Fibronectin Protein Levels Research Articles

Endothelin‐1 Induced Fibronectin Expression via c‐Jun

Glaucoma is an optic disease that is characterized by slow neurodegeneration of the optical nerves and steady loss of retinal ganglion cells. Over 70 million people suffer from glaucoma and it is estimated that over 118 million will suffer from it in 2040. A prime factor causing glaucoma is an increased in intraocular pressure (IOP) but the mechanism in how IOP contributes to glaucoma is still being studied. Blocking the aqueous humor through the trabecular meshwork (TM) is a major contributor for the IOP elevation. Endothelin (ET‐1) is a potent vasoconstrictor in which play's an important role in vascular system. Previous research has discovered that ET‐1 and its ETA/ETB receptors are also involved in the pathogenesis of glaucoma. The current study aims to test whether ET‐1 induced fibronectin deposition at TM. We hypothesize that ET‐1 induces over expression of fibronectin through c‐Jun‐mediated signaling pathway. Primary human TM cells were pretreated with c‐Jun siRNA, SP600125 (a JNK inhibitor to block activation of c‐Jun) or ET receptor antagonist's followed with ET‐1 treatment. Protein levels of fibronectin and c‐Jun were detected using immunoblotting. An increase of fibronectin and c‐Jun were detected in TM cells treated with ET‐1 for 24–72 hours. Fibronectin was increased as a result of c‐Jun being partially terminated by the knockdown of c‐Jun by C‐Jun siRNA. Blocking of ETA/ETB by BQ610/BQ788 reduced the expression of fibronectin. Our results preliminary demonstrate that ET‐1 induces the expression of fibronectin through c‐Jun mediated signaling pathway, suggesting that ET‐1 may cause the accumulation of fibronectin at TM contributing to IOP elevation.Support or Funding InformationThis work was supported by the Department of Health and Human Services, National Institute of Health, National Heart, Lung and Blood Institute, SMART Award Number 4R25HL007786‐25 to Harlan Jones, Ph.D This work was also supported by MARC U*STARGrant5T34GM008395 to MariaElena Zavala, Ph.DThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Meprin β deficiency associated with increased ECM protein buildup and reduced macrophage infiltration in mouse kidneys subjected to Unilateral Ureteral Obstruction (UUO)

Meprins are zinc dependent metalloproteases that are abundantly expressed in the brush border membranes (BBM) of proximal kidney tubules. Previous studies have implicated meprins in Ischemic/Reperfusion (IR) induced acute kidney injury (AKI), and diabetic nephropathy (DN). However, the mechanisms by which meprins modulate kidney injury are not fully understood. Known meprin substrates include extracellular matrix (ECM) proteins (e.g. Collagen, Fibronectin, Laminin, and Nidogen‐1) and modulators of inflammation such as interleukin 1β (IL‐1β), IL‐6, pro‐IL‐18, monocyte chemoattractant protein‐1 (MCP‐1), and thymosin β4 – leading to release of the anti‐inflammatory peptide Ac‐SDKP. Furthermore, meprins are expressed in leukocytes (monocytes, macrophages), and play a role in leukocyte infiltration to sites of inflammation in the small intestines. Inflammation is an underlying cause of renal injury in both IR‐induced AKI and DN. The objective of the current study was to determine how meprin deficiency impacts extracellular matrix protein build up and leukocyte infiltration in unilateral ureteral obstruction (UUO)‐induced inflammation. Surgical procedures were used to ligate one ureter in twelve‐week‐old wild‐type (WT) and meprin β knockout male mice. The contralateral kidney served as the control for each mouse. At 7 days post‐UUO, the kidneys were perfused with saline to remove leukocytes present in kidney blood vessels and kidneys were harvested. Sections of the kidney tissue were snap frozen for protein extraction and some were paraffin embedded and sectioned for microscopy. For immunofluorescence F4/80, was used as a macrophage maker. The images were captured and the number of cells that stained positive for F4/80 counted. Western blot analysis was used to evaluate the levels of ECM proteins (fibronectin, laminin, and nidogen‐1) in the kidney lysates. Statistical analysis utilized 2‐way ANOVA (Graphpad Prism 7 software). Our data demonstrates increases in fibronectin protein levels in both WT and βKO kidneys subjected to UUO. However, UUO‐induced fibronectin increases were higher in βKO than in WT (p=0.0016 vs p=0.004). The baseline levels of nidogen‐1 were higher in WT mice (p=0.01) when compared to βKOs. At 7 days post‐UUO, the levels of nidogen‐1 increased significantly in the kidneys of βKO subjected to UUO (p=0.001), but not in WT kidneys. There were no significant differences in the baseline or post‐UUO levels of laminin in either genotype. Results from immunofluorescence showed that WT and meprin βKO kidneys subjected to UUO had significant increases in the number of macrophages in tubulo‐interstitial tissue when compared to control counterparts (≤0.007 and ≤0.001 respectively). Taken together, our data suggest that meprin expression/activity reduces the buildup of specific ECM proteins (fibronectin and nidogen‐1), thus reducing the fibrosis observed in inflammation. Furthermore, meprins modulate inflammation via enhanced leukocyte infiltration of (monocytes/macrophages).Support or Funding InformationNIH/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and #SC1GM118271 to Elimelda Moige OngeriThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin-converting enzyme inhibitors attenuated advanced glycation end products-induced renal tubular hypertrophy via enhancing nitric oxide signaling.

Advanced glycation end products (AGE) and angiotensin II were closely correlated with the progression of diabetic nephopathy (DN). Nitric oxide (NO) is a protective mediator of renal tubular hypertrophy in DN. Here, we examined the molecular mechanisms of angiotensin-converting enzyme inhibitor (ACEI) and NO signaling responsible for diminishing AGE-induced renal tubular hypertrophy. In human renal proximal tubular cells, AGE decreased NO production, inducible NOS activity, guanosine 3',5'-cyclic monophosphate (cGMP) synthesis, and cGMP-dependent protein kinase (PKG) activation. All theses effects of AGE were reversed by treatment with ACEIs (captopril and enalapril), the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the PKG activator 8-para-chlorophenylthio-cGMPs (8-pCPT-cGMPs). In addition, AGE-enhanced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were clearly reduced by captopril, enalapril, SNAP, and 8-pCPT-cGMPs. The abilities of ACEIs and NO/PKG activation to inhibit AGE-induced hypertrophic growth were verified by the observation that captopril, enalapril, SNAP, and 8-pCPT-cGMPs decreased protein levels of fibronectin, p21 Waf1/Cip1 , and receptor for AGE. The results of the present study suggest that ACEIs significantly reduced AGE-increased ERK/JNK/p38 MAPK activation and renal tubular hypertrophy partly through enhancement of the NO/PKG pathway.

Increased oxidative stress, inflammation and fibrosis in perirenal adipose tissue of patients with cortisol-producing adenoma

ABSTRACT Although much is known about that corticosteroids affect the functions of adipose tissues, little genetic information is available for perirenal adipose tissue (peri-N) from patients with cortisol-producing adenoma (CPA). We conducted microarray analysis of peri-N from patients with CPA by using an Affymetrix human U133 plus 2.0 array. We also analysed the inflammation, fibrosis and oxidative stress in vitro. Compared with normotension (NT) group, CPA group has significantly higher protein levels of TNFα, IL-6, fibronectin (FN) and collagen I (COLI). The protein level of NADPH oxidase 4 (Nox4) significantly increased, while nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) levels were significantly reduced in the CPA group. Dexamethasone markedly induced fibrosis and adipogenesis-related gene expression in predifferentiated stromal vascular fraction (SVF) cells, 3T3-L1 preadipocytes and brown preadipocytes. Chronic exposure to endogenous glucocorticoids due to CPA increases peri-N oxidative stress, inflammation and fibrosis, which may contribute to the metabolic disturbances associated with hypercortisolism in these patients.

A non-invasive biomarker of type III collagen degradation reflects ischaemia reperfusion injury in rats.

Maintenance of kidney function in kidney allografts remains a challenge, as the allograft often progressively develops fibrosis after kidney transplantation. Fibrosis is caused by the accumulation of extracellular matrix proteins like type I and III collagen (COL I and III) that replace the functional tissue. We assessed the concentrations of a neo-epitope fragment of COL III generated by matrix metalloproteinase-9 cleavage (C3M) in two rat models resembling the ischaemic injury taking place following kidney transplantation. We measured C3M in urine (U-C3M) and plasma (P-C3M) samples of rats subjected to unilateral nephrectomy followed by sham operation (NTx) or ischaemia reperfusion injury (NTxIRI) as well as in rats subjected to bilateral ischaemia reperfusion injury (BiIRI). Levels of U-C3M were normalized to urinary creatinine and were correlated to plasma creatinine, blood urea nitrogen, messenger ribonucleic acid (mRNA) of markers of kidney injury, and mRNA and protein levels of markers of tissue repair and fibrosis. Levels of U-C3M were significantly elevated 7 days after ischaemia reperfusion in the NTxIRI. BiIRI animals showed higher levels of U-C3M after 7 and 14 days of reperfusion but not at 21 days. P-C3M did not change in any of the models. There was a significant correlation between U-C3M and mRNA levels of fibronectin, COL I alpha 1 chain (COL Ia1) and neutrophil gelatinase-associated lipocalin (NGAL), and protein levels of alpha smooth muscle actin (αSMA), fibronectin and COL III in NTxIRI but not in NTx animals. Levels of U-C3M increased significantly in the BiIRI animals subsequent to reperfusion, and mirrored the histological alterations. Furthermore, U-C3M was associated with the extent of fibrosis, and remained elevated even after plasma creatinine levels decreased. These results demonstrate that degradation of COL III increases after ischaemia reperfusion injury, and that U-C3M may be a non-invasive marker of tissue repair and fibrosis in the ischaemic kidney.

Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-β Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

Background & AimsTransdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in the pathogenesis of liver fibrosis. Transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) are canonical HSC activators after liver injury. The aim of this study was to analyze the epigenetic modulators that differentially control TGF-β and PDGF signaling pathways.MethodsWe performed a transcriptomic comparison of HSCs treated with TGF-β or PDGF-BB using RNA sequencing. Among the targets that distinguish these 2 pathways, we focused on the histone methyltransferase class of epigenetic modulators.ResultsEnhancer of zeste homolog 2 (EZH2) was expressed differentially, showing significant up-regulation in HSCs activated with TGF-β but not with PDGF-BB. Indeed, EZH2 inhibition using either a pharmacologic (GSK-503) or a genetic (small interfering RNA) approach caused a significant attenuation of TGF-β–induced fibronectin, collagen 1α1, and α-smooth muscle actin, both at messenger RNA and protein levels. Conversely, adenoviral overexpression of EZH2 in HSCs resulted in a significant stimulation of fibronectin protein and messenger RNA levels in TGF-β–treated cells. Finally, we conducted in vivo experiments with mice chronically treated with carbon tetrachloride or bile duct ligation. Administration of GSK-503 to mice receiving either carbon tetrachloride or bile duct ligation led to attenuated fibrosis as assessed by Trichrome and Sirius red stains, hydroxyproline, and α-smooth muscle actin/collagen protein assays.ConclusionsTGF-β and PDGF share redundant and distinct transcriptomic targets, with the former predominating in HSC activation. The EZH2 histone methyltransferase is preferentially involved in the TGF-β as opposed to the PDGF signaling pathway. Inhibition of EZH2 attenuates fibrogenic gene transcription in TGF-β–treated HSCs and reduces liver fibrosis in vivo. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE119606 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119606)

Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes.

Chronic exposure of tubular renal cells to high glucose contributes to tubulointerstitial changes in diabetic nephropathy. In the present study, we identified a new fibrosis gene called galectin-1 (Gal-1), which is highly expressed in tubular cells of kidneys of type 1 and type 2 diabetic mouse models. Gal-1 protein and mRNA expression showed significant increase in kidney cortex of heterozygous Akita+/- and db/db mice compared with wild-type mice. Mouse proximal tubular cells exposed to high glucose showed significant increase in phosphorylation of Akt and Gal-1. We cloned Gal-1 promoter and identified the transcription factor AP4 as binding to the Gal-1 promoter to up-regulate its function. Transfection of cells with plasmid carrying mutations in the binding sites of AP4 to Gal-1 promoter resulted in decreased protein function of Gal-1. In addition, inhibition of Gal-1 by OTX-008 showed significant decrease in p-Akt/AP4 and protein-promoter activity of Gal-1 and fibronectin. Moreover, down-regulation of AP4 by small interfering RNA resulted in a significant decrease in protein expression and promoter activity of Gal-1. We found that kidney of Gal-1-/- mice express very low levels of fibronectin protein. In summary, Gal-1 is highly expressed in kidneys of type 1 and 2 diabetic mice, and AP4 is a major transcription factor that activates Gal-1 under hyperglycemia. Inhibition of Gal-1 by OTX-008 blocks activation of Akt and prevents accumulation of Gal-1, suggesting a novel role of Gal-1 inhibitor as a possible therapeutic target to treat renal fibrosis in diabetes.-Al-Obaidi, N., Mohan, S., Liang, S., Zhao, Z., Nayak, B. K., Li, B., Sriramarao, P., Habib, S. L. Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes.

Progestin and AdipoQ Receptor 3 Upregulates Fibronectin and Intercellular Adhesion Molecule-1 in Glomerular Mesangial Cells via Activating NF-κB Signaling Pathway Under High Glucose Conditions.

Progestin and adipoQ receptor 3 (PAQR3), is a Golgi-anchored membrane protein containing seven transmembrane helices. It has been demonstrated that PAQR3 mediates insulin resistance, glucose and lipid metabolism, and inflammation. In addition, kidney inflammatory fibrosis is an important pathological feature of diabetic nephropathy (DN). Therefore, we aimed to investigate the role of PAQR3 in diabetic kidney fibrosis as well as inflammation in DN. The effect of PAQR3 on NF-κB signaling pathway, expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs) cultured by high glucose (HG) were examined. Diabetic mouse and rat models were induced by streptozotocin (STZ). GMCs were treated with HG and transfected with PAQR3 plasmids or small-interfering RNA targeting PAQR3 or NF-κB. The protein levels of FN and ICAM-1 were examined by Western blotting, and the transcriptional activity and DNA binding activity of NF-κB were measured by dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The interaction between PAQR3 and IKKβ (inhibitor of nuclear factor κB kinase β) was analyzed by co-immunoprecipitation. PAQR3 was increased in both STZ-induced diabetic models and HG-treated GMCs. PAQR3 overexpression further increased HG-induced FN and ICAM-1 upregulation. In contrast, silencing of PAQR3 suppressed the expressions of FN and ICAM-1. PAQR3 overexpression promoted the nuclear accumulation, DNA binding activity, and transcriptional activity of NF-κB. Mechanically, PAQR3 directly interacted with IKKβ. The upregulation effect of PAQR3 overexpression on the expressions of FN and ICAM-1 was abolished by the treatment of NF-κB siRNA or PDTC (ammonium pyrrolidinedithiocarbamate) in HG-treated GMCs. PAQR3 promotes the expressions of FN and ICAM-1 via activating NF-κB signaling pathway. Mechanistically, PAQR3 activates NF-κB signaling pathway to mediate kidney inflammatory fibrosis through direct interaction with IKKβ in DN.

Effects of δ-tocotrienol on ochratoxin A-induced nephrotoxicity in rats.

Ochratoxin A (OTA), is a natural contaminant of the food chain worldwide involved in the development of different type of cancers in animals and humans. Several studies suggested that oxidative damage might contribute to increase the cytotoxicity and carcinogenicity capabilities of OTA. The aim of this study was to evaluate the possible protective effect of δ-tocotrienol (Delta), a natural form of vitamin E, against OTA-induced nephrotoxicity. Male Sprague-Dawley rats were treated with OTA and/or Delta by gavage for 14 days. Our results shown that OTA treatment induced the increase of reactive oxigen species production correlated to a strong reduction of Glomerular Filtration Rate (GFR) and absoluted fluid reabsorption (Jv) with conseguent significant increase in blood pressure. Consistent, we noted in the kidney of rats treated with OTA, an increase in malondialdheyde and dihydroethidium production and a reduction of the activity of the catalase, superoxide dismutase, and glutathione peroxidase. Conversly, in the rat group subjected to the concomitant treatment OTA plus Delta, we observed the restored effect, compared the OTA treatment group, on blood pressure, GFR, Jv, and all activities of renal antioxidant enzymes. Finally, as far as concern the tissue damage induced by OTA and measured evaluating fibronectin protein levels, we observed that in OTA plus Delta group this effect is not restored. Our findings releval that a mechanism underlying the renal toxicity induced by OTA is the oxidative stress and provide a new rationale to use a Delta in order to protect, at least in part, against OTA-induced nephrotoxicity.

Essential role of connective tissue growth factor (CTGF) in transforming growth factor-\u03b21 (TGF-\u03b21)-induced myofibroblast transdifferentiation from Graves\u2019 orbital fibroblasts

Connective tissue growth factor (CTGF) associated with transforming growth factor-β (TGF-β) play a pivotal role in the pathophysiology of many fibrotic disorders. However, it is not clear whether this interaction also takes place in GO. In this study, we investigated the role of CTGF in TGF-β-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts. By Western blot analysis, we demonstrated that TGF-β1 induced the expression of CTGF, fibronectin, and alpha-smooth muscle actin (α-SMA) in Graves’ orbital fibroblasts. In addition, the protein levels of fibronectin and α-SMA in Graves’ orbital fibroblasts were also increased after treatment with a recombinant human protein CTGF (rhCTGF). Moreover, we transfected the orbital fibroblasts with a small hairpin RNA of CTGF gene (shCTGF) to knockdown the expression levels of CTGF, which showed that knockdown of CTGF significantly diminished TGF-β1-induced expression of CTGF, fibronectin and α-SMA proteins in Graves’ orbital fibroblasts. Furthermore, the addition of rhCTGF to the shCTGF-transfected orbital fibroblasts could restore TGF-β1-induced expression of fibronectin and α-SMA proteins. Our findings demonstrate that CTGF is an essential downstream mediator for TGF-β1-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts and thus may provide with a potential therapeutic target for treatment of GO.

Steroid Sulfatase activates the integrin signaling pathway in human cervical cancer cells

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a crucial function in the production of estrogens and androgens that induce the growth of hormone‐dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not elucidated clearly. To investigate the function of STS in cancer cell growth, we examined whether STS is able to enhance the integrin signaling pathway. We observed that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), the major metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. STS overexpression and DHEA treatment induced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Furthermore, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS enhances the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up‐regulation of integrin β1 expression and activation of FAK.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of SIRT2 Alleviates Fibroblast Activation and Renal Tubulointerstitial Fibrosis via MDM2

Background/Aims: Renal tubular epithelial cells and fibroblasts are the main sources of myofibroblasts, and these cells produce the extracellular matrix during tubulointerstitial fibrosis (TIF). Histone deacetylases (HDAC) inhibitors exert an antifibrogenic effect in the skin, liver and lung. Sirtuin 2 (SIRT2), which is a class III HDAC, is an important member of NAD⁺-dependent protein deacetylases. The current study evaluated the role of SIRT2 in renal TIF. Methods: Immunohistochemical staining and Western blot were performed to evaluate SIRT2 expression in TIF patients and unilateral urethral obstruction (UUO) mice. Western blot was used to assess the protein levels of SIRT2, α-SMA, collagen III, fibronectin, and MDM2 in tubular epithelial cells and fibroblasts. The specific inhibitor AGK2 was used to inhibit SIRT2 activity, and targeted siRNA was used to suppress SIRT2 expression. Results: SIRT2 expression increased in the tubulointerstitium of TIF patients and UUO mice. SIRT2 inhibition ameliorated TIF in UUO mice. SIRT2 expression in tubular cells was unchanged after exposure to TGF-β1. The SIRT2-specifc inhibitor AGK2 did not attenuate TGF-β1-induced tubular epithelial-mesenchymal transition. However, SIRT2 was upregulated in fibroblasts, and fibroblasts were activated after TGF-β1 treatment. Genetic knockdown and chemical inhibition of SIRT2 attenuated TGF-β1-induced fibroblast activation. We also explored the downstream signaling of SIRT2 during fibroblast activation. Genetic knockdown and chemical inhibition of SIRT2 suppressed TGF-β1-induced increase in MDM2 expression, and inhibition of the MDM2-p53 interaction using Nutlin-3 did not suppress SIRT2 upregulation. Conclusion: Our results suggest that SIRT2 participates in the activation of fibroblasts and TIF, which is mediated via regulation of the MDM2 pathway, and the downregulation of SIRT2 may be a therapeutic strategy for renal fibrosis.

Vangl2-dependent regulation of membrane protrusions and directed migration requires a fibronectin extracellular matrix.

During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells.

Recombinant truncated TGF‑β receptor II attenuates carbon tetrachloride‑induced epithelial‑mesenchymal transition and liver fibrosis in rats.

Liver fibrosis is a pathological process of chronic liver diseases. In particular, epithelial‑mesenchymal transition (EMT) is a major source of myofibroblast structure in liver fibrosis. The present study investigated the effects of recombinant truncated transforming growth factor‑ß receptor II (rtTGFβRII) on EMT and liver fibrosis in a carbon tetrachloride (CCl4)‑induced rat model. A total of 24 rats were randomly separated into three groups: Normal control (NC), model (CCl4) and treatment (CCl4 + rtTGFβRII) groups. Histological methods, including hematoxylin and eosin, Masson's trichrome and Sirius red staining were conducted. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic biochemical analyzer. The mRNA expression levels of fibroblast specific protein‑1 (FSP‑1), α‑smooth muscle actin (α‑SMA), fibronectin, collagen I, vimentin and E‑cadherin were detected using reverse transcription‑quantitative polymerase chain reaction analysis. The protein levels of fibronectin, collagen I, E‑cadherin, Smad2/3 and phosphorylated (p)‑Smad2/3 were detected using western blot analysis. The expression of α‑SMA, fibronectin, vimentin and E‑cadherin in the liver tissue was detected using immunofluorescence staining. The results demonstrated that invivo, rtTGFβRII significantly reduced the degree of liver injury, serum ALT and AST activities and liver fibrosis. These factors were associated with reduced expression of FSP‑1, α‑SMA, fibronectin, collagen I, vimentin and p‑Smad2/3, and increased expression of E‑cadherin. The results of the present study suggest that rtTGFβRII may inhibit EMT processes in CCl4‑induced liver fibrosis in rats and alter the expression of epithelial and myofibroblast markers. Therefore, rtTGFβRII may be considered a possible treatment for preventing liver fibrosis via EMT processes.

5-Aza-2′-deoxycytidine induces human Tenon's capsule fibroblasts differentiation and fibrosis by up-regulating TGF-β type I receptor

The principle reason of high failure rate of glaucoma filtration surgery is the loss of filtration function caused by postoperative scar formation. We investigated the effects of 5-aza-2′-deoxycytidine (5-Aza-dc), a DNA methyltransferases inhibitor, on human Tenon's capsule fibroblasts (HTFs) differentiation and fibrosis and its mechanism of action, especially in relation to transforming growth factor (TGF)-β1 signaling. TGF-β1 was used to induce differentiation of cultured HTFs. 5-Aza-dc suppressed DNA methyltransferases (DNMTs) activity 6 h after treatment with a course corresponding to that of TGF-β1-induced reduction of DNMT activity without affecting cell viability as measured by Cell Counting Kit-8 assay. 5-Aza-dc also reduced DNMT1 and DNMT3a protein expression from 24 to 48 h. HTFs migration evaluated by scratch-wound assay were significantly increased 24 h after 5-Aza-dc treatment, a time course similar to that of TGF-β1. Treatment with 5-Aza-dc significantly increased the mRNA and protein levels of α-smooth muscle actin (α-SMA), collagen-1A1 (Col1A1), fibronectin (FN) and TGF-β type I receptor (TGFβRI). Furthermore, the effects of 5-Aza-dc on DNMT activity suppression, cell migration, and fibrosis were all reversed by a TGFβRI inhibitor- SB-431542. Meanwhile, knockdown of DNMT1 upregulated TGFβRI expression and had the same fibrosis-inducing effect in HTFs, which was also inhibited by SB-431542. Thus, the results indicate that DNA hypomethylation induces HTFs differentiation and fibrosis through up-regulation of TGFβRI. DNA methylation status plays an important role in subconjunctival wound healing.

RETRACTED ARTICLE: Inhibition of microRNA-214 promotes epithelial–mesenchymal transition process and induces interstitial cystitis in postmenopausal women by upregulating Mfn2

Our study aims to investigate the roles that microRNA-214 (miR-214) plays in the epithelial mesenchymal transition (EMT) process and the development of interstitial cystitis (IC) in postmenopausal women by targeting Mitofusin 2 (Mfn2). IC bladder tissues and adjacent normal bladder tissues were collected from postmenopausal women. Immunohistochemistry (IHC) staining was conducted. The target relationship between miR-214 and Mfn2 was determined by a dual luciferase reporter gene assay. Adipose-derived mesenchymal stem cells (ADMSCs) were extracted from postmenopausal rats and assigned to the blank, mimics, miR-214 inhibitors, mimics negative control (NC), inhibitors NC, Mfn2 siRNA, miR-214 inhibitors and Mfn2 siRNA groups. Exosomes secreted by transfected ADMSCs were instilled into the bladders of postmenopausal rats. The expression of miR-214 and Mfn2 mRNA and EMT markers was assessed by qRT-PCR and western blotting. It was confirmed that Mfn2 was the target gene of miR-214 in IC. Compared with the normal bladder tissues, miR-214 decreased, but Mfn2 increased in IC bladder tissues. Compared with the blank group, the expression of miR-214 and the expression levels of N-cadherin, Fibronectin, Twist1, Snail and Vimentin mRNA and protein increased, whereas the expression levels of Mfn2, E-cadherin and ZO-1 mRNA and protein decreased in the miR-214 mimics and Mfn2 groups. The expression of MiR-214 and the expression levels of N-cadherin, Fibronectin, Twist1, Snail and Vimentin mRNA and protein decreased, whereas the expression levels of Mfn2, E-cadherin and ZO-1 mRNA and protein increased in the miR-214 inhibitors group. Our findings indicate that the inhibition of miR-214 promotes the EMT process and contributes to bladder wall fibrosis by up-regulating Mfn2, thus leading to the occurrence of IC in postmenopausal women.

Abstract 5212: Metabolic reprogramming of radiation fibrosis using adipose derived stromal cells

Abstract Background: Soft tissue radiation fibrosis (RF) affects up to 70% of cancer survivors post-radiotherapy. RF is an irreversible and progressive side effect of radiotherapy characterized by poor tissue elasticity and increased ECM deposition, clinically translating to increased morbidity due to hardening, distortion, and pain. Recent evidence has highlighted the potential role of metabolic alterations in the onset and progression of fibrosis. Adipose derived stromal cells (ADSCs) have been used effectively for enhancing complex wound repair in a number of clinical trials. The therapeutic effect of ADSCs has been attributed to its secretion of paracrine factors, which can regulate the metabolism of target cells. Even so, the metabolic effects of ADSCs on target cells is not well described. Methods and Results: Transcriptomic profiling and targeted metabolomics of human radiated tissue and a murine model of RF revealed that suppression of fatty acid oxidation (FAO) is hallmark of RF. TGF-B, a master regulator of fibrosis, has a large effect in suppressing FAO in primary human fibroblasts in vitro through downregulating genes in the PPAR pathway, a major regulatory pathway for FAO, and through inhibiting oxidation of the long chain fatty acid, palmitic acid (p<0.05, t-test). Treatment of TGF-B stimulated fibroblasts with ADSC conditioned media resulted in an improvement in FAO and a reduction in fibronectin, collagen-1, and PAI-1 protein levels, three of major contributors to fibrosis. Transplantation of ADSCs into murine RF resulted in a metabolic shift back to FAO and a significant reduction in RF both functionally (percentage leg contracture 36% vs 26%, 2-way ANOVA < 0.05) and histologically through image analysis of trichrome staining (p<0.01, t-test). Pathway analysis of significant genes altered by ADSCs revealed an upregulation of the PPAR pathway was the most significant effect of ADSC transplantation. Untargeted metabolomics confirmed that FAO metabolites were significantly upregulated with ADSC transplantation. To confirm the importance of metabolic regulation by ADSCs, we utilized a pharmacogenomics strategy to uncover a compound that mimicked the effect of ADSCs. Drug A ranked highest and replicated the anti-fibrotic and pro-FAO effects of ADSCs both on TGF-B stimulated primary fibroblasts and in murine RF. Etomoxir, a FAO inhibitor, inhibited the effect of ADSCs and Drug A in regulating protein levels of fibronectin and collagen, revealing that ADSCs and Drug A require an intact FAO pathway to exert their anti-fibrotic effects. Conclusions: Inhibition of FAO is a novel hallmark of RF. ADSC-mediated metabolic reprogramming resulted in an enhancement of FAO and a reduction in RF through upregulating the PPAR pathway. ADSC-directed pharmacogenomics uncovered Drug A, which mimicked the pro-FAO and anti-fibrotic effects of ADSCs. Our research has highlighted the importance of reversing metabolic aberrations to reduce RF. Citation Format: Xiao Zhao, Laleh Soltan Ghoraie, Pamela Psarianos, Kenneth Yip, Laurie Ailles, Benjamin Haibe-Kains, Fei-Fei Liu. Metabolic reprogramming of radiation fibrosis using adipose derived stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5212. doi:10.1158/1538-7445.AM2017-5212

Qi-Zhu-Xie-Zhuo-Fang reduces serum uric acid levels and ameliorates renal fibrosis in hyperuricemic nephropathy rats.

Hyperuricemia is associated with the development of chronic kidney disease. Epithelial-to-mesenchymal transition (EMT) induced by hyperuricemia is blamed for initiation of renal fibrosis, which is one of the main characters of hyperuricemic nephropathy. Qi-Zhu-Xie-Zhuo-Fang (QZXZF) has been employed clinically for many years to treat patients with hyperuricemic nephropathy, but the mechanism underlying the therapeutic potential remains unclear. In the present study, QZXZF was applied to rats treated with adenine (100mg/kg) and potassium oxonate (300mg/kg) and biochemical estimations, morphology and immunohistochemistry were performed to investigate whether QZXZF can improve hyperuricemia induced renal fibrosis and to explore the possible mechanisms. We found QZXZF significantly reduced serum uric acid, cystatinC and hepatic xanthine oxidase (XO) activities, meanwhile improved renal histopathologic changes of hyperuricemic nephropathy rats. Furthermore, QZXZF not only substantially decreased the protein levels of fibronectin and Collagen I but also downregulated E-cadherin and upregulated α-SMA in the kidneys of hyperuricemic nephropathy rats. In conclusion, QZXZF reduced serum uric acid levels and protected kidney against fibrosis in potassium oxonate and adenine induced hyperuricemic nephropathy rats. The mechanism might be associated with the inhibition of hepatic XO activity and the renal epithelial-to-mesenchymal transition.

Overexpression of ALK5 Induces Human Tenon’s Capsule Fibroblasts Transdifferentiation and Fibrosis In Vitro

ABSTRACTPurpose: To investigate the involvement of activin receptor-like kinase 5 (ALK5) in human Tenon’s capsule fibroblasts (HTFs) transdifferentiation and fibrosis.Methods: (1) Cultured HTFs were treated with transforming growth factor beta 1 (TGF-β1) at different concentrations for different durations, mRNA expression of ALK5 and plasminogen activator inhibitor-1 (PAI-1) was measured by quantitative polymerase chain reaction (PCR) while protein expression of ALK5, α-smooth muscle actin (α-SMA), and extracellular matrix deposition including fibronectin (FN) and collagen I (Col1) was assessed by western blot. HTFs with or without TGF-β1 were also treated with an ALK5 activity inhibitor, SB-431542, and fibrosis-related genes were assessed.(2) HTFs were transduced with ALK5 lentivirus (ALK5-OE group) or empty lentivirus (NC-OE) with or without the treatment of SB-431542. Protein expression of ALK5, α-SMA, FN, and Col1 was evaluated.(3) HTFs in the ALK5-OE group and NC-OE group were subjected to a scratch-wound assay and their migratory activities assessed.Results: (1) TGF-β1, in a concentration-dependent manner, upregulated ALK5 and PAI-1 expressions in the HTFs, which peaked between 24 and 36 h. These changes were associated with increases in protein levels of FN, Col1, and α-SMA. These TGF-β1 effects were blocked by the ALK5 inhibitor SB-431542.(2) Similarly, overexpression of ALK5 by lentiviral vector significantly increased protein expression of α-SMA, FN, and Col1. Addition of TGF-β1 to the ALK5-OE cells did not produce additional expression of any of the marker proteins. The upregulation of extracellular matrix and α-SMA can be reduced by SB-431542.(3) In ALK5-OE group, HTFs migration was significantly increased compared with normal control and TGF-β1 could still promote ALK5-OE cells migration.Conclusions: Our findings suggest that ALK5 is an important mediator of HTFs fibrosis. ALK5 is a potential therapeutic target to suppress scar formation after filtration surgery.

Emodin self-emulsifying platform ameliorates the expression of FN, ICAM-1 and TGF-β1 in AGEs-induced glomerular mesangial cells by promoting absorption

Emodin, a potential anti-diabetic nephropathy agent, is limited by its oral use due to the poor water solubility. The present study aimed to enhance the absorption and the suppressive effects of emodin on renal fibrosis by developing a self-microemulsifying drug delivery system (SMEDDS). Solubility studies, compatibility tests, pseudo-ternary phase diagrams analysis and central composite design were carried out to obtain the optimized formulation. The average droplet size of emodin-loaded SMEDDS was about 18.31±0.12nm, and the droplet size and zeta potential remained stable at different dilution ratios of water and different values of pH varying from 1.2 to 7.2. Enhanced cellular uptake in both the Caco-2 cells and glomerular mesangial cells (GMCs) is great advantageous for the formulation. The AUC0–24h of emodin-loaded SMEDDS was 1.87-fold greater than that of emodin suspension, which may be attributed to enhanced uptake in Caco-2 cells. Moreover, emodin-loaded SMEDDS showed better suppressive effects on the protein level of fibronectin (FN), transforming growth factor-beta 1 (TGF-β1) and intercellular adhesion molecule 1 (ICAM-1) than the crude emodin in advanced glycation-end products (AGEs)-induced GMCs and renal tubular epithelial cells (NRK-52E). Our study illustrated that developed SMEDDS improved the oral absorption of emodin, and attained better suppressive effects on the protein level of renal fibrosis compositions in AGEs-induced GMCs and NRK-52E cells.