate PQ-induced pulmonary fibrosis.

Circ_0123996 promotes the proliferation, inflammation, and fibrosis of mesangial cells by sponging miR-203a-3p to upregulate SOX6 in diabetic nephropathy.

Circular RNAhas been reported to participate in human diseases including diabetic nephropathy (DN). However, the role and mechanism of circ_0123996 in DN need to be further explored. Relative expression levels of circ_0123996, microRNA (miR)-203a-3p, SRY-box 6 (SOX6), and inflammatory cytokines were determined using quantitative real-time PCR. Western blot analysis was used to detect the protein expression of SOX6 and fibrosis-related markers. Cell proliferation was measured using the Cell Counting Kit 8assay. The interaction between miR-203a-3p and circ_0123996 or SOX6 was verified using the dual-luciferase reporter assay. The circ_0123996 and SOX6 expression wereincreased and the miR-203a-3p expression was decreased in high glucose-induced mesangial cells. Silenced circ_0123996 could hinder the proliferation, inflammation, and fibrosis of mesangial cells. In terms of mechanism, circ_0123996 could sponge miR-203a-3p to positively regulate SOX6 expression. Function experiments revealed that miR-203a-3p inhibitor could abolish the regulation of circ_0123996 silencing on mesangial cell proliferation, inflammation, and fibrosis. In addition, the knockdown of SOX6 could inhibit mesangial cell proliferation, inflammation, and fibrosis. Also, SOX6 overexpression could reverse the regulation of circ_0123996 silencing on mesangial cell progression. In summary, our data revealed that circ_0123996 promoted the proliferation, inflammation, and fibrosis of mesangial cells via modulating the miR-203a-3p/SOX6 axis, suggesting that circ_0123996 might be a target for alleviating DN progression.

Minimal Development of Liver Fibrosis in Adult Tolerant Liver Transplant Recipients Late After Immunosuppressive Drug Weaning and Transplantation

Operationally tolerant liver transplant (LTx)-recipients can be weaned off immunosuppressive (IS) drugs without development of graft rejection. However, it is feared that liver fibrosis might develop after complete IS weaning. The purpose of this small single-center study was to assess liver fibrosis in adult tolerant LTx recipients long after LTx and IS weaning. Liver fibrosis was assessed in adult tolerant LTx-recipients (n=9) using noninvasive transient elastography and measurements of multiple pro- and antifibrotic serum markers associated with liver fibrosis. The data was collected for 2 subsequent years; 8 and 9 years after IS weaning and 19 and 20 years after transplantation. Healthy individuals (n=9) matched for age and sex were included as a reference for fibrosis-related serum markers. This study was conducted in accordance with the Declaration of Helsinki and approved by the medical ethics committee of our institution. Transient elastography indicated that 7 of 9 tolerant LTx recipients had no or minimal liver fibrosis (F0-F1), whereas 2 recipients had moderate or severe liver fibrosis (F2-F3). Most fibrosis-related serum markers in tolerant LTx recipients were within or close to the range obtained for healthy individuals. The results from this small, single-center study indicated that most adult tolerant LTx recipients have no or minimal liver graft fibrosis long after transplantation and IS weaning, and their fibrosis-related serum marker profile indicates an absence of a profibrotic status.

MiR-488-5p mitigates hepatic stellate cell activation and hepatic fibrosis via suppressing TET3 expression

Background and aimsNumerous studies have demonstrated that hepatic fibrosis, a progressive condition as an endpoint of multiple chronic hepatic diseases, is largely characterized with the extensive activation of hepatic stellate cells (HSCs). The precise effect of miR-488-5p in HSCs during hepatic fibrosis has not been elucidated.MethodsIn our study, qRT‐PCR was applied to assess the level of miR-488-5p in activated HSCs stimulated by TGF-β1. We built murine liver fibrosis models with carbon tetrachloride (CCl4), high-fat diet (HFD) and bile duct ligation (BDL). In vitro, the effects of miR-488-5p in HSCs were examined through cell proliferation assay and apoptosis. Luciferase reporter assay was applied to identify the underlying target of miR-488-5p. In vivo, the effects of miR-488-5p were explored through mouse liver fibrosis models.ResultsThe reduction of miR-488-5p in the activated HSCs induced by TGF-β1 and three mouse hepatic fibrosis models were identified. The in vitro functional experimentations verified that miR-488-5p restrained expression of fibrosis-related markers and proliferative capacity in HSCs. Mechanically, we identified that miR-488-5p inhibited tet methylcytosine dioxygenase 3 (TET3) expression via straightly binding onto the 3′ UTR of its mRNA, which sequentially restrained the TGF-β/Smad2/3 pathway. TET3 inhibition induced by the overexpression of miR-488-5p reduced extracellular matrix deposition, which contributed to mitigating mouse liver fibrosis.ConclusionWe highlight that miR-488-5p restrains the activation of HSCs and hepatic fibrosis via targeting TET3 which is involved in the TGF-β/Smad2/3 signaling pathway. Collectively, miR-488-5p is identified as a potential therapeutic target for hepatic fibrosis.