Profibrotic Markers Research Articles

UDP-glucuronosyltransferases mediate coffee-associated reduction of liver fibrosis in bile duct ligated humanized transgenic UGT1A mice.

Coffee consumption has been shown to reduce the risk of liver fibrosis and is capable of inducing human UDP-glucuronosyltransferase (UGT) 1A genes. UGT1A enzymes act as indirect antioxidants catalyzing the elimination of reactive metabolites, which in turn are potent initiators of profibrotic mechanisms. The aim of this study was to analyze the role of UGT1A genes as effectors of the protective properties of coffee in bile duct ligation (BDL) induced liver fibrosis. Fourteen days BDL with and without coffee pre- and co-treatment was performed in htgUGT1A-WT and htgUGT1A-SNP mice. Hepatic UGT1A mRNA expression levels, serum bilirubin and aminotransferase activities were determined. Liver fibrosis was assessed by collagen deposition, computational analysis of Sirius red tissue staining and expression of profibrotic marker genes. Oxidative stress was measured by hepatic peroxidase concentrations and immunofluorescence staining. UGT1A transcription was differentially activated in the livers of htgUGT1A-WT mice after BDL, in contrast to a reduced or absent induction in the presence of SNPs. Co-treated (coffee + BDL) htgUGT1A-WT-mice showed significantly increased UGT1A expression and protein levels and a considerably higher induction compared to water drinking WT mice (BDL), whereas in co-treated htgUGT1A-SNP mice absolute expression levels remained below those observed in htgUGT1A-WT mice. Collagen deposition, oxidative stress and the expression of profibrotic markers inversely correlated with UGT1A expression levels in htgUGT1A-WT and SNP mice after BDL and coffee + BDL co-treatment. Coffee exerts hepatoprotective and antioxidative effects via activation of UGT1A enzymes. Attenuated hepatic fibrosis as a result of coffee-mediated UGT1A induction during cholestasis was detected, while the protective action of coffee was lower in a common low-function UGT1A SNP haplotype present in 10% of the Caucasian population. This study suggests that coffee consumption might constitute a potential strategy to support the conventional treatment of cholestasis-related liver diseases.

Connective-Tissue Growth Factor Contributes to TGF-β1-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and in the lung. Connective-tissue growth factor (CTGF) exacerbates pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we demonstrate upregulation of CTGF in a rat lung fibrosis model induced by an adenovirus vector encoding active TGF-β1 (AdTGF-β1). We show that CTGF is also upregulated in patients with idiopathic pulmonary fibrosis. Expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on Days 7 and 14 as well as endothelial cells sorted from fibrotic lungs on Days 14 and 28. These findings suggest contributions of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases profibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, the fibrotic extracellular matrix upregulated CTGF expression, compared with the normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for treatment of pulmonary fibrosis.

Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli.

Mounting evidence argues for the significant impact of sex in numerous cardiac pathologies, including myocarditis. Macrophage polarization and activation of cardiac fibroblasts play a key role in myocardial inflammation and remodeling. However, the role of sex in these processes is still poorly understood. In this study, we investigated sex-specific alterations in the polarization of murine bone marrow-derived macrophages (BMMs) and the polarization-related changes in fibroblast activation. Cultured male and female murine BMMs from C57/BL6J mice were polarized into M1 (LPS) and M2 (IL-4/IL-13) macrophages. Furthermore, male and female cardiac fibroblasts from C57/BL6J mice were activated with TNF-α, TGF-β, or conditioned medium from M1 BMMs. We found a significant overexpression of M1 markers (c-fos, NFκB, TNF-α, and IL-1β) and M2 markers (MCP-1 and YM1) in male but not female activated macrophages. In addition, the ROS levels were higher in M1 male BMMs, indicating a stronger polarization. Similarly, the pro-fibrotic markers TGF-β and IL-1β were expressed in activated cardiac male fibroblasts at a significantly higher level than in female fibroblasts. In conclusion, the present study provides strong evidence for the male-specific polarization of BMMs and activation of cardiac fibroblasts in an inflammatory environment. The data show an increased inflammatory response and tissue remodeling in male mice.

Abstract 11611: The Wnt Inhibitor Secreted Frizzled-Related Protein 3 Protects Mitral Valve Endothelium from Mi-Induced Endothelial to Mesenchymal Transition

Introduction: There are changes in the mitral valve (MV) leaflets seen within 2-6 months post-myocardial infarction (MI) that include endothelial to mesenchymal transition (EndMT) and fibrosis. Early modulation of EndMT and fibrosis in MV leaflets may limit ischemic mitral regurgitation and heart failure. The onset of EndMT and fibrosis in MV leaflets and the mechanism of their instigation are not known. Hypothesis: We hypothesized that circulating molecules present in plasma within days after MI incite EndMT and fibrotic processes in MV leaflets. Methods: Using flow cytometric analysis and immunofluorescence staining, we examined the onset of EndMT in 20 adult sheep: 8 sheep with IMI, 8 with sham surgery, and 4 naïve controls with immediate excision of hearts under general anesthesia. The effect of plasma on EndMT induction in primary MV endothelial cells (VECs) was assessed by qPCR, migration assays, and immunofluorescence staining. Plasma cytokine levels were assessed using a cytokine array. Immunoblotting, immunofluorescence staining, RNA sequencing and bioinformatic analysis were used to identify biochemical changes by post-MI plasma. Results: Ovine MVs harvested 8-10 days after inferior MI revealed an onset of EndMT, shown by increased CD31/α-SMA-positive cells in post-MI MV leaflets. In contract to sham plasma, post-MI plasma induced EndMT and pro-fibrotic markers and enhanced migration of primary mitral VECs. Analysis of sham versus post-MI plasma revealed a significant drop in the Wnt antagonist sFRP3 in post-MI plasma. Addition of recombinant sFRP3 to post-MI plasma reversed its EndMT-inducing effect on mitral VECs, measured by restored VE-cadherin, reduced α-SMA, and reduced TGFβ1-3 expression. RNA-seq analysis of mitral VECs exposed to post-MI showed upregulated FOXM1. Blocking FOXM1 with Siomycin A reduced EndMT and pro-fibrotic transcripts in mitral VECs treated with post-MI plasma. Finally, FOXM1 induced by post-MI plasma was downregulated by sFRP3. Conclusions: Reduced sFRP3 in post-MI plasma facilitates EndMT and fibrosis in mitral VECs by increasing the transcription factor FOXM1. Restoring sFRP3 levels and/or inhibiting FOXM1 may provide new strategies to minimize MI-induced maladaptive changes that occur in the MV.

Human Liver Stem Cell-Derived Extracellular Vesicles Target Hepatic Stellate Cells and Attenuate Their Pro-fibrotic Phenotype.

Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanosized membrane vesicles involved in intercellular communication. Our previous studies have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells of the liver with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in the liver. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-β1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of alpha-smooth muscle actin (α-SMA). Conversely, MSC-derived EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target pro-fibrotic transcripts. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and that treatment with HLSC-EVs increased miR-146a-5p expression in LX-2. In conclusion, this study demonstrates that HLSC-EVs can attenuate the activated phenotype of hepatic stellate cells and that their biological effect may be mediated by the delivery of anti-fibrotic miRNAs, such as miR-146a-5p.

Coagulation Factor Xa Induces Proinflammatory Responses in Cardiac Fibroblasts via Activation of Protease-Activated Receptor-1.

Coagulation factor (F) Xa induces proinflammatory responses through activation of protease-activated receptors (PARs). However, the effect of FXa on cardiac fibroblasts (CFs) and the contribution of PARs in FXa-induced cellular signalling in CF has not been fully characterised. To answer these questions, human and rat CFs were incubated with FXa (or TRAP-14, PAR-1 agonist). Gene expression of pro-fibrotic and proinflammatory markers was determined by qRT-PCR after 4 and 24 h. Gene silencing of F2R (PAR-1) and F2RL1 (PAR-2) was achieved using siRNA. MCP-1 protein levels were measured by ELISA of FXa-conditioned media at 24 h. Cell proliferation was assessed after 24 h of incubation with FXa ± SCH79797 (PAR-1 antagonist). In rat CFs, FXa induced upregulation of Ccl2 (MCP-1; >30-fold at 4 h in atrial and ventricular CF) and Il6 (IL-6; ±7-fold at 4 h in ventricular CF). Increased MCP-1 protein levels were detected in FXa-conditioned media at 24 h. In human CF, FXa upregulated the gene expression of CCL2 (>3-fold) and IL6 (>4-fold) at 4 h. Silencing of F2R (PAR-1 gene), but not F2RL1 (PAR-2 gene), downregulated this effect. Selective activation of PAR-1 by TRAP-14 increased CCL2 and IL6 gene expression; this was prevented by F2R (PAR-1 gene) knockdown. Moreover, SCH79797 decreased FXa-induced proliferation after 24 h. In conclusion, our study shows that FXa induces overexpression of proinflammatory genes in human CFs via PAR-1, which was found to be the most abundant PARs isoform in this cell type.

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice.

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor β (TGFβ), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1β (IL-1β) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

Attenuation of Circulating Trimethylamine N-Oxide Prevents the Progression of Cardiac and Renal Dysfunction in a Rat Model of Chronic Cardiorenal Syndrome.

Chronic heart failure (HF) frequently causes progressive decline in kidney function, known as cardiorenal syndrome-2 (CRS2). Current treatment options for CRS2 remain unacceptably limited. Trimethylamine-N-oxide (TMAO), a metabolite of gut microbiota, has recently been implicated in the pathogenesis of both HF and chronic kidney disease. Here we examined whether circulating TMAO is elevated in CRS2 and if so, whether attenuation of circulating TMAO would ameliorate the progression of CRS2. Sprague-Dawley rats underwent surgery for myocardial infarction (MI) or sham (week 0) followed by subtotal (5/6) nephrectomy (STNx) or sham at week 4 to induce CRS2 or control. At week 6, MI + STNx rats and control rats received vehicle or 1.0% 3,3-Dimethyl-1-butanol (DMB, a TMAO inhibitor) treatment for 8 weeks. Compared with control rats, MI + STNx rats exhibited elevated serum TMAO at week 6, which was increased further at week 14 but was attenuated by DMB treatment. MI + STNx rats showed cardiac dysfunction as assessed by echocardiography and renal dysfunction as evidenced by increased serum creatinine and urinary kidney injury molecule-1 and decreased creatinine clearance at week 6. The cardiac and renal dysfunction in MI + STNx rats was exacerbated at week 14 but was prevented by DMB treatment. Molecular and histological studies revealed myocyte hypertrophy and increases in interstitial myocardial fibrosis and gene expression of pro-hypertrophic and pro-fibrotic markers in both heart and kidney at week 14, which were accompanied by elevated gene expression of proinflammatory cytokines. The changes in molecular and histological parameters observed in MI + STNx rats were significantly reduced by DMB treatment. These findings suggest that rats with CRS2 have elevated circulating TMAO, which is associated with the exacerbation of cardiac and renal dysfunction. Attenuation of circulating TMAO can ameliorate cardiac and renal injury and prevents the progression of CRS2.

Features of changes in left ventricular mechanics in patients with epicardial obesity

Abstract Currently, there is no single non-invasive marker that can directly assess left ventricular (LV) diastolic function. Untwist of LV contributes to diastolic suction and early filling. Speckle-tracking imaging can be used to diagnose diastolic dysfunction at the preclinical stage. Objective To study the features of changes in the parameters of LV mechanics and their relationship with the levels of serum markers of myocardial fibrosis in patients with epicardial obesity (EO). Materials and methods The study included 125 men with general obesity. All patients underwent transthoracic echocardiography (ECG) to assess the thickness of epicardial adipose tissue (tEAT) as equivalent to visceral obesity, as well as to diagnose diastolic dysfunction (DD) of left ventricle (LV). According to the results of ECG, the patients were divided into 2 groups: EO(+) with epicardial fat thickness (tEAT) ≥7 mm (n=78); EO(−) with (tEAT) <7 mm (n=40). DD LV was detected in 7 patients, who were subsequently excluded from the analysis. Profibrotic markers in blood serum (MMP-3, collagen I, collagen III, TGF-β, VEGFA, PICP) were determined in all patients using enzyme immunoassay. Using speckle-tracking ECG, the mechanics of LV were studied (twist LV, peak twist ratio LV, time to peak twist of LV, peak untwist ratio LV, time to peak untwist of LV). The exclusion criteria were the presence of coronary pathology, arterial hypertension, and type 2 diabetes mellitus. Results In the group of patients with EO(+) statistically significant increase the level of all studied profibrotic markers was revealed. According to the results of speckle-tracking ECG in the EO(+) group an increase peak untwist ratio LV to −125.56 (−141.0; −117.0)deg/s (p=0.003) and an increase time to peak untwist of LV of 469.44 (509.0; 401.0) msec was determined in comparison with the EO(−) group (p=0.03). A weak statistically significant effect of tEAT on the peak untwist ratio LV in EO(+) group was revealed (r=0.28; p=0.03). In addition, there was a significant relationship between peak untwist ratio LV and markers of myocardial fibrosis MMP-3 (r=0.23; p=0.03) and collagen type III (r=0.25; p=0.04). Conclusion Thus, LV unwinding may be a new non-invasive marker of LV DD at the preclinical stage, since this parameter reflects the mechanical aspect of global diastolic function, especially the early phase of diastole. Funding Acknowledgement Type of funding sources: None.

L-Thyroxine induces left ventricular remodeling and fibrosis in rats by upregulating miR-21 in a reactive oxygen-dependent mechanism: a protective role of N-acetylcysteine

miR-21 is the most studied pro-fibrotic marker in the majority of mammalian tissues. The precise mechanism by which hyperthyroidism induces left ventricular LV fibrosis and remodeling remains unclear. In this study, we have investigated the role of miR-21 on l-thyroxine (l-Thy)-induced cardiac fibrosis in rats. Adult male Sprague–Dawley rats were divided into four groups as control, l-Thy, l-Thy + miR antagomir (inhibitor), and l-Thy + N-acetylcysteine (NAC/glutathione (GSH) precursor). Administration of l-Thy significantly increased mRNA levels of miR-21 in the LVs of the treated rats. Also, it impaired the LV systolic and diastolic function and increased the production of reactive oxygen species (ROS), the transactivation of NF-κB p65, the expression of NRLP3 inflammasome, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in these LVs. Concomitantly, l-Thy increased the ventricular collagen deposition, and stimulated the expression of collagen 1/3, alpha-smooth actin (α-SMA), transforming growth factor-β1, and Smad3/p-Smad3 but suppressed the expression of Smad7. All these effects were reversed by pre-treatment with miR-21 antagomir or co-administration of NAC. In conclusion, l-Thy-induced LV remodeling and fibrosis include a ROS-dependent upregulation of miR-21 which in turns activates NF-κB/NRLP3 inflammasome and suppresses SMad7.

Features of changes in the level of fibrosis markers in lipotoxic myocardial damage in patients with epicardial obesity

Abstract In visceral obesity, lipotoxic myocardial damage develops, which leads to the formation of myocardial fibrosis with impaired diastolic function and the development of heart failure (HF). Currently, the search for markers of early diagnosis of left ventricular (LV) diastolic dysfunction (DD) is relevant. Objective To study the features of changes in the level of fibrosis markers in lipotoxic myocardial damage in patients with epicardial obesity (EO). Materials and methods The study included 125 men with general obesity (BMI average 33.24±3.65 kg / sq. m.). All patients underwent transthoracic echocardiography (ECG) to assess the thickness of epicardial adipose tissue (tEAT) as the equivalent of visceral obesity, as well as to diagnose DD LV. According to the results of echocardiography, the patients were divided into 2 groups: EO(+) with epicardial fat thickness (tEAT) ≥7 mm (n=78); EO(−) with tEAT <7 mm (n=40). DD LV was detected in 7 patients, who were subsequently excluded from the analysis. All patients were assessed for profibrotic markers in the blood serum (MMP-3, collagen I, collagen III, TGF – β, VEGFA, PICP) using enzyme immunoassay. The level of free fatty acids (FFA), as early markers of lipotoxic myocardial damage, was also determined in all patients using enzymelinked immunosorbent assay. The peak untwist ratio LV was studied using speckle-tracking ECG. The exclusive criteria were the presence of coronary pathology, arterial hypertension, type 2 diabetes mellitus, as possible causes of lipotoxic myocardial damage. Results When studying the features of changes in the level of serum markers of myocardial fibrosis, a statistically significant increase in the level of all the studied markers was revealed in the EO(+) group compared to the EO(−) group, and a statistically significant increase in the level of FFA in the EO(+) group compared to the EO(−) group (0.84±0.02 mmol/l and 0.35±0.01 mmol/L, respectively, p=0.02). A statistically significant relationship was found between the levels of FFA and collagen III (r=0.32, p=0.03) and TGF – β (r=0.30, p=0.04). According to the results of speckle-tracking ECG in the EO(+) group, an increase in the rate of LV untwist to −125.56 (−141.0; −117.0) deg/s (p=0.003) was determined. When studying the effect of FFA as a marker of lipotoxic effect on the myocardium, a weak statistically significant relationship was revealed between the level of FFA and the rate of LV untwist (r=0.27; p=0.03) in the EO(+) group, while no such effect was found in the EO(−) group. Conclusion In EO, as a result of neurohumoral disorders, lipotoxic myocardial damage develops and progresses, leading to LV DD, the detection of which is probably possible with the help of the LV unwinding rate. Funding Acknowledgement Type of funding sources: None.

Exercise training induces benign right ventricular hypertrophy along with functional improvement and without pathological processes or arrhythmogenicity in a rodent model of athletes heart

Abstract Introduction Regular sport activity leads to the adaptation of cardiac structure and function, the so-called athlete's heart. Research projects over the last years have focused on exercise-induced adaptation of the right ventricle (RV), because the disproportionate load on the RV - when compared with the left ventricle - might lead to pathological consequences, such as myocardial interstital fibrosis or chamber dilation. Purpose We aimed at investigating comprehensively RV alterations induced by regular aerobic exercise training in a rat model of athlete's heart. Methods Young, adult rats were divided into control (Co) and exercised (Ex) groups (n=12–12). Exercised rats underwent a 12-week-long swim training program. In vivo electrophysiological study and in vitro cellular force assessments on isolated cardiomyocytes were carried out to investigate electrical and functional RV alterations, respectively. Molecular biological (qRT-PCR, Western-blot) and histological investigations were applied to reveal underlying mechanisms. Results Exercise training was associated with increased RV cardiomyocyte diameter (12.5±0.1 μm Co vs. 13.8±0.2 μm Ex, p<0.05), that was associated with hyperphosphorylation of protein kinase B (Akt). RV cardiomyocytes from exercised animals showed improved calcium sensitivity and increased maximal force development, that was associated with hypophosphorylation of troponin I. We found increased length of repolarization as reflected by prolonged QT interval and ventricular effective refracter period (VERP: 44.0±1.6 ms Co vs. 52.8±2.1 ms Ex, p<0.05) along with decreased gene expression of potassium channels (Kcnd2, Kcnj2). We could not induce ventricular arrhythmia by programmed stimulation. Picrosirius staining did not reveal fibrosis, that was associated with unchanged protein expression of connective tissue growth factor (CTGF) and gene expression of profibrotic markers (such as TGF-β). Gene expression of apoptotic markers (Bax, Bcl-2) and fetal gene program (such as β-MHC) did not differ between groups. Conclusions According to our data, regular swim training induced RV hypertrophy, that was associated with functional improvement (improved calcium sensitivity and maximal force), hypophosphorylation of troponin I and prolonged repolarization without characteristic pathological alterations or arrhythmogenicity of RV myocardial tissue. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office of Hungary

Human Liver Stem Cell Derived Extracellular Vesicles Alleviate Kidney Fibrosis by Interfering with the β-Catenin Pathway through miR29b.

Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with β-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The β-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of β-catenin was amplified both in vivo and in vitro, and β-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that β-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of β-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which β-catenin is down regulated by HLSC-EVs-induced miR29b expression.

SPARC knockdown attenuated TGF-β1-induced fibrotic effects through Smad2/3 pathways in human pterygium fibroblasts

PurposeSecreted protein acidic and rich in cysteine (SPARC), a matricellular glycoprotein, has been found to regulate processes involved in fibrotic diseases. The aim of this study was to investigate the anti-fibrotic effects of SPARC in primary human pterygium fibroblasts (HPFs) and elucidate the underlying mechanisms. MethodsThe expression of SPARC in HPFs was knocked down by RNA interference-based approach. Subsequently, we examined the expression of profibrotic markers induced by transforming growth factor-β1 (TGF-β1), including type 1 collagen (COL1), α-smooth muscle actin (α-SMA), and fibronectin (FN). The changes in signaling pathways and matrix metalloproteinases (MMPs) were also detected by western blotting. The cellular migration ability, proliferation ability, apoptosis, and contractile phenotype were detected using the wound healing assay, Cell Counting Kit-8 assay, flow cytometry, and collagen gel contraction assay, respectively. The interaction between SPARC and TGF-β RII was detected by Co-IP ResultsSilencing of SPARC inhibited the basal and TGF-β1-induced expression of COL1, α-SMA, and FN in HPFs, and suppressed the expression of p-Smad2, p-Smad3, Smad4 and MMP2, MMP9. The downregulation of SPARC also attenuated the cell migration and contractile phenotype of HPFs. SPARC could bind to TGF-βRII under TGF-β1 treatment. However, knockdown of SPARC did not affect the proliferation and apoptosis of HPFs. ConclusionSPARC knockdown attenuated the fibrotic effect induced by TGF-β1 at least in part by inactivating the Smad2/3 pathways in HPFs. Therefore, SPARC may be a promising therapeutic target for the treatment of pterygium.

Suppression of collagen IV alpha-2 subunit by prolyl hydroxylase domain inhibition via hypoxia-inducible factor-1 in chronic kidney disease.

Elevation of hypoxia‐inducible factor 1 protein has been shown to be protective in acute kidney injury and HIF1α enhancing drug therapies are currently in clinical trials for the treatment of anemia of chronic kidney disease. Despite its benefits, long‐term HIF1 elevation seems to be associated with additional effects in the kidneys such as tubulointerstitial fibrosis. To better understand the effects of prolonged HIF1 exposure, assessment of baseline and post‐therapy levels of HIF1α and other related biomarkers is essential. In this study, we assessed the effect of HIF1α enhancement using prolyl hydroxylase inhibitor (PHD‐I) DMOG, on a key profibrotic marker of kidney disease. In specific, we examined the change in expression of Collagen 4 subunit A2 in cultured urinary cells of CKD patients pre and post 24‐hour exposure to 1mM DMOG. Our results show that besides HIF1α enhancement, COL4A2 protein is suppressed in presence of DMOG. To determine if this effect is mediated by HIF1, we used HIF1α gene silencing in HEK293 cells and examined the effect of DMOG on protein and gene expression of COL4A2 post 24‐hour exposure. We showed that silencing HIF1α reverses and amplifies the expression of COL4A2 in HEK293 cells. Our data suggest that HIF1 directly regulates the expression of COL4A2 in kidney cells and that HIF1α enhancing therapy has suppressive effects on COL4A2 that may be clinically relevant and must be considered in determining the safety and efficacy of these drugs in the treatment of anemia.

Sleep Apnea in Idiopathic Pulmonary Fibrosis: A Molecular Investigation in an Experimental Model of Fibrosis and Intermittent Hypoxia.

Background: High prevalence of obstructive sleep apnea (OSA) is reported in incident and prevalent forms of idiopathic pulmonary fibrosis (IPF). We previously reported that Intermittent Hypoxia (IH), the major pathogenic element of OSA, worsens experimental lung fibrosis. Our objective was to investigate the molecular mechanisms involved. Methods: Impact of IH was evaluated on C57BL/6J mice developing lung fibrosis after intratracheal instillation of Bleomycin (BLM). Mice were Pre-exposed 14 days to IH before induction of lung fibrosis or Co-challenged with IH and BLM for 14 days. Weight loss and survival were daily monitored. After experimentations, lungs were sampled for histology, and protein and RNA were extracted. Results: Co-challenge or Pre-exposure of IH and BLM induced weight loss, increased tissue injury and collagen deposition, and pro-fibrotic markers. Major worsening effects of IH exposure on lung fibrosis were observed when mice were Pre-exposed to IH before developing lung fibrosis with a strong increase in sXBP1 and ATF6N ER stress markers. Conclusion: Our results showed that IH exacerbates BLM-induced lung fibrosis more markedly when IH precedes lung fibrosis induction, and that this is associated with an enhancement of ER stress markers.

Annexin A1 attenuates cardiac diastolic dysfunction in mice with inflammatory arthritis

Rheumatoid arthritis (RA) carries a twofold increased incidence of heart failure with preserved ejection fraction, accompanied by diastolic dysfunction, which can lead to death. The causes of diastolic dysfunction are unknown, and there are currently no well-characterized animal models for studying these mechanisms. Current medications for RA do not have marked beneficial cardio-protective effects. K/BxN F1 progeny and KRN control mice were analyzed over time for arthritis development, monitoring left ventricular diastolic and systolic function using echocardiography. Excised hearts were analyzed by flow cytometry, qPCR, and histology. In pharmacological experiments, K/BxN F1 mice were treated with human recombinant AnxA1 (hrAnxA1, 1 μg/mouse) or vehicle daily. K/BxN F1 mice exhibited fully developed arthritis with normal cardiac function at 4 wk; however, by week 8, all mice displayed left ventricular diastolic dysfunction with preserved ejection fraction. This dysfunction was associated with cardiac hypertrophy, myocardial inflammation and fibrosis, and inflammatory markers. Daily treatment of K/BxN F1 mice with hrAnxA1 from weeks 4 to 8 halted progression of the diastolic dysfunction. The treatment reduced cardiac transcripts of proinflammatory cytokines and profibrotic markers. At the cellular level, hrAnxA1 decreased activated T cells and increased MHC IIlow macrophage infiltration in K/BxN F1 hearts. Similar effects were obtained when hrAnxA1 was administered from week 8 to week 15. We describe an animal model of inflammatory arthritis that recapitulates the cardiomyopathy of RA. Treatment with hrAnxA1 after disease onset corrected the diastolic dysfunction through modulation of both fibroblast and inflammatory cell phenotype within the heart.

Key role for EphB2 receptor in kidney fibrosis.

Erythropoietin producing hepatocellular (Eph)–Eph receptor interacting (Ephrin) receptor–ligand signaling has been implicated in the development of tissue fibrosis, though it has not been well defined in the kidney. We detected substantial up-regulation of expression and phosphorylation of the EphB2 receptor tyrosine kinase in fibrotic kidney tissue obtained both from mice subjected to the unilateral renal ischemia–reperfusion (IR) model at 14 days and in patients suffering from chronic kidney disease (CKD). Knockout (KO) mice lacking EphB2 expression exhibited a normal renal structure and function, indicating no major role for this receptor in kidney development or action. Although IR injury is well-known to cause tissue damage, fibrosis, and renal dysfunction, we found that kidneys from EphB2KO mice showed much less renal tubular injury and retained a more preserved renal function. IR-injured kidneys from EphB2 KOs exhibited greatly reduced fibrosis and inflammation compared with injured wildtype (WT) littermates, and this correlated with a significant reduction in renal expression of profibrotic molecules, inflammatory cytokines, NADPH oxidases, and markers for cell proliferation, tubular epithelial-to-mesenchymal transition (EMT), myofibroblast activation, and apoptosis. A panel of 760 fibrosis-associated genes were further assessed, revealing that 506 genes in WT mouse kidney following IR injury changed their expression. However, 70.9% of those genes were back to or close to normal in expression when EphB2 was deleted. These data indicate that endogenous EphB2 expression and signaling are abnormally activated after kidney injury and subsequently contribute to the development of renal fibrosis via regulation of multiple profibrotic pathways.

Impact of High Serum Levels of MMP-7, MMP-9, TGF-β and PDGF Macrophage Activation Markers on Severity of COVID-19 in Obese-Diabetic Patients.

ObjectiveThe aim of this study was to identify an association between the severity of COVID-19 in obese-diabetic patients and altered serum levels of MMP-7, MMP-9, TGF-β, and PDGF macrophage activation markers.MethodologyThe study included 70 COVID-19 patients, divided into two groups: Group 1 included: Obese COVID-19 patients with type 2 diabetes mellitus (T2D, n=22 patients) and group 2 included; non-obese, non-diabetic COVID-19 patients as an age- and sex-matched control group (n=48 patients). Serum levels of the tested biomarkers were measured by ELISA at admission and after one weak follow-up.ResultsThere was a significant reduction in the serum levels of LBP in obese-diabetic COVID-19 patients versus the control group (8.34±3.94 vs 20.78±7.61) (p 0.0001). Significant elevation of MMP-7, MMP-9, PDGF and TGF-β was detected in obese diabetic COVID-19 patients compared to the non-obese non-diabetic group: 1044.7±519.6 vs 405.6±164.1, 483.05±46.5 vs 173.31±76.26, 154.5±62.78 vs 39.77±21.52, and 603.05±258.82 vs 180.29±97.17, respectively. The serum levels of macrophage activation markers in obese-diabetic patients one week after admission revealed that patients with acute respiratory distress syndrome (ARDS) had significantly higher serum levels of MMP-7 and MMP-9 than non-ARDS patients (p 0.02 and p 0.01 respectively).ConclusionMacrophages were mainly polarized towards the M2 phenotype in obese-diabetic COVID-19 patients with significant upregulation of the pro-fibrotic markers MMP-7, MMP-9, PDGF, and TGF-β. Thus, high levels of MMP-7 and MMP-9 are associated with ARDS in severe COVID-19 disease among obese-diabetic patients.

Vitamin D3 Attenuates Viral-Induced Inflammation and Fibrotic Responses in Bronchial Smooth Muscle Cells.

Toll-like receptor 3 (TLR3) activation by viral infections plays a key role in promoting inflammatory immune responses that contribute to pulmonary fibrosis in chronic inflammatory respiratory diseases. Vitamin D3 has been shown to be beneficial to patients with asthma and chronic obstructive pulmonary disease (COPD) through its anti-inflammatory and anti-fibrotic properties. Smooth muscle cells are one of the major contributors to airway remodeling in asthma and COPD. We therefore aimed to investigate the effect of vitamin D3 treatment on viral-induced TLR3 responses in Bronchial Smooth Muscle Cells (BSMCs) as a mechanism contributing to pulmonary fibrosis in asthma and COPD. Primary BSMCs from patients with asthma (n=4), COPD (n=4), and healthy control subjects (n=6) were treated with polyinosinic: polycytidylic acid (polyI:C), TLR3 agonist in the presence or absence of vitamin D3 (1,25D3). Here we report the mRNA expression and protein levels of pro-inflammatory and pro-fibrotic markers (IL-6, IFN-β1, CCL2/MCP-1, fibronectin 1 and type I collagen) among BSMCs groups: asthma, COPD, and healthy controls. We show that at the baseline, prior to polyI:C stimulation, asthma and COPD BSMCs presented increased pro-inflammatory and pro-fibrotic state compared to healthy control subjects, as measured by quantitative PCR and immunoassays (ELISA/Flow Cytometry. Ligation of TLR3 by polyI:C in BSMCs was associated with increased TLR3 mRNA expression, and 1,25D3 treatment significantly reduced its expression. In addition, 1,25D3 decreased the expression of IL-6, IFN-β1, CCL2, FN1 and COL1A1 induced by polyI:C in BSMCs. The regulatory effect of 1,25D3 treatment on polyI:C-stimulated BSMCs was further confirmed at protein levels. Our findings suggest that vitamin D3 attenuates TLR3 agonist-induced inflammatory and fibrotic responses in BSMCs and support the clinical relevance of vitamin D3 supplementation in patients with viral infections having chronic respiratory diseases, such as asthma and COPD.