Inhibition of Sirt2 Alleviates Fibroblasts Activation and Pulmonary Fibrosis via Smad2/3 Pathway

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with unknown cause and limited treatment options. Its mechanism needs to be further explored. Sirtuin2 (Sirt2), a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, has been proved to be involved in the fibrosis and inflammation in the liver, kidney and heart. In this study, we aimed to evaluate the role of Sirt2 in pulmonary fibrosis. We found that Sirt2 expression was upregulated in transforming growth factor-β1 (TGF-β1) treated human embryonic lung fibroblasts. Sirt2 inhibitor AGK2 or the knockdown of Sirt2 expression by targeting small interfering RNA (siRNA) suppressed the fibrogenic gene α-SMA and Fibronectin expression in TGF-β1 treated fibroblasts and primary lung fibroblasts derived from patients with IPF. In addition, Sirt2 inhibition suppresses the phosphorylation of Smad2/3. Co-immunoprecipitation (Co-IP) showed that there is interaction between Sirt2 and Smad3 in the TGF-β1 treated lung fibroblasts. In bleomycin-induced pulmonary fibrosis in mice, AGK2 treatment significantly mitigated the degree of fibrosis and decreased the phosphorylation of Smad2/3. These data suggest that Sirt2 may participate in the development of IPF via regulating the Smad2/3 pathway. Inhibition of Sirt2 would provide a novel therapeutic strategy for this disease.

Hyperphosphatemia-Induced Oxidant/Antioxidant Imbalance Impairs Vascular Relaxation and Induces Inflammation and Fibrosis in Old Mice

Aging impairs vascular function, but the mechanisms involved are unknown. The aim of this study was to analyze whether aging-related hyperphosphatemia is implied in this effect by elucidating the role of oxidative stress. C57BL6 mice that were aged 5 months (young) and 24 months (old), receiving a standard (0.6%) or low-phosphate (0.2%) diet, were used. Isolated mesenteric arteries from old mice showed diminished endothelium-dependent vascular relaxation by the down-regulation of NOS3 expression, increased inflammation and increased fibrosis in isolated aortas, compared to those isolated from young mice. In parallel, increased Nox4 expression and reduced Nrf2, Sod2-Mn and Gpx1 were found in the aortas from old mice, resulting in oxidant/antioxidant imbalance. The low-phosphate diet improved vascular function and oxidant/antioxidant balance in old mice. Mechanisms were analyzed in endothelial (EC) and vascular smooth muscle cells (SMCs) treated with the phosphate donor ß-glycerophosphate (BGP). In EC, BGP increased Nox4 expression and ROS production, which reduced NOS3 expression via NFκB. BGP also increased inflammation in EC. In SMC, BGP increased Collagen I and fibronectin expression by priming ROS production and NFκB activity. In conclusion, hyperphosphatemia reduced endothelium-dependent vascular relaxation and increased inflammation and vascular fibrosis through an impairment of oxidant/antioxidant balance in old mice. A low-phosphate diet achieved improvements in the vascular function in old mice.

Use of Platelet-Rich Plasma Plus Suramin, an Antifibrotic Agent, to Improve Muscle Healing After Injuries

Background: The increasing use of platelet-rich plasma (PRP) to treat muscle injuries raises concerns because transforming growth factor–beta (TGF-β) in PRP may promote fibrosis in the injured muscle and thus impair muscle regeneration. Purpose: To investigate whether suramin (a TGF-β inhibitor) can reduce muscle fibrosis to improve healing of the injured muscle after PRP treatment and identify the underlying molecular mechanism. Study Design: Controlled laboratory study. Methods: Myoblasts isolated from the gastrocnemius muscle of Sprague Dawley rats were treated with PRP or PRP plus suramin. MTT assays were performed to evaluate cell viability. The expression of fibrosis-associated proteins (such as type I collagen and fibronectin), Smad2, and phosphorylated Smad2 was determined using Western blot analysis and immunofluorescent staining. An anti–TGF-β antibody was employed to verify the role of TGF-β in fibronectin expression. Gastrocnemius muscles were injured through a partial transverse incision and then treated using PRP or PRP plus suramin. Hematoxylin and eosin staining was conducted to evaluate the healing process 7 days after the injury. Immunofluorescent staining was performed to evaluate fibronectin expression. Muscle contractile properties—fast-twitch and tetanic strength—were evaluated through electric stimulation. Results: PRP plus 25 μg/mL of suramin promoted myoblast proliferation. PRP induced fibronectin expression in myoblasts, but suramin reduced this upregulation. The anti–TGF-β antibody also reduced the upregulation of fibronectin expression in the presence of PRP. The upregulation of phosphorylated Smad2 by PRP was reduced by either the anti–TGF-β antibody or suramin. In the animal study, no significant difference was discovered in muscle healing between the PRP versus PRP plus suramin groups. However, the PRP plus suramin group had reduced fibronectin expression at the injury site. Fast-twitch strength and tetanic strength were significantly higher in the injured muscle treated using PRP or PRP plus suramin. Conclusion: Simultaneous PRP and suramin use reduced fibrosis in the injured muscle and promoted healing without negatively affecting the muscle’s contractile properties. The underlying molecular mechanism may be associated with the phosphorylated Smad2 pathway. Clinical Relevance: Simultaneous PRP and suramin use may reduce muscle fibrosis without compromising muscle contractile properties and thus improve muscle healing.

Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

Fibronectin is an extracellular matrix glycoprotein with a regulatory role in fundamental cellular processes. Recent reports on the cardioprotective effect of fibronectin inhibition in a setting of myocardial injury suggest a role for fibronectin in cardiac fibroblast function, which remains largely unexplored. This study probed the molecular basis and functional implications of fibronectin gene expression in cardiac fibroblasts exposed to Angiotensin II, a potent pro-fibrotic factor in the myocardium. Using gene knockdown and over-expression approaches, western blotting and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-stimulated transcriptional up-regulation of fibronectin expression by Yes-activated Protein in cardiac fibroblasts. Further, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-dependent expression of anti-apoptotic cIAP2 and promoted cell death under oxidative stress. Fibronectin was also found to mediate Angiotensin II-stimulated collagen type I expression. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of its receptor, AT1R, which would link ECM signalling and Angiotensin II signalling in cardiac fibroblasts. Moreover, the regulatory role of DDR2-dependent fibronectin expression in Ang II-stimulated cIAP2, collagen type I and AT1R expression was mediated by Integrin-β1-integrin-linked kinase signalling. The pro-survival role of fibronectin in cardiac fibroblasts and its regulatory role in collagen and AT1R expression, downstream of DDR2, could be critical determinants of cardiac fibroblast-mediated wound healing following myocardial injury. Our findings point to a complex mechanism of regulation of cardiac fibroblast function involving two major extracellular matrix proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-β1.

Long Noncoding RNA SNHG10 Inhibits Renal Fibrosis By Negatively Regulating MiR-378b Expression

Background: LncRNA have been increasingly shown that plays pivotal roles in the development of various diseases, including renal fibrosis. Nevertheless, the pathological function of Long non-coding RNA SNHG10 (SNHG10) in the renal fibrosis remains obscure.Methods: We detected the expression levels of SNHG10 in the tissue samples and cell lines via RT-qPCR analysis. The functions of SNHG10 on the progression of renal fibrosis were examined by CCK-8, EdU, dual luciferase reporter and immunofluorescence analyses.Results: In the present study, SNHG10, production of extracellular matrix (ECM), including α-SMA and Fibronectin levels were significantly increased in HK-2 cells after TGF-β stimulation. Ectopic of SNHG10 inhibited cell proliferation and inhibits theα-SMA and Fibronectin expression of TGF-β1-induced HK-2 cells. In addition, bioinformatics analysis and dual luciferase reporter assay indicated that miR-378b was a target gene of SNHG10. Mechanistically, miR-378b overexpression abolished the repressive effects of SNHG10 on TGF-β1-induced HK-2 cells.Conclusion: SNHG10 plays an anti-fibrotic effect through suppression of miR-378b expression in renal fibrosis, which provides a promising therapeutic target for the treatment of renal fibrosis.

The Methyl Ester of 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid Reduces Endometrial Lesions Development by Modulating the NFkB and Nrf2 Pathways

Endometriosis is a common gynecological disease. Here, we aimed to investigate the anti-fibrotic, anti-inflammatory, and anti-oxidative role of the methyl ester of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO-Me) on endometriosis. An endometriosis rat model was constructed by intraperitoneally injecting recipient rats with an equivalent of tissue from the uterus of a donor animal. Endometriosis was allowed to develop for seven days. CDDO-Me was administered on the 7th day and for the next 7 days. On day 14, rats were sacrificed, and peritoneal fluid and endometriotic implants were collected. CDDO-Me displayed antioxidant activity by activating the Nfr2 pathway and the expression of antioxidant mediators such as NQO-1 and HO-1. Moreover, it reduced lipid peroxidation and increased glutathione (GSH) levels and superoxide dismutase (SOD) activity. CDDO-Me also showed anti-inflammatory activity by decreasing the expression of pro-inflammatory cytokines in peritoneal fluids and NFkB activation. It, in turn, reduced cyclooxygenase-2 (COX-2) expression in the endometriotic loci and prostaglandin E2 (PGE2) levels in the peritoneal fluids, leading to increased apoptosis and reduced angiogenesis. The reduced oxidative stress and pro-inflammatory microenvironment decreased implants diameter, area, and volume. In particular, CDDO-Me administration reduced the histopathological signs of endometriosis and inflammatory cells recruitment into the lesions, as shown by toluidine blue staining and myeloperoxidase (MPO) activity. CDDO-Me strongly suppressed α-SMA and fibronectin expression and collagen deposition, reducing endometriosis-associated fibrosis. In conclusion, CDDO-Me treatment resulted in a coordinated and effective suppression of endometriosis by modulating the Nrf2 and NFkB pathways.

Nicotine, Smoking, Podocytes and Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (EC). Podocytes (POD) are essential to preserve the structure and function of the glomerular filtration barrier and strong evidence indicates that early POD loss promotes DN progression. We performed studies in human POD and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and EC activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by COX2, known to induce podocyte injury; downregulation of AMPK, important for maintaining cellular energy stores and antioxidation and upregulation of CD36 that increased lipid uptake and promoted apoptosis. In diabetic mice NIC increased proteinuria, a recognized marker of CKD progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of POD cytoskeleton and increased fibronectin expression. These novel studies critically implicate NIC itself as a contributor to DN progression in CS and EC users.

Inhibition of fibrosis with multi-agent therapy in pulmonary fibrosis: Results of a drug library screening

AimsSuccessful management of IPF will likely require multi-drug therapy as its pathogenesis is thought to be both driven by both pro-inflammatory and pro-fibrotic pathways. We hypothesized that the available anti-fibrotic agents, pirfenidone and nintedanib, may exhibit synergy in suppressing lung fibroblast extracellular matrix protein generation when administered in combination with other orally active agents.Materials and MethodsA fibroblastic cell line (AKR-2B) was stimulated with TGF-β1 and used to screen a library of over 1500 FDA approved drugs. Extracellular matrix protein generation was assessed via fibronectin ELISA assay and maintenance of cell viability confirmed with XTT assay.ResultsThe screening revealed sixty-two drugs from the repurposed drug-screening library that were shown to significantly suppress fibronectin expression and not result in cell death. Specifically drugs within the category of NSAIDs, steroids, azole antifungal agents, and antipyrine were associated with significant suppression of fibronectin on ELISA analysis. Surprisingly, we observed anti-fibrotic activity across a number of the azole antifungal compounds. We next assessed whether combination of azoles would exhibit synergy when combined with current anti-fibrotic therapies in the stimulated fibroblasts. As proof of concept, we demonstrated in vitro synergy between oxiconazole and nintedanib in suppressing fibroblast generation of extracellular matrix fibronectin.ConclusionsThese results suggest an approach to identify potential combinations of therapy that may improve patient outcomes by reducing cost and potential toxicities during treatment.

P0996THE INVOLVEMENT OF PRO-INFLAMMATORY IL-1 CYTOKINES IN TUBULAR INJURY IN DIABETIC KIDNEY DISEASE

Background and Aims Pro-inflammatory cytokines are one of several factors which contribute to the progression of diabetic kidney disease (DKD), a condition characterized by chronic kidney inflammation which results in the tubulointerstitial fibrosis which contributes to the progression of DKD. Interleukin 1 (IL-1) two main agonists IL-1α and IL-1β activate a pro-inflammatory cascade in response to different inflammatory stimuli, including hyperglycemia. It was previously shown that a deficiency of NLRP3 which is required for the conversion of IL-1 to its active state, protects mice from the development and progression of DKD. We hypothesize that the chronic hyperglycemia in diabetic patients triggers the activation and release of IL1α and/or IL-1β from renal tubular cells and that this activation leads to the tissue fibrosis. We aim to assess Il-1 and fibronectin expression in an immortalized proximal tubule epithelial cell line from normal adult human kidney (HK-2). In addition, we evaluate the influence of Anakinra™, a pharmaceutical inhibitor of the Il-1 receptor, currently indicated mainly for rheumatoid diseases, on the levels of fibronectin expression in this model. Methods HK-2 cells were cultured and treated with either physiological glucose concentration (5.5mM), high glucose (30mM) or 30mM mannitol as osmotic control for 24 hours to evaluate their effects on Il-1 expression and fibronectin expression. mRNA levels of IL-1α, IL-1β and fibronectin were assessed in q-PCR, and protein expression levels were quantified by western blotting. Immunofluorescence was used to visually demonstrate the presence of IL-1α and IL-1β upon stimulation. Finally, Anakinra™ was added to the tissue cultures in a range of physiologic prescribed concentrations and its effect on cell fibrosis was assessed by the measurement of fibronectin expression 24 hours later by western blotting. Results mRNA and protein expression of IL-1α but mostly IL-1β was elevated in HK-2 cells under hyperglycemic conditions but not in physiological glucose environment or under high osmotic conditions. Fibronectin levels were elevated in the high glucose treated cells compared with control. Finally, Anakinra™ was found to attenuate fibronectin expression under high glucose conditions, compared with the untreated cells. Conclusion Proinflammatory IL-1α and IL-1β cytokines are expressed by HK-2 cells upon stimulation with glucose and result in the fibrosis on the cells measured by the production of fibronectin. The addition of Anakinra™, an IL-1 receptor blocker, to the cell culture attenuate the expression of fibronectin by the tubular cells. Our research is the first to describe a causation between hyperglycemia, IL-1 elevated levels and fibrosis in HK-2 cells, as demonstrated by the beneficial effect of Anakinra™ on lowering fibronectin expression.