Extracellular Collagen Deposition Research Articles

Repurposing mesalazine against cardiac fibrosis in vitro

Cardiovascular diseases are exacerbated and driven by cardiac fibrosis. TGFβ induces fibroblast activation and differentiation into myofibroblasts that secrete excessive extracellular matrix proteins leading to stiffening of the heart, concomitant cardiac dysfunction, and arrhythmias. However, effective pharmacotherapy for preventing or reversing cardiac fibrosis is presently unavailable. Therefore, drug repurposing could be a cost- and time-saving approach to discover antifibrotic interventions. The aim of this study was to investigate the antifibrotic potential of mesalazine in a cardiac fibroblast stress model. TGFβ was used to induce a profibrotic phenotype in a human cardiac fibroblast cell line. After induction, cells were treated with mesalazine or solvent control. Fibroblast proliferation, key fibrosis protein expression, extracellular collagen deposition, and mechanical properties were subsequently determined. In response to TGFβ treatment, fibroblasts underwent a profound phenoconversion towards myofibroblasts, determined by the expression of fibrillary αSMA. Mesalazine reduced differentiation nearly by half and diminished fibroblast proliferation by a third. Additionally, TGFβ led to increased cell stiffness and adhesion, which were reversed by mesalazine treatment. Collagen 1 expression and deposition—key drivers of fibrosis—were significantly increased upon TGFβ stimulation and reduced to control levels by mesalazine. SMAD2/3 and ERK1/2 phosphorylation, along with reduced nuclear NFκB translocation, were identified as potential modes of action. The current study provides experimental pre-clinical evidence for antifibrotic effects of mesalazine in an in vitro model of cardiac fibrosis. Furthermore, it sheds light on possible mechanisms of action and suggests further investigation in experimental and clinical settings.

Platelet-derived growth factor B restores vascular barrier integrity and diminishes permeability in ovarian hyperstimulation syndrome.

Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) have been introduced, complete prevention is not yet possible. Previously, we and other authors have shown that vascular endothelial growth factor, angiopoietins (ANGPTs) and sphingosine-1-phosphate are involved in OHSS etiology. In addition, we have demonstrated that ovarian protein levels of platelet-derived growth factor (PDGF) ligands -B and -D decrease in an OHSS rat model, whilst PDGFR-β and ANGPT2 remain unchanged. In the present work, we investigated the role of PDGF-B in OHSS by evaluating ligand protein levels in follicular fluid (FF) from women at risk of developing OHSS and by using an immature rat model of OHSS. We demonstrated that PDGF-B and PDGF-D are lower in FF from women at risk of developing OHSS compared to control patients (P < 0.05). In the OHSS rat model, PDGF-B (0.5 µg/ovary) administration decreased ovarian weight (P < 0.05), reduced serum progesterone (P < 0.05) and lowered the percentage of cysts (P < 0.05), compared to untreated OHSS rats, but had no effect on the proportion of follicles or corpora lutea (CL). PDGF-B treatment also restored the expression of steroidogenic acute regulatory protein (P < 0.05) and P450 cholesterol side-chain cleavage enzyme (P < 0.01) to control levels. In addition, PDGF-B increased the peri-endothelial cell area in CL and cystic structures, and reduced vascular permeability compared to untreated OHSS ovaries. Lastly, PDGF-B increased the levels of junction proteins claudin-5 (P < 0.05), occludin (P < 0.05) and β-catenin (P < 0.05), while boosting the extracellular deposition of collagen IV surrounding the ovarian vasculature (PP < 0.01), compared to OHSS alone. In conclusion, our findings indicate that PDGF-B could be another crucial mediator in the onset and development of OHSS, which may lead to the development of novel prediction markers and therapeutic strategies.

Fat-Soluble Vitamins Affect Composition of Extracellular Matrix Deposited by Human Aortic Smooth Muscle and Endothelial Cells In Vitro

Atherosclerotic cardiovascular disease is accompanied by changes in arterial connective tissue. We evaluated the effects of fat-soluble vitamins A, D, and E individually and in combinations on the composition of extracellular matrix produced and deposited by arterial wall cells, human aortic smooth muscle cells, and endothelial cells. Individually, vitamins D and E stimulated collagen type I extracellular matrix deposition in human aortic smooth muscle cell cultures. However, vitamins A, D, and E reduced collagen type IV deposition by human aortic smooth muscle cell, counteracting the stimulatory effects of vitamin C. The extracellular matrix deposition of heparan sulfate by human aortic smooth muscle cells increased by vitamin C and its combination (C+D+E). β-carotene + D + C induced the extracellular matrix deposition of collagen I by endothelial cells. Vitamin E with other vitamins resulted in either induction (E+C+A) or inhibition (E+D). The extracellular matrix deposition of type IV collagen and elastin by human aortic endothelial cells was not affected by test vitamins, except the extracellular matrix type IV collagen decrease by combinations (A+E), (A+D+E), and (C+D+E). The extracellular matrix deposition of all tested glycosaminoglycans was reduced by vitamin A and its combination (A+C+D+E). Therefore, the fat-soluble vitamins applied individually or in combination—both with each other or with ascorbic acid—can affect extracellular matrix deposition of type I and IV collagens, and key glycosaminoglycans by cultured human aortic arterial wall cells.

Potential Mechanism of Dermal Wound Treatment With Preparations From the Skin Gel of Arabian Gulf Catfish: A Unique Furan Fatty Acid (F6) and Cholesta-3,5-Diene (S5) Recruit Neutrophils and Fibroblasts to Promote Wound Healing.

Preparations from Arabian Gulf catfish (Arius bilineatus, Val) epidermal gel secretion (PCEGS) effectively heal chronic wounds in diabetic patients. However, specific lipid components of PCEGS that are responsible for various aspects of wound healing are unknown. Here, we report for the first time that, i) a unique preparation containing only proteins and lipids (Fraction B, FB), derived from the PCEGS accelerated the healing of experimental dermal wounds in female rats (transdermal punch biopsy) in vivo. Histological analyses showed that topical treatment of these wounds with FB promoted the migration of fibroblasts, facilitated the production of extracellular matrix (collagen, fibronectin), induced capillary formation and recruitment of immune cells, and accelerated overall wound healing by day 4 (tested at 1, 2, 3, 4, and 10 days; n=15 for vehicle; n=15 for FB treatment), ii) the lipids responsible for different stages of wound healing were separated into a protein-free bioactive lipid fraction, Ft, which contained a few common long-chain fatty acids, a unique furan fatty acid (F6) and a cholesterol metabolite, cholesta-3,5-diene (S5). Ft (the partially purified lipid fraction of PCEGS), and F6 and S5 present in Ft, proved to be bioactive for wound healing in human dermal fibroblasts. Ft increased the production and extracellular deposition of collagen and fibronectin, ex vivo, iii) Ft and its subcomponents, pure F6 and S5, also promoted human dermal fibroblast migration into the scratch wound gaps, ex vivo, iv) Ft, F6, and S5 promoted the recruitment of neutrophils (Green fluorescence protein labeled) to the site of injury in the transected tailfins of transgenic zebrafish, in vivo, v) Ft, but not F6 or S5, promoted the regeneration of tissues at the wound site in the transgenic zebrafish tailfin, in vivo. Therefore, we conclude that lipid fraction Ft from PCEGS contains the components necessary to promote complete wound healing, and F6 and S5 are responsible for promoting fibroblast and neutrophil recruitment to the site of wounds.

A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity

A key feature in the pathogenesis of heart failure is cardiac fibrosis, but effective treatments that specifically target cardiac fibrosis are currently not available. A major impediment to progress has been the lack of reliable in vitro models with sufficient throughput to screen for activity against cardiac fibrosis. Here, we established cell culture conditions in micro-well format that support extracellular deposition of mature collagen from primary human cardiac fibroblasts – a hallmark of cardiac fibrosis. Based on robust biochemical characterization we developed a high-content phenotypic screening platform, that allows for high-throughput identification of compounds with activity against cardiac fibrosis. Our platform correctly identifies compounds acting on known cardiac fibrosis pathways. Moreover, it can detect anti-fibrotic activity for compounds acting on targets that have not previously been reported in in vitro cardiac fibrosis assays.Taken together, our experimental approach provides a powerful platform for high-throughput screening of anti-fibrotic compounds as well as discovery of novel targets to develop new therapeutic strategies for heart failure.

Repeated Remote Ischemic Conditioning Reduces Doxorubicin-Induced Cardiotoxicity

ObjectivesThis study investigated the cardioprotective effect of repeated remote ischemic preconditioning (rRIC) on doxorubicin-induced cardiotoxicity in mice. BackgroundDoxorubicin is an effective chemotherapeutic agent for a wide range of tumor types but its use and dosing are limited by acute and chronic cardiotoxicity. Remote ischemic conditioning (RIC) is cardioprotective in multiple cardiovascular injury models, but the effectiveness of rRIC in doxorubicin-induced cardiotoxicity has not been fully elucidated. MethodsrRIC was performed on mice before and after doxorubicin administration. Cardiac function was assessed by echocardiography and myocardial biology was tested by molecular approaches. ResultsDoxorubicin administration induced acute cardiotoxicity, as indicated by reduced cardiac function, reduced myocyte cross-section area and increased extracellular collagen deposition, increased circulating cardiac muscle damage markers, and decreased heart weight. Doxorubicin also adversely affected other organs, including the kidney, liver, and spleen, as evaluated by circulating markers or organ weight loss. rRIC not only abrogated doxorubicin-induced cardiotoxicity (left ventricular ejection fraction, doxorubicin 47.5 ± 1.1%, doxorubicin + rRIC 51.6 ± 0.7%, p = 0.017), but also was associated with multiorgan protection. Within the myocardium, rRIC attenuated doxorubicin-induced cardiomyocyte apoptosis, reduced inflammation, and increased autophagy signaling. ConclusionsrRIC may be a promising approach to reduce doxorubicin-induced cardiotoxicity.

Protective Effect of Low-Molecular-Weight Fucoidan on Radiation-Induced Fibrosis Through TGF-β1/Smad Pathway-Mediated Inhibition of Collagen I Accumulation.

Radiation-induced fibrosis (RIF) occurs after radiation therapy in normal tissues due to excessive production and deposition of extracellular matrix proteins and collagen, possibly resulting in organ function impairment. This study investigates the effects of low-molecular-weight fucoidan (LMF) on irradiated NIH3T3 cells. Specifically, we quantified cellular metabolic activity, fibrosis-related mRNA expression, transforming growth factor beta-1 (TGF-β1), and collagen-1 protein expression, and fibroblast contractility in response to LMF. LMF pre + post-treatment could more effectively increase cellular metabolic activity compared with LMF post-treatment. LMF pre + post-treatment inhibited TGF-β1 expression, which mediates negative activation of phosphorylated Smad3 (pSmad3) and Smad4 complex formation and suppresses downstream collagen I accumulation. In addition, LMF pre + post-treatment significantly reduced actin-stress fibers in irradiated NIH3T3 cells. LMF, a natural substance obtained from brown seaweed, may be a candidate agent for preventing or inhibiting RIF.

Lack of Thy1 defines a pathogenic fraction of cardiac fibroblasts in heart failure

In response to heart injury, inflammation, or mechanical overload, quiescent cardiac fibroblasts (CFs) can become activated myofibroblasts leading to pathological matrix remodeling and decline in cardiac function. Specific targeting of fibroblasts may thus enable new therapeutic strategies to delay or reverse the progression of heart failure and cardiac fibrosis. However, it remains unknown if all CFs are equally responsive to specific pathological insults and if there exist sub-populations of resident fibroblasts in the heart that have distinctive pathogenic phenotypes. Here, we show that in response to transverse aortic constriction (TAC)-induced heart failure, previously uncharacterized Thy1neg (Thy1-/MEFSK4+/CD45-/CD31-) fraction of mouse ventricular fibroblasts became more abundant and attained a more activated, pro-fibrotic myofibroblast phenotype compared to Thy1Pos fraction. In a tissue-engineered 3D co-culture model of healthy cardiomyocytes and freshly isolated CFs, Thy1neg CFs from TAC hearts significantly decreased cardiomyocyte contractile function and calcium transient amplitude, and increased extracellular collagen deposition yielding a profibrotic heart tissue phenotype. In vivo, mice with global knockout of Thy1 developed more severe cardiac dysfunction and fibrosis in response to TAC-induced heart failure than wild-type mice. Taken together, our studies identify cardiac myofibroblasts lacking Thy1 as a pathogenic CF fraction in cardiac fibrosis and suggest important roles of Thy1 in pathophysiology of heart failure.

The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71-257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase-dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase-positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

Abstract TMIM-081: THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING

Abstract High grade serous ovarian cancer (HGSOC) is a leading cause of cancer related deaths among women. The primary cause of morbidity and mortality in HGSOC patients is metastasis to organs within the peritoneal cavity. Mesothelial cells line the surface of peritoneal organs and are a key stromal cell in HGSOC metastatic niche. However, the mechanisms by which mesothelial cells promote ovarian cancer peritoneal metastasis remain largely unknown. Here we demonstrate that tumor associated mesothelial cells promote tumor invasion by increasing collagen deposition and remodeling. Mechanistically, we demonstrate the tumor-mesothelial niche is hypoxic where both tumor cells and mesothelial cells express the hypoxia inducible factors HIF-1 and HIF-2 and their downstream target gene lysyl oxidase (LOX). LOX is an enzyme that crosslinks collagen fibrils to promote collagen remodeling. In tumor-mesothelial co-culture experiments, we demonstrate that hypoxia enhances extracellular fibrillar collagen deposition by mesothelial cells. Conditioned media from hypoxic tumor-mesothelial co-cultures promotes collagen remodeling and HGSOC tumor cell invasion. Genetic inactivation of either HIF-1 and HIF-2 or LOX reduces the hypoxic induction of collagen remodeling and tumor cell invasion. Importantly, pharmacologic inhibition of HIF-1 and HIF-2 with digoxin reduces the colonization of disseminated HGSOC cells to the omentum, the preferential migration site of peritoneal cancer metastasis. Moreover, pharmacologic inhibition of LOX with BAPN (beta-aminopropionitrile) is sufficient to inhibit metastatic tumor burden and collagen remodeling at metastatic sites in preclinical models of HGSOC metastasis. These data reveal a novel role for mesothelial cells in the production of type I collagen and collagen remodeling in the HGSOC metastatic microenvironment. Furthermore, these studies demonstrate a role for the HIF/LOX signaling axis in the HGSOC tumor-mesothelial niche that can be therapeutically targeted to inhibit collagen remodeling and ovarian cancer metastatic progression. Citation Format: Suchitra Natarajan, Katie Foreman, Michaela Soriano, Hussein Shehade, Daniel Fregoso, Joshua Eggold, Ninna S. Rosen, Sarah Heilshorn, Adam J. Krieg, Venkatesh Krishnan, Oliver Dorigo, Subarna Sinha, Katherine C. Fuh, Erinn B. Rankin. THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-081.

Carvacrol Downregulates Lysyl Oxidase Expression and Ameliorates Oxidative Stress in the Liver of Rats with Carbon Tetrachloride-Induced Liver Fibrosis.

In the current study, we aimed to investigate the effect of carvacrol on the suppression of liver fibrosis progression through targeting lysyl oxidase (LOX) expression. The rats received carbon tetrachloride (CCl4) intraperitoneally and carvacrol orally for 10weeks. Liver damage was evaluated by measuring the serum level of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase and hepatic oxidative stress parameters including total antioxidant capacity, total thiol group and total oxidant status spectrophotometry and malondialdehyde fluorometrically. Extracellular deposition of collagen was detected using Masson's trichrome standing. Furthermore the gene expression of lysyl oxidase homolog 2 (Loxl2) was analyzed using quantitative reverse transcription-polymerase chain reaction. And then the protein level of LOX was detected in liver tissue by western blot method. Carvacrol administration normalized serum biochemical parameters and improved oxidative stress status in liver homogenate of CCl4 treated rats. Collagen fiber bundles in interlobular spaces were decreased remarkably by carvacrol treatment. Also, carvacrol downregulated hepatic gene expression of Loxl2 and protein level of LOX. Our data clearly revealed that carvacrol suppresses progression of liver fibrosis development via attenuating of liver damage and oxidative stress status as well as via downregulation of hepatic gene expression of Loxl2 and protein level of LOX.

LncRNA PFAR contributes to fibrogenesis in lung fibroblasts through competitively binding to miR-15a.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, debilitating disease with unknown etiopathogenesis. Previous reports have reported that long non-coding RNAs (lncRNAs) were involved in various pathophysiological processes. However, the role of lncRNAs in IPF has not been fully described. We aimed to explore the relationship between miR-15a and lncRNA PFAR and its function in pulmonary fibrosis. Biological information analysis and luciferase were used to identify targeted binding of lncRNA PFAR and miR-15a. Western blot, quantitative reverse transcription-PCR (qRT-PCR) and immunofluorescence staining were conducted to detect fibrosis-related factors. Fibroblasts proliferation were analyzed using 5-ethynyl-2′-deoxyuridine (EdU) staining and fibroblasts migration ability were measured using wound-healing scratch assay. We identified that lncRNA PFAR has a binding site with miR-15a and luciferase reporter assays demonstrated their combinative relationship. Our results showed that silencing PFAR attenuated TGF-β1 induced fibrogenesis in primary lung fibroblasts. And miR-15a antagonized the function of PFAR and inhibited PFAR induced extracellular collagen deposition, fibroblasts proliferation, migration and differentiation. In conclusion, our results revealed that PFAR functions as a competitive endogenous RNA (ceRNA) by acting as a sponge for miR-15a, revealing a potential regulatory network involving PFAR and miR-15a with a role in the modulation of YAP1-Twist expression. This mechanism may contribute to a better understanding of pulmonary fibrosis pathogenesis and treatment method.

The Role of MicroRNA-181a in Myocardial Fibrosis Following Myocardial Infarction in a Rat Model.

BackgroundThe role of miR-181a in the development of cardiac disease and in particular, myocardial fibrosis following myocardial infarction (MI) remains unknown. The aim of this study was to explore the role of miR-181a in myocardial fibrosis in a rat model of MI and the expression of TGF-β receptor III (TβRIII).Material/MethodsForty adult male Wistar rats were randomly divided into an MI model group (n=30) and a control group with (n=10). The rat MI model involved ligating the left anterior descending (LAD) coronary artery in the model group; the control group was treated with a sham operation. Cardiac function was assessed using cardiac ultrasound. Myocardial fibroblasts were extracted from the rat hearts and transfected with a miR-mimic or miR-inhibitor, and cell growth was measured using an MTT assay. The level of miR-181a expression was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blots.ResultsmiR-181a expression was significantly increased during the progression of MI (P<0.05). Over-expression of miR-181a was associated with increased deposition of extracellular matrix (ECM) components, collagen I and fibronectin. This effect was reversed with the use of a miR-181a inhibitor (P<0.05). Upregulation of miR-181a suppressed the expression of TGF-β receptor III (TβRIII) by binding with 3′-UTR.ConclusionsIn this rat model of MI, the findings were that miR-181a had a role in the progression of myocardial fibrosis. The findings require further studies to determine whether miR-181a might provide a novel therapeutic target to limit myocardial fibrosis following MI.

Progress in non-invasive detection of liver fibrosis.

Liver fibrosis is an important pathological precondition for hepatocellular carcinoma. The degree of hepatic fibrosis is positively correlated with liver cancer. Liver fibrosis is a series of pathological and physiological process related to liver cell necrosis and degeneration after chronic liver injury, which finally leads to extracellular matrix and collagen deposition. The early detection and precise staging of fibrosis and cirrhosis are very important for early diagnosis and timely initiation of appropriate therapeutic regimens. The risk of severe liver fibrosis finally progressing to liver carcinoma is >50%. It is known that biopsy is the gold standard for the diagnosis and staging of liver fibrosis. However, this method has some limitations, such as the potential for pain, sampling variability, and low patient acceptance. Furthermore, the necessity of obtaining a tissue diagnosis of liver fibrosis still remains controversial. An increasing number of reliable non-invasive approaches are now available that are widely applied in clinical practice, mostly in cases of viral hepatitis, resulting in a significantly decreased need for liver biopsy. In fact, the non-invasive detection and evaluation of liver cirrhosis now has good accuracy due to current serum markers, ultrasound imaging, and magnetic resonance imaging quantification techniques. A prominent advantage of the non-invasive detection and assessment of liver fibrosis is that liver fibrosis can be monitored repeatedly and easily in the same patient. Serum biomarkers have the advantages of high applicability (>95%) and good reproducibility. However, their results can be influenced by different patient conditions because none of these markers are liver-specific. The most promising techniques appear to be transient elastography and magnetic resonance elastography because they provide reliable results for the detection of fibrosis in the advanced stages, and future developments promise to increase the reliability and accuracy of the staging of hepatic fibrosis. This article aims to describe the recent progress in the development of non-invasive assessment methods for the staging of liver fibrosis, with a special emphasize on computer-aided quantitative and deep learning methods.

Activation of cannabinoid receptor type 2 by JWH133 alleviates bleomycin-induced pulmonary fibrosis in mice.

Activation of cannabinoid receptor type 2 has been shown to have anti-fibrosis function in skin and heart. However, whether activating cannabinoid receptor type 2 inhibits pulmonary fibrosis remains elusive. Lung fibroblasts and TGF-β1 are key players in the pathogenesis of pulmonary fibrosis. In this research, we aimed to investigate the role of cannabinoid receptor type 2 in pulmonary fibrosis in vitro and in vivo. In lung fibroblasts stimulated by TGF-β1, preincubated by cannabinoid receptor type 2 agonist JWH133 not only reduced the elevated levels of collagen I and α-SMA, but also inhibited fibroblasts’ proliferation and migration. The dosage of JWH133 had no clear cytotoxic activity, and all these JWH133 effects were partially abrogated by cannabinoid receptor type 2 antagonist SR144528. In bleomycin-induced mice pulmonary fibrosis model, CT images of the lung tissue revealed an extensive ground-glass opacity, reticular pattern and fibrosis stranding. Notably, JWH133 treatment controlled the ongoing fibrotic process (showed by decreased lung density and fibrosis score). Meanwhile, lung histological results revealed that JWH133 treatment suppressed both the inflammatory response and extracellular collagen deposition. SR144528 may increase the pulmonary fibrosis, but no statistically significant difference was proved. Importantly, JWH133 reduced serum profibrotic cytokines levels of TGF-β1 and inhibited TGF-β1/Smad2 pathway in vitro and in vivo. Our research indicated that activating cannabinoid receptor type 2 by a pharmacological method might be a potential strategy for pulmonary fibrosis.

Loss of \u03b2-catenin in resident cardiac fibroblasts attenuates fibrosis induced by pressure overload in mice

Cardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Cardiac pressure overload resulting from trans-aortic constriction in mice leads to cardiac fibrosis and increased Wnt/β-catenin signaling in cardiac fibroblasts. Here, we conditionally induce β-catenin loss of function in resident cardiac fibroblasts using Tcf21MerCreMer or in activated cardiac fibroblasts using periostin (Postn)MerCreMer. We show that β-catenin loss of function in cardiac fibroblasts after trans-aortic constriction significantly preserves cardiac function, and reduces interstitial fibrosis but does not alter the numbers of activated or differentiated cardiac fibroblasts in vivo. However, β-catenin is specifically required in resident cardiac fibroblasts for fibrotic excessive extracellular matrix gene expression and binds Col3a1 and Postn gene sequences in cultured cardiac fibroblasts after induction of Wnt signaling. Moreover, cardiomyocyte hypertrophy is blunted with cardiac fibroblast-specific loss of β-catenin after trans-aortic constriction in vivo. Thus, Wnt/β-catenin signaling in resident cardiac fibroblasts is required for excessive extracellular matrix gene expression and collagen deposition after trans-aortic constriction.

Rutin alleviates diabetic cardiomyopathy and improves cardiac function in diabetic ApoEknockout mice

Rutin, a natural bioflavonoid, has demonstrated anti-diabetic and anti-oxidative bioactivity. Oxidative stress is a potential therapeutic target for diabetic cardiomyopathy. We investigated whether rutinadministration (60mg/kg body weight) reduces diabetic cardiomyopathy in a diabetic ApoE knock out mouse model. Diabetes was induced in ApoEknockout mice (male, C57BL/6 background) with a high fat diet combined with injection of streptozotocin. Cardiac function was evaluated by echocardiography and cardiac catheter hemodynamic analysis. Cardiac myocardial hypertrophy, myocardial fibrosis, lipid content, myocardial capillary density, and oxidative stress were detected by a series of histopathological analyses, western blotting, and reactive oxygen species analysis. Diabetic mice showed myocardial hypertrophy, lipid accumulation, myocardial fibrosis, elevated collagen content, deteriorating oxidative stress, and left ventricular systolic and diastolic dysfunction. Rutin reversed the myocardial hypertrophy, alleviated extracellular collagen deposition, and lipid accumulation, but increased capillary density in diabetic myocardial tissues. Moreover, rutin substantially improved cardiac function while decreasing blood glucose and lipid content. Therapeutic rutin administration reduced cardiac remodeling and improved myocardial function in diabetic mice, at least in part by reducing oxidative damage and ectopic lipid deposition, inhibiting fibrosis, and promoting angiogenesis. Thus, rutin may represent a potential therapeutic agent for diabetic cardiomyopathy.

Hypertrophy and Fibrosis of the Ligamentum Flavum in Lumbar Spinal Stenosis is Associated With Increased Expression of LPA and LPAR1.

Histologic, immunohistochemical (IHC), and enzyme-linked immunosorbent assay analysis of the human ligamentum flavum (LF). The purpose of this study was to determine the level of expression of lysophosphatidic acid (LPA) in the LF from lumbar spinal stenosis (LSS) patients and to analyze the relationship among LPA, LPA receptors (LPARs), and LF hypertrophy. LF hypertrophy and fibrosis are important causes of LSS. LPA is involved in the fibrotic process in multiple organ systems. Therefore, we hypothesized that LPA and its receptors might also play a role in degeneration of the LF in LSS patients. Forty-one LF samples were enrolled in this study. The thickness of the LF was measured by magnetic resonance imaging. Histologic analysis using hematoxylin and eosin and Masson trichrome stain was performed for each LF to evaluate the architecture of the extracellular matrix. The content of LPA and connective tissue growth factor (CTGF) in LF samples was analyzed using enzyme-linked immunosorbent assay. The expression of LPAR1 was determined by IHC. Degeneration of the LF was characterized by an increase in disorganized elastic fibers and fibrotic transformation by extracellular collagen deposition. The thickness of the LF and the concentration of LPA and CTGF in the hypertrophic LF group were significantly higher than the control group. Furthermore, the LPA and CTGF concentrations had a positive correlation with the LF thickness (r=0.91, P<0.001 and r=0.943, P<0.001, respectively). On the basis of IHC analysis, the expression of LPAR1 was increased in the hypertrophy group. The increased expression of LPA and LPAR1 is associated with the fibrosis and hypertrophy of the LF in patients with LSS. Further study on the mechanism underlying LF fibrosis may lead to new therapies for LF hypertrophy and fibrosis.

Inhibition of the KCa3.1 Channel Alleviates Established Pulmonary Fibrosis in a Large Animal Model.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of increasing prevalence marked by poor prognosis and limited treatment options. Ca2+-activated KCa3.1 potassium channels have been shown to play a key role in the aberrant activation and responses to injury in both epithelial cells and fibroblasts, both considered key drivers in the fibrotic process of IPF. Pharmacological inhibition of IPF-derived fibroblasts is able to somewhat prevent TGF-β- and basic fibroblast growth factor-dependent profibrotic responses. In the current study, we investigated whether blockade of the KCa3.1 ion channel in vivo with a selective inhibitor, Senicapoc, was able to attenuate both histological and physiological outcomes of early fibrosis in our large animal (sheep) model for pulmonary fibrosis. We also determined whether treatment was targeting the profibrotic activity of sheep lung fibroblasts. Senicapoc was administered in established fibrosis, at 2 weeks after bleomycin instillation, and drug efficacy was assessed 4 weeks after treatment. Treatment with Senicapoc improved pre-established bleomycin-induced changes compared with vehicle control, leading to improved lung compliance, reduced extracellular matrix and collagen deposition, and a reduction in both α-smooth muscle actin expression and proliferating cells, both in vivo and in vitro. These studies show that inhibiting the KCa3.1 ion channel is able to attenuate the early fibrogenic phase of bleomycin-dependent fibrosis and inhibits profibrotic behavior of primary sheep lung fibroblasts. This supports the previous research conducted in human IPF-derived fibroblasts and suggests that inhibiting KCa3.1 signaling may provide a novel therapeutic approach for IPF.

Therapeutic efficacy of a co-blockade of IL-13 and IL-25 on airway inflammation and remodeling in a mouse model of asthma

Repeated airway inflammation and unremitting remodeling provoke irreversible pulmonary dysfunction and resistance to current drugs in patients with chronic bronchial asthma. Interleukin (IL)-13 and IL-25 play an important role in airway inflammation and remodeling in asthma. We aimed to investigate whether co-inhibiting IL-13 and IL-25 can effectively down-regulate allergen-induced airway inflammation and remodeling in mice. Mice with asthma induced by chronic exposure to ovalbumin (OVA) were given soluble IL-13 receptor α2 (sIL-13R) or soluble IL-25 receptor (sIL-25R) protein alone and in combination to neutralize the bioactivity of IL-13 and IL-25, and relevant airway inflammation and remodeling experiments were performed. We found that the co-blockade of IL-13 and IL-25 with sIL-13R and sIL-25R was more effective than either agent alone at decreasing inflammatory cell infiltration, airway hyperresponsiveness (AhR) and airway remodeling including mucus production, extracellular collagen deposition, smooth muscle cell hyperplasia and angiogenesis in mice exposed to OVA. These results suggest that the combined inhibition of IL-13 and IL-25 may provide a novel therapeutic strategy for asthma, especially for patients who are resistant to current treatments.