Profibrotic Markers Research Articles

Direct Effects of Empagliflozin on Extracellular Matrix Remodelling in Human Cardiac Myofibroblasts: Novel Translational Clues to Explain EMPA-REG OUTCOME Results

BackgroundEmpagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. MethodsCardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. ResultsEmpagliflozin significantly attenuated transforming growth factor β1–induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 μmol/L, 1 μmol/L, and 5 μmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. ConclusionsWe provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

Moderate hyperoxia induces senescence in developing human lung fibroblasts.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.

New treatments/targets for primary biliary cholangitis.

SummaryPrimary biliary cholangitis (PBC) is an autoimmune, cholestatic, chronic liver disease that ultimately progresses towards cirrhosis and liver failure if untreated. While ursodeoxycholic acid has been established as standard of care for PBC in the last few decades, significant advances in second-line treatment options have recently been made and new therapeutic developments are currently under evaluation. The purpose of this article is to provide the clinician with an overview of the current treatment options and future opportunities for patients with PBC.

WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Abstract 518: Pharmacological Inhibition of Human Antigen R (HuR) Blunts fibroblast Activation and Cardiac Fibrosis

Myocardial fibrosis is associated with many cardiac diseases and often leads to a worsened prognosis. In a healthy heart, cardiac fibroblasts are responsible for maintaining the extracellular matrix (ECM). In response to stress or injury, however, the fibroblasts activate to myofibroblasts and begin proliferating, migrating, and depositing excess ECM. This leads to decreased compliance and contractility of the myocardium, furthering the progression to heart failure. Dampening the chronic activation of fibroblasts, therefore, is an attractive target for future cardiac therapeutics. Human Antigen R (HuR) is an RNA binding protein known to stabilize mRNA through binding to AU rich regions in the 3’UTR. Our lab has previously utilized a myocyte-specific knockout of HuR to show that HuR plays an important role in cardiac fibrosis through mediating TGFβ gene expression in the myocytes. Additionally, pharmacological inhibition of HuR markedly reduced interstitial cardiac fibrosis in an 11-week transverse aortic constriction (TAC) mouse model of heart failure, quantifying the fibrosis with Masson’s Trichrome Stains, western blots, and qPCR of profibrotic markers. The goal of this work is to identify the functional role of HuR in mediating pro-fibrotic gene expression and ECM remodeling activity in cardiac fibroblasts. Our results demonstrate that HuR inhibition blunts the activation and wound healing activity of primary adult cardiac fibroblasts. Specifically, utilizing an in vitro scratch/wound healing assay we show that HuR inhibition significantly prolongs wound closure time and blunts the expression of profibrotic genes (e.g. collagen1a1, periostin, TGFb, fibronectin). Ongoing studies will determine the effect of HuR inhibition in a two-week isoproterenol pump (30mg/kg/day) model. Quantification of fibrosis and upstream signaling of fibroblast activation, such as TGFβ-dependent phosphorylation of SMAD2/3, will be assessed to determine the role of HuR in the initiation of fibrotic signaling.

SUN-306 ESTABLISHING EQUIVALENT DIABETES IN MALE AND FEMALE NOS3 DEFICIENT MICE RESULTS IN SIMILAR ONSET OF DIABETIC KIDNEY DISEASE

Streptozotocin (STZ)-induced diabetes in male mice lacking endothelial nitric oxide synthase (Nos3-/-) is a widely used model for examining the development of diabetic kidney disease (DKD). Female mice are partially protected from developing STZ-induced diabetes due to their abundance of 17β-estradiol and lack of testosterone, and are not normally used for studies of DKD. In this study, we sought to identify whether male and female Nos3-/- mice with equivalent diabetes develop comparable renal injury, facilitating the potential to use both sexes in studies of DKD. Male and female Nos3-/- mice were made diabetic with 5 or 6 intraperitoneal injections of STZ (55mg/kg/day), respectively. Groups of male and female mice with equivalent diabetes at week 3 after STZ were assessed for diabetic kidney injury (albuminuria, podocyte loss, inflammation, renal function impairment, glomerulosclerosis, tubular injury) at week 8. STZ-treated male and female Nos3-/- mice maintained similar elevated blood glucose levels between weeks 3 and 8 and had equivalent levels of glycated hemoglobin (HbA1c) and hypertension. At week 8, Urine albumin/creatinine levels were increased 8-fold in both diabetic males and females compared to non-diabetic, and were accompanied by a similar (17-20%) loss of podocytes. In diabetic males and females, plasma cystatin-C levels were increased by 33% at week 8, demonstrating similar renal function impairment. Glomerular area of collagen IV immunostaining was also similarly increased by 30% in both genders, signifying equal development of glomerulosclerosis. In addition, kidney mRNA expression of proinflammatory markers (CD68, TNF-α, CCL2), profibrotic markers (TGF-β, Collagen I, Collagen IV, Fibronectin) and KIM-1 were equally elevated in males and females, indicating similar renal inflammation, renal fibrosis and tubular injury. Our study shows that equivalent diabetes induces a similar onset of DKD in male and female Nos3-/- mice, demonstrating it is possible to include males and females together in studies of DKD.

Recombinant human granulocyte colony-stimulating factor alleviates liver fibrosis in bile duct-ligated mice

Introduction: Biliary atresia (BA) is the leading cause of liver fibrosis and failure in neonates with surgical jaundice, leading to poor outcome. Clinical and animal studies showing that granulocyte colony-stimulating factor (GCSF) treatment could improve liver fibrosis and cirrhosis suggest that GCSF may be offered as a low-cost intervention to improve the course of BA. This study aims to test the hypothesis that 10 µg/kg/day x 5 days of GCSF could improve liver function, reduce molecular pro-fibrotic markers and decrease liver fibrosis in a mouse model of bile duct ligation (BDL).&#x0D; Methods: Balb/c mice underwent Sham surgery, or BDL for seven days followed by subcutaneous GCSF administration at 10 µg/kg/day for five consecutive days. Twelve days post-operation, blood samples were taken from the facial vein for leukocyte/neutrophil count and for measurement of serum enzymatic activities. The median lobe of the liver was acquired for total RNA and protein extraction. Moreover, the median liver lobe was used for hematoxylin-eosin staining, sirius red staining, and for visualization by immunohistochemistry (IHC).&#x0D; Results: Twelve days post-operation, GCSF-treated bile duct-ligated (BDL) mice had a higher survival rate than that of placebo-treated mice (hazard ratio=1.88, p=0.084). The GCSF-treated mice had diminished liver serum transaminase activities (AST: 228.92 ± 222.67 vs. 313.46 ± 164.80 IU/L; ALP: 573.24 ± 177.89 IU/L vs. 471.75 ± 117.92 IU/L). GCSF treatment also reduced fibrosis with down-regulation of expression of pro-fibrotic markers including TGF-β1 (-2.61-fold mRNA), α-SMA (-2.46-fold mRNA; -1.88-fold protein, p&lt;0.001) and collagen (-3.28-fold mRNA; -1.79-fold collagen deposit, p=0.0055). Moreover, GCSF treatment led to an improvement of histological grade and a reduction of extension of ductular structures caused by cholestasis (-1.77-fold CK7-positive bile ducts, p&lt;0.0001; -2.33-fold CK7 positivity, p&lt;0.0001).&#x0D; Conclusion: Administration of GCSF (10 μg/kg/day) for five consecutive days improved the pathological condition of BDL mice. In this study, the positive effect of GCSF could be eventually surpassed due to end-stage liver disease caused from BDL in the mouse model. Further experiments are required to elucidate the effects and mechanisms of GCSF on bile obstruction.&#x0D;

Exposure to the Harmful Algal Bloom (HAB) Toxin Microcystin-LR (MC-LR) Prolongs and Increases Severity of Dextran Sulfate Sodium (DSS)-Induced Colitis

Inflammatory Bowel Disease (IBD) represents a collection of gastrointestinal disorders resulting from genetic and environmental factors. Microcystin-leucine arginine (MC-LR) is a toxin produced by cyanobacteria during algal blooms and demonstrates bioaccumulation in the intestinal tract following ingestion. Little is known about the impact of MC-LR ingestion in individuals with IBD. In this study, we sought to investigate MC-LR’s effects in a dextran sulfate sodium (DSS)-induced colitis model. Mice were separated into four groups: (a) water only (control), (b) DSS followed by water (DSS), (c) water followed by MC-LR (MC-LR), and (d) DSS followed by MC-LR (DSS + MC-LR). DSS resulted in weight loss, splenomegaly, and severe colitis marked by transmural acute inflammation, ulceration, shortened colon length, and bloody stools. DSS + MC-LR mice experienced prolonged weight loss and bloody stools, increased ulceration of colonic mucosa, and shorter colon length as compared with DSS mice. DSS + MC-LR also resulted in greater increases in pro-inflammatory transcripts within colonic tissue (TNF-α, IL-1β, CD40, MCP-1) and the pro-fibrotic marker, PAI-1, as compared to DSS-only ingestion. These findings demonstrate that MC-LR exposure not only prolongs, but also worsens the severity of pre-existing colitis, strengthening evidence of MC-LR as an under-recognized environmental toxin in vulnerable populations, such as those with IBD.

Antibodies against specific extractable nuclear antigens (ENAs) as diagnostic and prognostic tools and inducers of a profibrotic phenotype in cultured human skin fibroblasts: are they functional?

BackgroundThe importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become recognized by their incorporation into the 2013 ACR/EULAR classification criteria. Clear prognostic and phenotypic associations with cutaneous subtype and internal organ involvement have been also described. However, little is known about the potential of autoantibodies to exert a direct pathogenic role in SSc. The aim of the study is to assess the pathogenic capacity of anti-DNA-topoisomerase I (anti-Topo-I) and anti-centromeric protein B (anti-Cenp-B) autoantibodies to induce pro-fibrotic markers in dermal fibroblasts.MethodsDermal fibroblasts were isolated from unaffected and affected skin samples of (n = 10) limited cutaneous SSc (LcSSc) patients, from affected skin samples of diffuse cutaneous (DcSSc) patients (n = 10) and from healthy subjects (n = 20). Fibroblasts were stimulated with anti-Topo-I, anti-Cenp-B IgGs, and control IgGs in ratios 1:100 and 1:200 for 24 h. Cells were also incubated with 10% SSc anti-Topo-I+ and anti-Cenp-B+ whole serum and with 10% control serum for 24 h. Viability was assessed by MTT test, while apoptosis was assessed by flow cytometry. Activation of pro-fibrotic genes ACTA2, COL1A1, and TAGLN was evaluated by quantitative real-time PCR (qPCR), while the respective protein levels alpha-smooth-muscle actin (α-SMA), type-I-collagen (Col-I), and transgelin (SM22) were assessed by immunocytochemistry (ICC).ResultsMTT showed that anti-Cenp-B/anti-Topo-I IgGs and anti-Cenp-B+/anti-Topo-I+ sera reduced viability (in a dilution-dependent manner for IgGs) for all the fibroblast populations. Apoptosis is induced in unaffected LcSSc and control fibroblasts, while affected LcSSc/DcSSc fibroblasts showed apoptosis resistance. Basal mRNA (ACTA2, COL1A1, and TAGLN) and protein (α-SMA, Col-1, and SM22) levels were higher in affected LcSSc/DcSSc fibroblasts compared to LcSSc unaffected and to control ones. Stimulation with anti-Cenp-B/anti-Topo-I IgGs and with anti-Cenp-B+/anti-Topo-I+ sera showed a better induction in unaffected LcSSc and control fibroblasts. However, a statistically significant increase of all pro-fibrotic markers is reported also in affected LcSSc/DcSSc fibroblasts upon stimulation with both IgGs and sera.ConclusionsThis study suggests a pathogenic role of SSc-specific autoantibodies to directly induce pro-fibrotic activation in human dermal fibroblasts. Therefore, besides the diagnostic and prognostic use of those autoantibodies, these data might further justify the importance of immunosuppressive drugs in the early stages of the autoimmune disease, including SSc.

Chronic treatment with C-type natriuretic peptide impacts differently in the aorta of normotensive and hypertensive rats.

The aim of this study was to determine whether exogenous administration of C-type natriuretic peptide (CNP) induces functional and morphological vascular changes in spontaneously hypertensive rats (SHR) compared with normotensive rats. Male 12-week-old normotensive Wistar and SHR were administered with saline (NaCl 0.9%) or CNP (0.75μg/h/100g) for 14days (subcutaneous micro-osmotic pumps). Systolic blood pressure (SBP) was measured in awake animals and renal parameters were evaluated. After decapitation, the aorta was removed, and vascular morphology, profibrotic markers, and vascular reactivity were measured. In addition, nitric oxide (NO) system and oxidative stress were evaluated. After 14-days of treatment, CNP effectively reduced SBP in SHR without changes in renal function. CNP attenuated vascular remodeling in hypertensive rats, diminishing both profibrotic and pro-inflammatory cytokines. Also, CNP activated the vascular NO system and exerted an antioxidant effect in aortic tissue of both groups, diminishing superoxide production and thiobarbituric acid-reactive substances, and increasing glutathione content. These results show that chronic treatment with CNP attenuates the vascular damage development in a model of essential hypertension, inducing changes in fibrotic, inflammatory, oxidative, and NO pathways that could contribute to beneficial long-term effects on vascular morphology, extracellular matrix composition, and function. The knowledge of these effects of CNP could lead to improved therapeutic strategies to not only control BP but also reduce vascular damage, primarily responsible for the risk of cardiovascular events.

MiR-455-3p Alleviates Hepatic Stellate Cell Activation and Liver Fibrosis by Suppressing HSF1 Expression

Liver fibrosis is a common pathological process of end-stage liver diseases. However, the role of microRNA (miRNA) in liver fibrosis is poorly understood. The activated hepatic stellate cells (HSCs) are the major source of fibrogenic cells and play a central role in liver fibrosis. In this study, we investigated the differential expression of miRNAs in resting and transforming growth factor β1 (TGF-β1) activated HSCs by microarray analysis and found that miR-455-3p was significantly downregulated during HSCs activation. In addition, the reduction of miR-455-3p was correlated with liver fibrosis in mice with carbon tetrachloride (CCl4), bile duct ligation (BDL), and high-fat diet (HFD)-induced liver fibrosis. Our functional analyses demonstrated that miR-455-3p inhibited expression of profibrotic markers and cell proliferation in HSCs in vitro. Moreover, miR-455-3p regulated heat shock factor 1 (HSF1) expression by binding to the 3′ UTR of its mRNA directly. Overexpression of HSF1 facilitated HSCs activation and proliferation by promoting heat shock protein 47 (Hsp47) expression, leading to activation of the TGF-β/Smad4 signaling pathway. To explore the clinical potential of miR-455-3p, we injected ago-miR-455-3p into mice with CCl4-, BDL-, and HFD-induced hepatic fibrosis in vivo. The overexpression of miR-455-3p suppressed HSF1 expression and reduced fibrosis marker expression, which resulted in alleviated liver fibrosis in mice. In conclusion, our present study suggests that miR-455-3p inhibits the activation of HSCs through targeting HSF1 involved in the Hsp47/TGF-β/Smad4 signaling pathway. Therefore, miR-455-3p might be a promising therapeutic target for liver fibrosis.

Culture enhanced mesenchymal stromal cells with increased potency

Background & Aim Mesenchymal stromal cells (MSCs) have been pursued for treatment of many inflammatory and fibrotic diseases due to their capacity to modulate immune cell (such as monocyte/macrophage, MΦ) phenotype and to dampen fibrosis. With the goal of developing an MSC product with increased potency, the Viswanathan lab has pioneered a strategy to generate 3D MSC aggregates with enhanced immunomodulatory functions (US62/397,572). The objective of this work is to assess the ability of culture enhanced MSCs to reduce inflammation and fibrosis, and to investigate mechanism of action by probing extracellular vesicle (EV) release and microRNA (miR) expression in 3D versus 2D MSCs. In parallel, hypoxic MSC cultures will also be investigated. Methods, Results & Conclusion MSCs were isolated from human bone marrow and subject to a serum-free 3D cell aggregation protocol (US62/397,572) with conventional 2D culture in serum-free medium or with 10% fetal bovine serum serving as controls. To evaluate MSC potency, gene expression analysis (quantitative PCR) was performed on MSCs licensed with tumour necrosis factor-α (TNFα) or interferon-γ (IFNγ) for 24 h. Results showed that TNFα-licensed 3D MSCs displayed highest expression of the anti-inflammatory marker TNFα-stimulated gene 6 ( TSG6 ) (Fig. 1) which has been correlated to MSC anti-inflammatory function. IFNγ-licensed 3D MSCs displayed reduced transcript levels of pro-inflammatory markers chemokine (C-X-C motif) ligand 8 ( CXCL8 ) and cyclooxygenase-2 ( COX2 ), as well as the pro-fibrotic marker transforming growth factor-β ( TGFβ ). Co-culture of MSCs with human peripheral blood-derived monocytes demonstrated that MSCs promote a shift toward a more homeostatic “M2-like” MΦ phenotype with higher expression of CD163 and CD206 relative to M1 markers CD86 and HLA-DR measured by flow cytometry (Fig. 2). A trend toward increased expression of CD163 was observed in 3D MSC co-cultures relative to serum 2D MSCs (p=0.0802) but no other statistical differences were noted. Taken together, these results demonstrate that 3D MSCs may have improved immunomodulatory capacity and are less fibrotic and inflammatory relative to MSCs cultured in 2D. In ongoing work, we are evaluating EV release and miR expression in enhanced MSCs (3D and hypoxic cultures) and will examine the role of these factors in mediating MΦ polarization and fibroblast phenotype in vitro . The results will aid in developing MSC therapies with increased potency for treatment of inflammatory and fibrotic conditions.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis

HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Influence of inflammasome pathway activation in macrophages on the matrix metalloproteinase expression of human hepatic stellate cells

Inflammasomes are protein complexes that produce IL-1β in response to damage or pathogens. As such, inflammasomes are involved in several types of hepatic fibrosis. However, the mechanisms by which these complexes drive the liver's fibrogenic status remain unclear. We co-cultured differentiated macrophages (the THP-1 cell line or human monocyte-derived macrophages (MDMs)) with human hepatic fibroblasts (either the LX-2 cell line or primary human hepatic stellate cells (HSCs)). The inflammasome pathway was activated with lipopolysaccharide (LPS) and monosodium urate (MSU) crystals, and the HSCs' responses were analyzed. Our results show that co-culture of HSCs with THP-1 cells upregulated transcription of the genes coding for metalloproteinase (MMP)-3 and MMP-9. After inflammasome pathway activation, the HSCs' phenotype was the same in the presence of THP-1 cells or MDMs (i.e. upregulation of MMP-3, MMP-9, and the pro-inflammatory cytokine IL-1β). We found that two cytokines were involved in these changes: IL-1β regulated MMP-3 and IL-1β mRNA expression, whereas TNF-α regulated MMP-9 mRNA expression. Experiments with primary cells revealed that a general inflammatory environment is responsible for the downregulation of pro-fibrotic markers. Our present results suggest that inflammasome pathway activation in macrophages leads to a pro-inflammatory environment for HSCs leading to MMP/TIMP imbalance and enhanced fibrolytic properties.

15-Deoxy-Δ-12, 14-prostaglandin J2 acts cooperatively with prednisolone to reduce TGF-β-induced pro-fibrotic pathways in human osteoarthritis fibroblasts

Background/AimsSynovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear. MaterialsHuman OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays. ResultsPrednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β. ConclusionsPrednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern.

Gender Differences in Cardiac Remodeling Induced by a High-Fat Diet and Lifelong, Low-Dose Cadmium Exposure.

Childhood obesity, which is prevalent in developed countries, is a metabolic risk factor for cardiovascular disease. Cadmium (Cd), a ubiquitous environmental toxic metal, also has deleterious effects on the cardiovascular system. However, the combined effects of a high-fat diet (HFD) and lifelong, low-dose Cd exposure on cardiac remodeling remain unclear. This study aims to determine the effects of combined HFD and Cd exposure on cardiac remodeling, as well as gender-specific differences in the response. C57BL/6J mice were exposed to Cd at a low dose (L-Cd, 0.5 ppm) or high dose (H-Cd, 5 ppm) via drinking water from conception to sacrifice. After being weaned, the offspring mice were fed with a HFD (42% kcal from fat) for an additional 10 weeks. H-Cd exposure significantly increased Cd accumulation in the hearts of both parents and their offspring; a HFD showed no added effects on cardiac Cd content. H-Cd exposure increased cardiac metallothionein protein levels only in female mice, regardless of dietary intake. Histological analysis revealed that H-Cd exposure combined with a HFD induced cardiac hypertrophy and fibrosis only in female mice. This was further supported by elevated expression of ANP and COL1A1 protein levels along with COL1A1, COL1A2, and COL3A1 mRNA levels. Profibrotic markers PAI-1, CTGF, and FN were also elevated in HFD/H-Cd-exposed female mice. Levels of the oxidative stress marker 3-NT significantly increased in the hearts of HFD-fed female mice, whereas Cd exposure showed no additional effects. Of all the antioxidant markers examined, levels of CAT significantly increased in mice fed a HFD, regardless of gender and Cd exposure. In summary, a HFD combined with lifelong, low-dose Cd exposure induces cardiac hypertrophy and fibrosis in female but not male mice, a response that is independent of oxidative stress.

Mesenchymal stem cell therapy inhibited inflammatory and profibrotic pathways induced by partial bladder outlet obstruction and prevented high-pressure urine storage

Partial bladder outlet obstruction (pBOO) is characterized by an initial inflammatory response that progresses to smooth muscle hypertrophyand fibrosis. Current treatment modalities carry high risk of morbidity. Mesenchymal stem cells (MSCs) are undifferentiated adult cells with reparative, immunomodulatory, and anti-inflammatory capacities. The ability of MSCs to inhibit inflammatory and profibrotic pathways in bladder cells has been recently reported. This study aimed to investigate the therapeutic effects of MSCs on pBOO-induced inflammatory, profibrotic signaling pathways and end-organ physiology. Twenty Sprague Dawley rats were randomly assigned to 5 groups: unobstructed controls, pBOO for 2 and 4 weeks, pBOO+MSCs for 2 and 4 weeks. Partial bladder outlet obstruction was surgically induced followed by intravenous injection of MSCs. Endpoint urodynamics was performed, and bladder tissueharvested for analysis. Reverse transcription real time polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to study gene and protein expression of major inflammatory and profibrotic markers. Partial bladder outlet obstruction resulted in an upregulation of transforming growth factor beta (TGFβ1), mothers against decapentaplegic homolog 2/3 (SMAD2/3), hypoxia inducible factor 1 alpha (HIF1α), hypoxia inducible factor 3 alpha (HIF3α), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNFα), mechanistic target of rapamycin (mTOR), p70 ribosomal S6 protein kinase (p70 S6K), collagen 1 (COL1), and collagen 3 (COL3) expression in a time-dependent manner. This was coupled with a downregulation of interleukin (IL)-10 expression. Increase of bladder fibrosis was directly related to the duration of pBOO and associated with high urine storage pressure. Injected MSCs were identified in the bladder 4 weeks after therapy. The immunomodulatory effect of MSCs(defined by reduced TNFα and increased IL-10 and VEGF) was most predominant 2 weeks after therapy. Significant downregulation of profibrotic genes occurred 4 weeks after therapy. End filling pressure, hypertrophy, and fibrosis were significantly reduced after MSCtherapy (P<0.05). Mesenchymal stem cell therapy led to a profound systematic improvement of the obstructed bladder. This included an initial anti-inflammatory response and a subsequent antifibrotic reaction. Essentially, both phases were associated with a reduction of urine storage pressure. The intravenously injected MSCs were tracked in the bladder. However, their presence in non-target organs such as the lungs, spleen, and liver was not tracked. Partial bladder outlet obstruction induced significant upregulation of hypoxic, inflammatory, and profibrotic markers. Mesenchymal stem cell therapy potently inhibited these pathways and improved bladder function.

CT-1 (Cardiotrophin-1)-Gal-3 (Galectin-3) Axis in Cardiac Fibrosis and Inflammation.

Myocardial fibrosis is a main contributor to the development of heart failure (HF). CT-1 (cardiotrophin-1) and Gal-3 (galectin-3) are increased in HF and associated with myocardial fibrosis. The aim of this study is to analyze whether CT-1 regulates Gal-3. Proteomic analysis revealed that Gal-3 was upregulated by CT-1 in human cardiac fibroblasts in parallel with other profibrotic and proinflammatory markers. CT-1 upregulation of Gal-3 was mediated by ERK (extracellular signal-regulated kinase) 1/2 and Stat-3 (signal transducer and activator of transcription 3) pathways. Male Wistar rats and B6CBAF1 mice treated with CT-1 (20 µg/kg per day) presented higher cardiac Gal-3 levels and myocardial fibrosis. In CT-1-treated rats, direct correlations were found between cardiac CT-1 and Gal-3 levels, as well as between Gal-3 and perivascular fibrosis. Gal-3 genetic disruption in human cardiac fibroblasts and pharmacological Gal-3 inhibition in mice prevented the profibrotic and proinflammatory effects of CT-1. Dahl salt-sensitive hypertensive rats with diastolic dysfunction showed increased cardiac CT-1 and Gal-3 expression together with cardiac fibrosis and inflammation. CT-1 and Gal-3 directly correlated with myocardial fibrosis. In HF patients, myocardial and plasma CT-1 and Gal-3 were increased and directly correlated. In addition, HF patients with high CT-1 and Gal-3 plasma levels presented an increased risk of cardiovascular death. Our data suggest that CT-1 upregulates Gal-3 which, in turn, mediates the proinflammatory and profibrotic myocardial effects of CT-1. The elevation of both molecules in HF patients identifies a subgroup of patients with a higher risk of cardiovascular mortality. The CT-1/Gal-3 axis emerges as a candidate therapeutic target and a potential prognostic biomarker in HF.

MicroRNA‑181 exerts an inhibitory role during renal fibrosis by targeting early growth response factor‑1 and attenuating the expression of profibrotic markers.

Progressive renal fibrosis is a common complication of chronic kidney disease that results in end‑stage renal disorder. It is well established that several microRNAs (miRs) function as critical regulators implicated in fibrotic diseases. However, the role of miR‑181 in the development and progression of renal fibrosis remains unclear, and the precise mechanism has not yet been fully defined. The present study identified the functional implications of miR‑181 expression during renal fibrosis. miR‑181 exhibited significantly reduced expression in the serum of renal fibrosis patients and in the kidneys of mice with unilateral ureteral obstruction (UUO). In addition, miR‑181 downregulated the expression of human α‑smooth muscle actin (α‑SMA) in response to angiotensin II stimulation. Transfection with miR‑181 mimics significantly suppressed the expression levels of α‑SMA, connective tissue growth factor, collagen type I α1 (COL1A1) and collagen type III α1 (COL3A1) in NRK49F cells. Notably, early growth response factor‑1 (Egr1) was identified as a direct target gene of miR‑181. Furthermore, invivo experiments revealed that treatment with miR‑181 agonist strongly rescued kidney impairment induced by UUO, as supported by Masson's trichrome staining of kidney tissues and reverse transcription‑quantitative polymerase chain reaction analysis of COL1A1 and COL3A1 mRNA levels. Therefore, miR‑181 may be regarded as an important mediator in the control of profibrotic markers during renal fibrosis via binding to Egr1, and may be a promising new target in the diagnosis and therapy of renal fibrosis.

Prenatal Alcohol Exposure Causes Adverse Cardiac Extracellular Matrix Changes and Dysfunction in Neonatal Mice.

Fetal alcohol syndrome (FAS) is the most severe condition of fetal alcohol spectrum disorders (FASD) and is associated with congenital heart defects. However, more subtle defects such as ventricular wall thinning and cardiac compliance may be overlooked in FASD. Our studies focus on the role of cardiac fibroblasts in the neonatal heart, and how they are affected by prenatal alcohol exposure (PAE). We hypothesize that PAE affects fibroblast function contributing to dysregulated collagen synthesis, which leads to cardiac dysfunction. To investigate these effects, pregnant C57/BL6 mice were intraperitoneally injected with 2.9g EtOH/kg dose to achieve a blood alcohol content of approximately 0.35 on gestation days 6.75 and 7.25. Pups were sacrificed on neonatal day 5 following echocardiography measurements of left ventricular (LV) chamber dimension and function. Hearts were used for primary cardiac fibroblast isolation or protein expression analysis. PAE animals had thinner ventricular walls than saline exposed animals, which was associated with increased LV wall stress and decreased ejection fraction. In isolated fibroblasts, PAE decreased collagen I/III ratio and increased gene expression of profibrotic markers, including α-smooth muscle actin and lysyl oxidase. Notch1 signaling was assessed as a possible mechanism for fibroblast activation, and indicated that gene expression of Notch1 receptor and downstream Hey1 transcription factor were increased. Cardiac tissue analysis revealed decreased collagen I/III ratio and increased protein expression of α-smooth muscle actin and lysyl oxidase. However, Notch1 signaling components decreased in whole heart tissue. Our study demonstrates that PAE caused adverse changes in the cardiac collagen profile and a decline in cardiac function in the neonatal heart.