Expression Of Fibrotic Markers Research Articles

Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis.

Does activin A contribute to testicular fibrosis under inflammatory conditions? Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n= 6), 50 (n= 6) and 80 (n= 5) days after first immunization. Age-matched mice injected with adjuvant alone (n= 14) and untreated mice (n= 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n= 3) or vector with an empty cassette (empty vector controls; n= 2) 30days prior to the first immunization, as well as appropriate adjuvant (n= 2) and untreated (n = 2) controls, were also examined.Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n= 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n= 7).Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. Production of activin A by SC was stimulated by 25 and 50ng/ml TNF (P< 0.01, P< 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P< 0.05), accompanied by increased total collagen (P< 0.01) and fibronectin deposition. Total testicular collagen (P< 0.0001) and fibronectin protein expression (P< 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r= 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P< 0.05) and the production of collagen I (P< 0.001; P< 0.01) and fibronectin (P< 0.05). Moreover, activin A also increased collagen IV mRNA (P< 0.05) in PTC, while αSMA mRNA (P< 0.01) and protein (P< 0.0001) were significantly increased by activin A in NIH 3T3 cells. N/A. A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government's Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1-2) on `Molecular pathogenesis on male reproductive disorders' funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

Combined treatment of low-level laser therapy and phloroglucinol for inhibition of fibrosis.

Fibrosis is a highly prevalent disease, which is responsible for 45% of deaths through pathological effects in developed countries. Previous studies have reported that low-level laser therapy (LLLT) can modulate fibrotic activity, but significant enhancement of therapeutic efficacy is still required for clinical translation. The aim of this study is to evaluate the feasible effect of LLLT combined with phloroglucinol (PHL) on the inhibition of fibrosis in vitro. NIH/3T3 murine embryonic fibroblasts cells were cultured and transforming growth factor-β1 (TGF-β1) was treated for transition of fibroblasts. After TGF-β1 treatment, LLLT and PHL were used, respectively, and in combination to suppress fibrosis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and BrdU assays were performed to estimate the cell viability and proliferation. To evaluate the expression of fibrotic markers, we used confocal immunofluorescence and western blot. When compared with respectively treated groups, the group with the combined treatment of LLLT and PHL significantly reduced cell viability and proliferation. Immunofluorescence staining showed that the combined group minimized more α-smooth muscle actin (α-SMA) and type I collagen than the other groups. Western blot analysis showed that the combined treatment had significant decreases in α-SMA, TGF-β1, and type I collagen. PHL-assisted LLLT may be an effective treatment to inhibit fibrosis due to its additive effects. The combined treatment has a potential to be an alternative treatment for fibrosis. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Role of Calbindin-D28k in Diabetes-Associated Advanced Glycation End-Products-Induced Renal Proximal Tubule Cell Injury

Diabetes-associated advanced glycation end-products (AGEs) can increase extracellular matrix (ECM) expression and induce renal fibrosis. Calbindin-D28k, which plays a role in calcium reabsorption in renal distal convoluted tubules, is increased in a diabetic kidney. The role of calbindin-D28k in diabetic nephropathy still remains unclear. Here, calbindin-D28k protein expression was unexpectedly induced in the renal tubules of db/db diabetic mice. AGEs induced the calbindin-D28k expression in human renal proximal tubule cells (HK2), but not in mesangial cells. AGEs induced the expression of fibrotic molecules, ECM proteins, epithelial-mesenchymal transition (EMT) markers, and endoplasmic reticulum (ER) stress-related molecules in HK2 cells, which could be inhibited by a receptor for AGE (RAGE) neutralizing antibody. Calbindin-D28k knockdown by siRNA transfection reduced the cell viability and obviously enhanced the protein expressions of fibrotic factors, EMT markers, and ER stress-related molecules in AGEs-treated HK2 cells. Chemical chaperone 4-Phenylbutyric acid counteracted the AGEs-induced ER stress and ECM and EMT markers expressions. Calbindin-D28k siRNA in vivo delivery could enhance renal fibrosis in db/db diabetic mice. These findings suggest that inducible calbindin-D28k protects against AGEs/RAGE axis-induced ER stress-activated ECM induction and cell injury in renal proximal tubule cells.

Tongxinluo Attenuates Myocardiac Fibrosis after Acute Myocardial Infarction in Rats via Inhibition of Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndMT) is an essential mechanism in myocardial fibrosis (MF). Tongxinluo (TXL) has been confirmed to protect the endothelium against reperfusion injury after acute myocardial infarction (AMI). However, whether TXL can inhibit MF after AMI via inhibiting EndMT remained unknown. This study aims to identify the role of EndMT in MF after AMI as well as the protective effects and underlying mechanisms of TXL on MF. The AMI model was established in rats by ligating left anterior descending coronary artery. Then, rats were administered with high- (0.8 g·kg−1·d−1), mid- (0.4 g·kg−1·d−1), and low- (0.2 g·kg−1·d−1) dose Tongxinluo and benazepril for 4 weeks, respectively. Cardiac function, infarct size, MF, and related indicators of EndMT were measured. In vitro, human cardiac microvascular endothelial cells (HCMECs) were pretreated with TXL for 4 h and then incubated in hypoxia conditions for 3 days to induce EndMT. Under this hypoxic condition, neuregulin-1 (NRG-1) siRNA were further applied to silence NRG-1 expression. Immunofluorescence microscopy was used to assess expression of endothelial marker of vWF and fibrotic marker of Vimentin. Related factors of EndMT were determined by Western blot analysis. TXL treatment significantly improved cardiac function, ameliorated MF, reduced collagen of fibrosis area (types I and III collagen) and limited excessive extracellular matrix deposition (mmp2 and mmp9). In addition, TXL inhibited EndMT in cardiac tissue and hypoxia-induced HCMECs. In hypoxia-induced HCMECs, TXL increased the expression of endothelial markers, whereas decreasing the expression of fibrotic markers, partially through enhanced expressions of NRG-1, phosphorylation of ErbB2, ErbB4, AKT, and downregulated expressions of hypoxia inducible factor-1a and transcription factor snail. After NRG-1 knockdown, the protective effect of TXL on HCMEC was partially abolished. In conclusion, TXL attenuates MF after AMI by inhibiting EndMT and through activating the NRG-1/ErbB- PI3K/AKT signalling cascade.

Disconnect between Fibrotic Response and Right Ventricular Dysfunction.

Rationale: Remodeling and fibrosis of the right ventricle (RV) may cause RV dysfunction and poor survival in patients with pulmonary hypertension. Objectives: To investigate the consequences of RV fibrosis modulation and the accompanying cellular changes on RV function. Methods: Expression of fibrotic markers was assessed in the RV of patients with pulmonary hypertension, the murine pulmonary artery banding, and rat monocrotaline and Sugen5416/hypoxia models. Invasive hemodynamic and echocardiographic assessment was performed on galectin-3 knockout or inhibitor-treated mice. Measurements and Main Results: Established fibrosis was characterized by marked expression of galectin-3 and an enhanced number of proliferating RV fibroblasts. Galectin-3 genetic and pharmacologic inhibition or antifibrotic treatment with pirfenidone significantly diminished RV fibrosis progression in the pulmonary artery banding model, without improving RV functional parameters. RV fibrotic regions were populated with mesenchymal cells coexpressing vimentin and PDGFRα (platelet-derived growth factor receptor-α), but generally lacked αSMA (α-smooth muscle actin) positivity. Serum levels of galectin-3 were increased in patients with idiopathic pulmonary arterial hypertension but did not correlate with cardiac function. No changes of galectin-3 expression were observed in the lungs. Conclusions: We identified extrapulmonary galectin-3 as an important mediator that drives RV fibrosis in pulmonary hypertension through the expansion of PDGFRα/vimentin-expressing cardiac fibroblasts. However, interventions effectively targeting fibrosis lack significant beneficial effects on RV function.

C1q/tumor necrosis factor-related protein-3 improves renal fibrosis via inhibiting notch signaling pathways.

C1q/tumor necrosis factor-related protein-3 (CTRP3) has been extensively reported as an important role involved in antifibrosis, antiapoptosis, and anti-inflammation. However, the role of CTRP3 involved in renal fibrosis remains unclear. Our current study explored the role of CTRP3 in renal fibrosis and its underlying mechanisms by using serums and renal biopsy specimens from renal fibrosis patients and control subjects, rats models with the surgery of unilateral ureteral obstruction (UUO) and human renal proximal tubular epithelial cells (HRPTEpiCs). We found that circulating levels of CTRP3 had no significant difference between renal fibrosis patients and healthy subjects; however, renal CTRP3 expression was markedly downregulated in the fibrotic region with an abundant expression of collagen-I. In UUO rat models, circulating levels of CTRP3 have not changed with the prolonged obstruction of the kidney; renal CTRP3 expression was decreased with the severity of renal fibrosis; adenovirus-mediated CTRP3 treatment inhibited renal interstitial fibrosis. In vitro experiments revealed that CTRP3 attenuates TGF-β1 induced tubular epithelial cells fibrotic changes; CTRP3 knockdown facilitates the expression of fibrotic markers in TGF-β1-induced HRPTEpiCs; recombinant CTRP3 or adenovirus-mediated CTRP3 overexpression significantly inhibited the Notch signaling pathway-associated factors, and knockdown of CTRP3 increased TGF-β1-mediated activation of the Notch signaling pathways. Collectively, our current study found that CTRP3 could improve renal fibrosis, to some extent, through inhibiting the Notch pathway.

Alamandine attenuates long‑term hypertension‑induced cardiac fibrosis independent of blood pressure.

Cardiac fibrosis secondary to long-term hypertension is known to promote cardiac dysfunction; however, few therapeutic agents are available for the treatment of this condition in clinical practice. The heptapeptide alamandine (Ala) has recently been identified as a component of the renin-angiotensin system (RAS), which exerts a protective effect against cardiac hypertrophy; however, it is unknown whether Ala may also be useful for the treatment of cardiac fibrosis. In the present study, the potential therapeutic effects of Ala on long-term hypertension-induced cardiac fibrosis were investigated in an aged, spontaneous hypertensive rat model. Weekly blood pressure (BP) measurements revealed that daily Ala treatment significantly decreased the systolic, diastolic and mean arterial BP compared with the control. Of note, the observed reduction in BP in Ala-treated animals markedly differed to that observed in rats treated with hydralazine (Hyd). Echocardiography further demonstrated that Ala treatment decreased the ratio of left ventricle mass to body weight, and alleviated structural and functional parameters associated with cardiac fibrosis, including left ventricular volume, ejection fraction and fractional shortening compared with the control and Hyd-treated groups. Furthermore, Ala deceased the density of cardiac fibrosis, as assessed by Masson and Sirius red staining; reduced expression of fibrotic proteins, including connective tissue growth factor, collagen I (COL1A1) and matrix metalloproteinase 9, was also observed. In addition, Ala treatment further decreased the expression of angiotensin II-induced fibrotic markers at the mRNA and protein levels in cultured cardiac fibroblasts; Ala-mediated inhibition of COL1A1 expression and Akt phosphorylation was inhibited via the Mas-related G protein receptor antagonist, PD123319. Collectively, the findings of the present study suggest that Ala is an effective anti-hypertensive peptide that can attenuate cardiac dysfunction and fibrosis induced by chronic hypertension, independent of BP.

The antifibrotic and anti-inflammatory effects of icariin on the kidney in a unilateral ureteral obstruction mouse model

BackgroundThe pathology change of renal tubulointerstitial fibrosis is a critical feature of chronic kidney disease (CKD), regardless of the primary insults. The infiltration of inflammatory cells and the consecutive secretion of profibrotic factors are frequently and conspicuously observed during the development of renal fibrosis. Icariin, an active polyphenol of the Epimedium genus, has been found to alleviate the symptoms of chronic diseases like diabetes, neurodegeneration, and heart and renal diseases. The effect and mechanism of icariin on the prevention of CKD-associated renal fibrosis still needed clarification. PurposeThe aims of this study were to investigate whether icariin treatment improves the development of CKD-associated renal fibrosis and its possible mechanism. MethodsAn experimental unilateral ureteral obstruction (UUO)-induced chronic renal fibrosis mouse model was used. Mice were orally administered with icariin (20 mg/kg/day) for 3 consecutive days before and 14 consecutive days after UUO surgery. ResultsThe pathological changes, collagen deposition, and protein expressions of profibrotic factors (transforming growth factor-β and connective tissue growth factor) and fibrotic markers (α-smooth muscle actin and fibronectin), which were significantly elevated in the kidneys of UUO mice, could be significantly reversed by icariin treatment. Icariin treatment also significantly inhibited the increased Smad2/3 and decreased E-cadherin protein expressions in the kidneys of UUO mice. Icariin treatment prominently mitigated the protein expression of proinflammatory factors like nuclear factor-κB, cyclooxygenase-2, interleukin 1-β and prooxidative enzyme (NADPH oxidase-4), and it increased the protein expression of antioxidative enzymes (superoxide dismutase and catalase). ConclusionIcariin treatment protects against CKD-associated renal fibrosis via its antifibrotic and anti-inflammatory properties. Icariin may serve as a therapeutic agent in the prevention of CKD-associated renal fibrosis.

A Dual Farnesoid X Receptor Agonist /Soluble Epoxide Hydrolase Inhibitor Prevents Non‐Alcoholic Steatohepatitis in Mice

Non‐alcoholic steatohepatitis (NASH) is becoming the most prevalent metabolic liver disorder and is a serious global health burden. Farnesoid X receptor (FXR) agonists are in late stage clinical development to counter the disease and promise therapeutic efficacy. However, the multi‐factorial nature of NASH pathogenesis and progression indicate the need for a therapeutic approach that will target multiple pathophysiological aspects of NASH. We have developed a dual FXR agonist/soluble epoxide hydrolase (sEH) inhibitor (FXRA/sEHI) and investigated its ability to prevent NASH in toxin‐ and diet‐induced mouse NASH models. Toxin‐induced NASH was achieved in male C57BL/6 mice by injecting 200 μg streptozotocin (STZ) solution two days after birth and feeding a high‐fat diet after 4 weeks of age. Diet‐induced NASH was carried out in adult (8–12 weeks old) male C57BL/6J mice fed a methionine‐choline deficient diet (MCD; D518810) and was compared to mice fed a composition‐matched methionine‐choline replete control diet (D518754) for 4 weeks. In toxin‐induced NASH model, mice were orally treated with vehicle or FXRA/sEHI (10 mg/kg, thrice daily) or an FXR agonist obeticholic acid (OCA, 10 mg/kg, once daily) for 4 weeks. The mice from diet‐induced NASH model treated with FXRA/sEHI (10 mg/kg/d) or vehicle given in drinking water for 4 weeks. At the end of the 4‐week treatment period, blood was collected for biochemical analysis, liver tissue was collected for mRNA expression and histopathological analysis. Mice of the toxin‐induced NASH model had higher liver enzyme, hyperlipidemia, liver fibrosis, and steatosis. FXRA/sEHI treatment reduced these parameters (by 20–50%, P&lt;0.05), while OCA displayed weak or no beneficial effect on NASH (P&gt;0.05). In diet‐induced NASH model, the mice exhibited 2‐fold higher hepatic fibrosis, 2–6‐fold higher hepatic mRNA expressions of inflammatory and fibrotic markers, and marked hepatic steatosis (P&lt;0.05). The dual modulator FXRA/sEHI robustly prevented hepatic steatosis, fibrosis, and inflammation (by 50–80%, P&lt;0.05) compared to vehicle. Our results demonstrate strong potential for dual FXRA/sEHI modulators as a novel therapeutic strategy in NASH and advocate multi‐targeted approaches in NASH treatment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blockade of Cardiac Hypertrophy and Fibrosis by TGF‐Beta 1 Receptor Antagonist in Npr1 Gene‐Knockout Mice

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) gene (Npr1) in mice triggers cardiac hypertrophy, fibrosis, and remodeling associated with heart failure. However, the specific mechanisms of cardiac defects in Npr1 null mutant mice is not well understood. The objective of this study was to determine the mechanisms regulating the development of cardiac hypertrophy and fibrosis in Npr1 gene‐knockout mice. The Npr1 null mutant (Npr1−/−, 0‐copy), heterozygous (Npr1+/−, 1‐copy), and wild‐type (Npr1+/+, 2‐copy) mice were orally administered with transforming growth factor‐beta 1 (TGF‐β1) receptor antagonist GW788388 (1 mg/kg/day) for 28 days, the hearts were isolated, weighted, and used for quantification of hypertrophic and fibrotic markers using real time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and Western blot analyses. Together, systolic blood pressure (SBP), heart weight‐to‐body weight (HW/BW) ratio, and cardiac fibrosis were analyzed. HW/BW ratio was significantly higher in Npr1 knockout mice than wild‐type control mice. The Npr1−/− mice showed significant induction of cardiac hypertrophy and fibrosis with markedly induced expression of hypertrophic marker collagen‐1α (3.5‐fold) and fibrotic markers monocyte chemoattractant protein (4‐fold), connective tissue growth factor (CTGF, 5‐fold), alpha‐smooth muscle actin (α‐SMA, 4‐fold), and SMAD proteins (SMAD‐2, 5‐fold; SMAD‐3, 3‐fold) compared with wild‐type control mice. In addition, the expression of phosphorylated extracellular‐regulated kinase 1/2 (pERK1/2) proteins were also increased in Npr1−/−mice. On the other hand, Npr1−/−mice treated with GW788388 showed significant inhibition of cardiac hypertrophy and fibrosis and reduced expression of fibrotic markers and SMAD proteins compared to vehicle‐treated wild‐type mice. In contrast, the expressions of pERK1/2 proteins were unaffected in drug‐treated animals. The results of the present study suggest that development of cardiac hypertrophy and fibrosis in Npr1−/− mice is regulated independently through TGF‐β1‐mediated SMAD‐dependent pathways.Support or Funding InformationThis work was supported by NIH grants (HL057531 and HL062147).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wedelolactone Attenuates Pulmonary Fibrosis Partly Through Activating AMPK and Regulating Raf-MAPKs Signaling Pathway.

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, there is currently no effective clinical drug treatment. It has been reported that the ethanol extract of Eclipta prostrata L. can improve the lung collagen deposition and fibrosis pathology induced by bleomycin (BLM) in mice. In the present study, we studied whether wedelolactone (WEL), a major coumarin ingredient of E. prostrata, provided protection against BLM-induced pulmonary fibrosis. ICR or C57/BL6 strain mice were treated with BLM to establish lung fibrosis model. WEL (2 or 10 mg/kg) was given daily via intragastric administration for 2 weeks starting at 7-day after intratracheal instillation. WEL at 10 mg/kg significantly reduced BLM-induced inflammatory cells infiltration, pro-inflammatory factors expression, and collagen deposition in lung tissues. Additionally, treatment with WEL also impaired BLM-induced increases in fibrotic marker expression (collagen I and α-SMA) and decrease in an anti-fibrotic marker (E-cadherin). Treatment with WEL significantly prevented BLM-induced increase in TGF-β1 and Smad2/3 phosphorylation in the lungs. WEL administration (10 mg/kg) also significantly promoted AMPK activation compared to model group in BLM-treated mice. Further investigation indicated that activation of AMPK by WEL can suppressed the transdifferentiation of primary lung fibroblasts and the epithelial mesenchymal transition (EMT) of alveolar epithelial cells, the inhibitive effects of WEL was significantly blocked by an AMPK inhibitor (compound C) in vitro. Together, these results suggest that activation of AMPK by WEL followed by reduction in TGFβ1/Raf-MAPK signaling pathways may have a therapeutic potential in pulmonary fibrosis.

Spirulina supplementation in a mouse model of diet-induced liver fibrosis reduced the pro-inflammatory response of splenocytes.

Treatment of liver fibrosis is very limited as there is currently no effective anti-fibrotic therapy. Spirulina platensis (SP) is a blue-green alga that is widely supplemented in healthy foods. The objective of this study was to determine whether SP supplementation can prevent obesity-induced liver fibrosis in vivo. Male C57BL/6J mice were randomly assigned to a low-fat or a high-fat (HF)/high-sucrose/high-cholesterol diet or an HF diet supplemented with 2·5 % SP (w/w) (HF/SP) for 16 or 20 weeks. There were no significant differences in body weight, activity, energy expenditure, serum lipids or glucose tolerance between mice on HF and HF/SP diets. However, plasma alanine aminotransferase level was significantly reduced by SP at 16 weeks. Expression of fibrotic markers and trichrome stains showed no differences between HF and HF/SP. Splenocytes isolated from HF/SP fed mice had lower inflammatory gene expression and cytokine secretion compared with splenocytes from HF-fed mice. SP supplementation did not attenuate HF-induced liver fibrosis. However, the expression and secretion of inflammatory genes in splenocytes were significantly reduced by SP supplementation, demonstrating the anti-inflammatory effects of SP in vivo. Although SP did not show appreciable effect on the prevention of liver fibrosis in this mouse model, it may be beneficial for other inflammatory conditions.

Oxalate induces type II epithelial to mesenchymal transition (EMT) in inner medullary collecting duct cells (IMCD) in vitro and stimulate the expression of osteogenic and fibrotic markers in kidney medulla in vivo.

EMT occurs in response to a number of stresses conditions as mechanical stretch, cancer, hypoxia, oxidative stress (ROS), among others. EMT describes a phenotypical change induced in epithelial cells. It is characterized by increases in motility, extracellular matrix synthesis, proliferation, and invasiveness. The present study analyzed if oxalate ions (Ox) could induce EMT in IMCD cells. Ox (0.5 mM) and transforming growth factor beta (TGF-β1 20 ng/mL) exposition during 48 hours increased migration and invasiveness, increased mesenchymal marker expression (Vimentin, alpha-smooth muscle actin: α-SMA, TGF-β1) and decreased epithelial marker expression (E-cadherin). IMCD stimulated with Ox and TGF-β1 and then exposed to the osteogenic medium during 15 days significantly increased early osteogenic markers (RUNX-2 and Alkaline Phosphatase) expression. Hyperoxaluric mice fed with trans-4-hydroxy-L-proline (HPL) presented calcium oxalate crystal excretion, increased in TGF-β1 expression and collagen fibers deposition and increased early osteogenic markers (RUNX-2 and Alkaline Phosphatase) at 60 days. Our in vitro and in vivo results suggest that oxalate induces EMT in inner medulla collecting duct cells and it may be involved in fibrotic tissue development, osteogenic differentiation and calcium crystal production both implicated in nephrolithiasis.

RAAVrh74.MCK.GALGT2 Protects against Loss of Hemodynamic Function in the Aging mdx Mouse Heart.

Dilated cardiomyopathy is a common cause of death in patients with Duchenne muscular dystrophy (DMD). Gene therapies forDMD must, therefore, have a therapeutic impact in cardiac as well as skeletal muscles. Our previous studies have shown that GALGT2 overexpression in mdx skeletal muscles can prevent muscle damage. Here we have tested whether rAAVrh74.MCK.GALGT2 gene therapy in mdx cardiac muscle can prevent the loss of heart function. Treatment of mdx hearts with rAAVrh74.MCK.GALGT2 1day after birth did not negatively alter hemodynamic function, tested at 3months of age, and it prevented early left ventricular remodeling and expression of fibrotic gene markers. Intravenous treatment of mdx mice with rAAVrh74.MCK.GALGT2 at 2months of age significantly improved stroke volume and cardiac output compared to mock-treated mice analyzed at 17months, both at rest and after stimulation with dobutamine. rAAVrh74.MCK.GALGT2 treatment of mdx heart correlated with increased glycosylation of α-dystroglycan with the CT glycan and increased utrophin protein expression. These data provide the first demonstration that GALGT2 overexpression can inhibit the loss of cardiac function in the dystrophin-deficient heart and, thus, may benefit both cardiac and skeletal muscles in DMD patients.

SIRT1 Attenuates Kidney Disorders in Male Offspring Due to Maternal High-Fat Diet.

Maternal obesity has been associated with kidney disorders in male offspring. Our previous studies have demonstrated that Sirtuin (SIRT)1, an essential regulator of metabolic stress responses, is suppressed in the offspring as the result of maternal high-fat diet (HFD) consumption, which is likely to underpin the adverse metabolic and renal outcomes. To examine if SIRT1 overexpression or activation early in life can protect the offspring kidney, wild-type (WT) and transgenic (Tg) offspring were born to the same diet-induced obese female C57BL/6 mice through breeding with hemizygous SIRT1-transgenic (Tg) male mice and examined for renal pathological changes. In separate experiments, SIRT1 activator SRT1720 (25 mg/kg/2 days i.p) was administrated in WT offspring over 6 weeks of postnatal high-fat diet exposure. The results show that offspring born to obese dams have increased kidney weight, higher levels of renal triglycerides, and increased expression of oxidative stress, inflammatory, and fibrotic markers, as well as increased albuminuria compared to offspring of control dams. Both SIRT1 overexpression and SRT1720 treatment attenuated renal lipid contents and expression of lipogenesis, oxidative stress, and inflammatory markers; however, fibrosis was modestly reduced and albuminuria was not affected. The findings suggest that SIRT1 therapy can ameliorate some pathological mechanisms of kidney programming due to maternal obesity but may not be sufficient to prevent the resulting chronic kidney injury.

NLR Family Pyrin Domain-Containing 3 Inflammasome Activation in Hepatic Stellate Cells Induces Liver Fibrosis in Mice.

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC).Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.

Calcitriol and Punica Granatum Extract Concomitantly Attenuate Cardiomyopathy of Diabetic Mother Rats and Their Neonates via Activation of Raf/MEK/ERK Signalling and Mitigation of Apoptotic Pathways

We investigated the detrimental effects of diabetes on myocardium of pregestational streptozotocin (STZ)-diabetic mother rats and their neonates via evaluations of oxidative redox, inflammatory and apoptotic pathways, also aiming to characterize whether calcitriol and/or pomegranate peel extract confer myocardial protection in hyperglycaemic dams and their foetuses via modulation of the Raf/ MEK/ERK cascade. Sixty Sprague-Dawley female rats were randomized into five groups (N = 12): control, diabetic, diabetic treated with calcitriol and/or pomegranate peel extract (PPE), and mated with non-diabetic healthy males. After confirmation of pregnancy, treatments were kept until gestational day (E-18). Serum and cardiac tissues of mothers and foetuses were collected and processed for biochemical, histopathological, and molecular assessments. We observed that, compared to the control, diabetic mothers showed dramatically increased hyperglycaemia and hyperlipidaemia associated with decreased myocardial functions and disrupted maternal performance. Also, diabetic mothers and their neonates exhibited elevated levels of myocardial injury (troponin I, endothelin 1, creatine kinase-MB, lactate dehydrogenase), with increased pro-inflammatory cytokines (interleukin 1, interleukin 1β, transforming growth factor β) and oxidative redox. Concurrently, the MAPK pathway was significantly down-regulated with increased myocardial apoptotic activity. Furthermore, mRNA expression of angiogenic and fibrotic markers was significantly increased. Paradoxically, calcitriol and/or pomegranate peel extract alleviated these diabetic myocardial insults and normalized the aforementioned assayed parameters. Our findings hypothesized that calcitriol and/or pomegranate peel extract exerted cardioameliorative impacts due to their unique anti-oxidative and anti-inflammatory properties, and thus may be a promising treatment that directly targets the secondary myocardial complications of diabetes in dams and their offspring.

Potential Anti-Fibrotic Effect of Direct Acting Antiviral Drugs on CCl4 Induced Hepatic Fibrosis in Rats

Background: Hepatic fibrosis is a hall mark of chronic liver diseases such as chronic HCV. Direct acting antiviral (DAA) regimens such as Sofosbuvir (SOF) and daclatasvir (DAC) have been found to be associated with decreased fibrotic markers in HCV patients. It is not clear however the reported antifibrotic effect is antiviral dependent or not. Aim: This study investigated the effect of SOF and DAC in hepatic fibrosis induced by CCl4 in rats. Method: Hepatic fibrosis was induced by (0.5 ml/kg) CCl4 IP twice a week for six weeks. SOF (20 mg/kg/d) and DAC (30 mg/kg/d) were added in the last four weeks of treatments. Liver functions, fibrotic markers such as Hyaluronic acid and metalloproteinase-9 were detected using immunoassay. Liver tissues were examined by different stains. Results: SOF and DAC induced marked inhibitions in fibrotic markers expression significantly (P≤0.001). Moreover, the drugs protected liver tissues from progressed fibrosis. Conclusion: SOF/DAC antifibrotic effect is independent on its antiviral activity.

Mesenchymal Stem Cell-Derived Exosomes Ameliorated Diabetic Nephropathy by Autophagy Induction through the mTOR Signaling Pathway

Background: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus and a common cause of end-stage renal disease. Autophagy has a defensive role against kidney damage caused by hyperglycemia. Mesenchymal stem cell (MSC)-derived exosomes are currently considered as a new promising therapy for chronic renal injury. However, the renal-protective mechanism of exosomes on DN is not completely understood. We examined the potential role of MSC-derived exosomes for enhancement of autophagy activity and their effect on DN. In our study, we used five groups of rats: control; DN; DN treated with exosomes; DN treated with 3-methyladenine (3-MA) and chloroquine (inhibitors of autophagy); and DN treated with 3-methyladenine (3-MA), chloroquine, and exosome groups. We assessed renal function, morphology, and fibrosis. Moreover, ratios of the autophagy markers mechanistic target of rapamycin (mTOR), Beclin-1, light chain-3 (LC3-II), and LC3-II/LC3-I were detected. Additionally, electron microscopy was used for detection of autophagosomes. Results: Exosomes markedly improved renal function and showed histological restoration of renal tissues, with significant increase of LC3 and Beclin-1, and significant decrease of mTOR and fibrotic marker expression in renal tissue. All previous effects were partially abolished by the autophagy inhibitors chloroquine and 3-MA. Conclusion: We conclude that autophagy induction by exosomes could attenuate DN in a rat model of streptozotocin-induced diabetes mellitus.

Abstract 17087: Competent p53 Stabilized iPS-Cardiomyocyte Transplantation in Ischemic Myocardium of Mice Reduce Fibrosis Through Exosomal miRNA-26a

Introduction: With the growing field of myocardial repair through transplantation of cardiomyocytes (CM) derived from human induced pluripotent stem cells (iPSC) calls for a method for reducing fibrotic tissue development at the site of cell transplantation. We tested the hypothesis whether transplanting CMs derived from p53 stabilized (p53S) iPSCs could reduce the recruitment of fibroblast and reduce fibrosis at the site of transplantation. Methods: Transcription factor p53 functions are inflammation dependent: inducing apoptosis in inflammatory and transformed cells while promoting cell cycle in normal cells. Utilizing this discriminating nature of p53 we selected iPSCs that express less inflammatory and more anti-inflammatory markers and differentiated them into CM. Control CM (ctrl-CM) and p53S-CMs were tested for transplantation induced fibrosis in a mice model. Results and Discussion: The inflammatory markers such as accumulation of ROS, superoxide and DNA damage were significantly lesser in p53S cells. RNA sequencing revealed that, in p53S-CMs, the anti-inflammatory and anti-fibrosis signaling pathways are upregulated compared to ctrl-CMs. Transplantation of both CMs into the myocardium of ischemia induced mice resulted in cell engraftment and fibrosis around the transplantation site. However, fibrotic area was significantly reduced in p53S-CM transplanted sites evidenced by COL1 and COL4 accumulation and Sirus red staining of fibrotic tissue. In vitro treatment of human fibroblast with p53S-CM exosomes (p53S-Exo) resulted in reduced expression of fibrotic markers than ctrl-CM exosomes (Ctrl-Exo) treatment. miRNA analysis of exosomes showed that the anti-inflammatory miRNA26a is upregulated and the inflammatory miRNA101 is downregulated in p53S-Exo. Overexpression of miRNA26a mimic in ctrl-Exo abrogated the fibrosis inducing property of ctrl-Exo. Interestingly, in migration study, fibroblasts preferentially migrated towards the ctrl-CMs. Conclusion: Our results clearly shows that anti-inflammatory cells can be isolated from a population of heterogeneous cells by activating p53. Transplanting p53S-CMs attracted reduced fibroblast to the engraft site and oppose fibrosis through exosomal expression of miRNA26a.