Profibrotic Growth Factor Research Articles

Bronchoalveolar-Lavage-Derived Fibroblast Cell Line (B-LSDM7) as a New Protocol for Investigating the Mechanisms of Idiopathic Pulmonary Fibrosis.

Background: The use of BAL to study ILDs has improved our understanding of IPF pathogenesis. BAL fluid is routinely collected and can be considered a clinical and research tool. The procedure is well tolerated and minimally invasive. No specific cell lines from BAL or immortalized cell lines from IPF patients are available commercially. A method to quickly isolate and characterize fibroblasts from BAL is an unmet research need. Materials and methods: Here we describe a new protocol by which we isolated a cell line from IPF. The cell line was expanded in vitro and characterized phenotypically, morphologically and functionally. Results: This culture showed highly filamentous cells with an evident central nucleus. From the phenotypic point of view, this cell line displays fibroblast/myofibroblast-like features including expression of alpha-SMA, vimentin, collagen type-1 and fibronectin. The results showed high expression of ROS in these cells. Oxidative stress invariably promotes extracellular matrix expression in lung diseases directly or through over-production of pro-fibrotic growth factors. Conclusions: Our protocol makes it possible to obtain fibroblasts BAL that is a routine non-invasive method that offers the possibility of having a large sample of patients. Standardized culture methods are important for a reliable model for testing molecules and eventual novel development therapeutic targets.

Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)β1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFβ1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFβ-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

Defective perlecan-associated basement membrane regeneration and altered modulation of transforming growth factor beta in corneal fibrosis.

In the cornea, the epithelial basement membrane (EBM) and corneal endothelial Descemet's basement membrane (DBM) critically regulate the localization, availability and, therefore, the functions of transforming growth factor (TGF)β1, TGFβ2, and platelet-derived growth factors (PDGF) that modulate myofibroblast development. Defective regeneration of the EBM, and notably diminished perlecan incorporation, occurs via several mechanisms and results in excessive and prolonged penetration of pro-fibrotic growth factors into the stroma. These growth factors drive mature myofibroblast development from both corneal fibroblasts and bone marrow-derived fibrocytes, and then the persistence of these myofibroblasts and the disordered collagens and other matrix materials they produce to generate stromal scarring fibrosis. Corneal stromal fibrosis often resolves completely if the inciting factor is removed and the BM regenerates. Similar defects in BM regeneration are likely associated with the development of fibrosis in other organs where perlecan has a critical role in the modulation of signaling by TGFβ1 and TGFβ2. Other BM components, such as collagen type IV and collagen type XIII, are also critical regulators of TGF beta (and other growth factors) in the cornea and other organs. After injury, BM components are dynamically secreted and assembled through the cooperation of neighboring cells-for example, the epithelial cells and keratocytes for the corneal EBM and corneal endothelial cells and keratocytes for the corneal DBM. One of the most critical functions of these reassembled BMs in all organs is to modulate the pro-fibrotic effects of TGFβs, PDGFs and other growth factors between tissues that comprise the organ.

Inhibition of Vascular Endothelial Growth Factor Receptors 1 and 2 Attenuates Natural Killer Cell and Innate Immune Responses in an Experimental Model for Obliterative Bronchiolitis

Obliterative bronchiolitis (OB) after lung transplantation is a nonreversible, life-threatening complication. Herein, the role of vascular endothelial growth factor receptor (Vegfr)-1 and -2 was investigated in the development of obliterative airway disease (OAD), an experimental model for OB. The nonimmunosuppressed recipients underwent transplantation with fully major histocompatibility complex mismatched heterotopic tracheal allografts and received Vegfr1 and -2-specific monoclonal antibodies either alone or in combination, or rat IgG as a control. The treatment with Vegfr1- or -2-blocking antibody significantly decreased intragraft mRNA expression of natural killer cell activation markers early after transplantation. This was followed by reduced infiltration of Cd11b+ cells and Cd4+ T cells as well as down-regulated mRNA expression of proinflammatory chemokines and profibrotic growth factors. However, blocking of both Vegfr1 and -2 was necessary to reduce luminal occlusion. Furthermore, concomitant inhibition of the calcineurin activation pathway almost totally abolished the development of OAD. This study proposes that blocking of Vegf receptors blunted natural killer cell and innate immune responses early after transplantation and attenuated the development of OAD. The results of this study suggest that further studies on the role of Vegfr1 and -2 blocking in development of obliterative airway lesions might be rewarding.

Platelet Transcriptome Yields Progressive Markers in Chronic Myeloproliferative Neoplasms and Identifies Putative Targets of Therapy

Predicting disease progression remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the spectrum of chronic myeloproliferative neoplasms (MPNs), as a model, we identify the blood platelet transcriptome as a proxy strategy for highly sensitive progression biomarkers that also enables prediction of advanced disease via machine learning algorithms. Using RNA sequencing (RNA-seq), we derive disease-relevant gene expression in purified platelets from 120 peripheral blood samples constituting two time-separated cohorts of patients diagnosed with one of three MPN subtypes at sample acquisition - essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or post ET/PV secondary myelofibrosis, MF (n=42), and healthy donors (n=21).The MPN platelet transcriptome reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy and discriminates each clinical phenotype. Differential markers in each of ET, PV and MF also highlight candidate genes as potential mediators of the pro-thrombotic and pro-fibrotic phenotypes in MPNs. In ET and PV, a strong thromboinflammatory profile is revealed by the upregulation of several interferon inducible transmembrane genes (IFITM2, IFITM3, IFITM10, IFIT3, IFI6, IFI27L1, IFI27L2), interleukin receptor accessory kinases/proteins (IRAK1, IL15, IL1RAP, IL17RC) and several solute carrier family genes (SLC16A1, SLC25A1, SLC26A8, SLC2A9) as glucose and other metabolic transport proteins, and coagulation factor V (F5). In MF, fibrosis-specific markers were identified by an additional focused comparison of MF patients versus ET and PV, showing increased expression of several pro-fibrotic growth factors (FGFR1, FGFR3, FGFRL1), matrix metalloproteinases (MMP8, MMP14), vascular endothelial growth factor A (VEGFA), insulin growth factor binding protein (IGFBP7), and cell cycle regulators (CCND1, CCNA2, CCNB2, CCNF).Also, focusing on the JAK-inhibitor ruxolitinib/RUX-specific signatures, we not only confirm previous observations on its anti-inflammatory and immunosuppressive effects (e.g. downregulation in our RUX-treated cohort of IL1RAP, CXCR5, CPNE3, ILF3) but also identify new gene clusters responsive to RUX - e.g. inhibition of type I interferon (e.g. IFIT1, IFIT2, IFI6), chromatin regulation (HIST2H3A/C, HIST1H2BK, H2AFY, SMARCA4, SMARCC2), epigenetic methylation in mitochondrial genes (ATP6, ATP8, ND1-6 and NDUFA5), and other proliferation, and proteostasis-associated markers as putative targets for MPN-directed therapy.Mechanistic insights from our data highlight impaired protein homeostasis as a prominent driver of MPN evolution, with a persistent integrated stress response. Preliminary ex vivo data on MPN patient bone-marrow-derived CD34+ cells and cultured megakaryocytes validate a proteostasis-focused subset of our peripheral platelet RNA-seq signatures.Further leveraging this substantive dataset, and in particular a progressive expression gradient across MPN, we develop a machine learning model (Lasso-penalized regression) predictive of the advanced subtype MF at high accuracy and under two conditions of validation: i) temporal Stanford internal (AUC-ROC of 0.96) and ii) geographic external cohorts (AUC-ROC of 0.97, using independently published data of an additional n=25 MF and n=46 healthy donors). Lasso-derived signatures offer a robust core set of < 5 MPN progression markers.Together, our platelet transcriptome snapshot of chronic MPNs demonstrates a methodological avenue for disease risk stratification and progression beyond genetic data alone, with potential utility in a wide range of age-related disorders.Part of the work contributing to this abstract has been posted as a preprint at this link: https://www.biorxiv.org/content/10.1101/2021.03.12.435190v2 [Display omitted] DisclosuresGotlib: Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Kartos: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; PharmaEssentia: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cogent Biosciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair for the Eligibility and Central Response Review Committee, Research Funding; Allakos: Consultancy.

Anti-inflammatory and anti-fibrotic effects of modafinil in nonalcoholic liver disease

Small- and intermediate-conductance Ca2+-activated K+ channels, KCa2.3 and KCa3.1, are involved in cellular signaling processes associated with inflammation and fibrosis. KCa2.3 and KCa3.1 are upregulated by proinflammatory cytokines and profibrotic growth factors. Cyclic AMP, which downregulates KCa2.3 and KCa3.1, is elevated by modafinil in cells; accordingly, we investigated whether modafinil exerts anti-inflammatory and anti-fibrotic responses via KCa2.3- and KCa3.1-mediated pathways in high-fat diet (HFD)- or thioacetamide-induced liver disease models in mice. Modafinil was administered orally in the form of a racemate, (R)-isomer, or (S)-isomer. We also determined whether the treatment targeted the profibrotic activity of hepatic stellate cells using immortalized human hepatic stellate cells (LX-2 cells). Modafinil improved HFD- or thioacetamide-induced changes compared to the control, leading to a reduced inflammatory response, collagen deposition, and α-smooth muscle actin expression both in vivo and in vitro. However, modafinil did not relieve HFD-induced steatosis. There were no significant differences in the effects of the (R)- and (S)-isomers of modafinil. KCa2.3 and KCa3.1 were upregulated and catalase was downregulated in liver tissues from thioacetamide- or HFD-induced liver disease models or in TGF-β-treated LX-2 cells. TGF-β-induced upregulation of KCa2.3, KCa3.1, collagen, and α-smooth muscle actin and downregulation of catalase were reversed by modafinil, polyethylene glycol catalase, N-acetylcysteine, siRNA against KCa2.3 or KCa3.1, and Epac inhibitors. Our investigation revealed that modafinil attenuated inflammatory and fibrotic progression via KCa2.3- and KCa3.1-mediated pathways in nonalcoholic hepatitis, suggesting that inhibiting KCa2.3- and KCa3.1-mediated signaling may serve as a novel therapeutic approach for inflammatory and fibrotic liver diseases.

Hop and artichoke extracts inhibit expression of extracellular matrix components in uterine leiomyoma cells

To screen 14 different plant extracts for their antifibrotic effect on human primary leiomyoma and healthy myometrial cells. Preclinical study. University research laboratory. Human uterine leiomyoma and matched myometrial tissues were obtained from Caucasian premenopausal women with symptomatic uterine fibroids at the time of hysterectomy. Primary human leiomyoma and myometrial cells were cultured in the absence or presence of the plant extracts. Quantification of the expression of extracellular matrix components, such as fibronectin 1 (FN1), collagen type I alpha 1 (COL1A1), and versican (VCAN), and the profibrotic growth factor activin A or inhibin beta A subunit (INHBA). The cells were treated with the 14 extracts for 48 hours, and we measured FN1 messenger RNA (mRNA) expression. Of the 14 extracts, about (ABO) ABO-2 (hop) and ABO-9 (artichoke) significantly reduced FN1 expression in both the cell types. Next, we evaluated the effect of fractions of these 2 extracts on the mRNA expression of FN1 and other extracellular matrix components, such as COL1A1, VCAN, and INHBA, in leiomyoma and myometrial cells. We found that ABO-2 (hop) and ABO-9 (artichoke) as well as their fractions, ABO-AR-2016-015 (fraction of ABO-2) and ABO-AR-2014-168 (fraction of ABO-9), reduced the mRNA expression of FN1, COL1A1, VCAN, and INHBA in primary leiomyoma cells. In primary myometrial cells, the mRNA expression of FN1, COL1A1, VCAN, and INHBA was not greatly affected. These results suggest that the hop and artichoke extracts possess antifibrotic properties and support additional evaluation using invivo models.

High glucose-induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model.

Background: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis. Methods: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-β1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. Results: Only the combination of high D-glucose and TGF-β1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this rise was significantly reduced by both SGLT2 and MEK inhibition. Conclusions: We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.

MO620ANTIFIBROTIC EFFECTS OF SGLT2 INHIBITION IN THE KIDNEY AND PROXIMAL TUBULAR CELLS*

Abstract Background and Aims Diabetic kidney disease (DKD) is a major cause of chronic kidney disease and end stage renal disease, therefore identification of novel therapeutic strategies that reduce the risk of DKD is a research priority. Recent large clinical trials suggest that improved renal outcomes by sodium-glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects. Enhanced glucose reabsorption in diabetes leads to tubular hypoxia triggering fibrotic response. Hyperglycemia is in strong association with increased protein O-GlcNAcylation, a post-translational modification contributing to renal fibrosis. Considering the proximal tubular involvement in DKD pathogenesis and the key role of SGLT2 in glucose metabolism, here we investigated the effects of SGLT2i on tubular hypoxia and O-GlcNAcylation. Method Diabetes (D) was induced by streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day, po.). Metabolic parameters and renal function were evaluated. Novel urinary biomarkers of extracellular matrix remodeling (Pro-C3, uC3M, tumstatin) and profibrotic growth factors (TGF-β, CTGF, PDGF) were determined. Histological evaluation of glomerular damage (PAS), tubulointerstitial fibrosis (Masson’s trichrome, Picrosirius red) and fibronectin accumulation were performed. The effect of hyperglycemia was tested in human proximal tubular epithelial cells (HK-2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions for 24 hours. HG cells were treated with 10 µM DAPA. O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 µM DAPA and were placed in a hypoxic chamber (1% O2) for 2 hours. Hypoxic injury was investigated using three different methods (qRT-PCR, Western blot, immunofluorescence analysis). HIF-1α, EPO, VEGFA and profibrotic factors were measured. Results DAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p&amp;lt;0.05) and improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p&amp;lt;0.01). In parallel, novel urinary biomarkers of extracellular matrix remodeling, profibrotic growth factor expressions and extensive fibrotic tissue accumulation were reduced in the kidney. DAPA minimized hyperglycemia-induced total protein O-GlcNAcylation in HK-2 cells. Hypoxia-induced HIF-1α elevation was suspended by DAPA treatment. Moreover, DAPA treatment prevented HIF-1α translocation to the nucleus, thereby confirming abolished HIF-1α activation. EPO, VEGFA and profibrotic factor levels were also increased in hypoxia and DAPA prevented EPO, TGFB and PDGF elevation. Conclusion These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia and fibrosis, a vicious trio, which seem to be targeted by SGLT2i. All these mechanisms are important parts in the puzzle of the complex system behind the protective effect of SGLT2i. OTKA-FK124491-K135398, 2017-1.3.1-VKE-2017-00006, 2020-4.1.1.-TKP2020-6183169273, 2020-4.1.1.-TKP2020-6183069269

MO010COLLAGEN TYPE VI PRODUCTION BY KIDNEY FIBROBLASTS IS DEPENDENT ON THE RIGHT STIMULI AND MAY SELF-PERPETUATE FIBROSIS

Abstract Background and Aims Accumulation of extracellular matrix (ECM) proteins is a hallmark of kidney fibrosis, which can lead to altered tissue homeostasis, kidney failure, and ultimately death. Many different cell types are involved in this process, but fibroblasts are the main source of ECM proteins such as collagen type I (COL I), III (COL III), and VI (COL VI). Recently, it was suggested that a fragment of COL VI, released during collagen maturation, is a bioactive molecule (endotrophin; ETP) with signaling potential, indicating that collagens are not just passive structural proteins. In this study, we investigated the effect of different pro-fibrotic stimulants on COL VI production and the effect of ETP itself on human kidney fibroblasts in the scar-in-a-jar (SiaJ) cell model. Method Cells were seeded in 48-well plates at 30.000 cells/well and incubated for 24h in DMEM + 10% FBS for adherence. Cells were then starved by incubating them for further 24h in DMEM + 0.4% FBS. To induce fibrogenesis, fresh medium was added at day 0 with 225/150 mg/mL Ficoll 70/400 and 1% ascorbic acid, containing either 7-, 0.7-, or 0.07 nM PDGF-AA, 8-, 0.8-, or 0.08 nM PDGF-BB, 4-, 0.4-, or 0.04 nM PDGF-CC, 7-, 0.7-, or 0.07 nM PDGF-DD, 0.9-, or 0.09 nM CTGF, 0.02 nM TGF-β or 30 nM ETP. Medium was changed and collected on days 3, 6, 10, and 13. Biomarkers of COL I (PRO-C1), III (PRO-C3), and VI (PRO-C6) formation were assessed in the medium by enzyme-linked immunosorbent assays developed at Nordic Bioscience. Results The stimulation of kidney fibroblasts with PDGF-AA, -BB, -CC, and -DD caused an increase in PRO-C6 compared to the unstimulated cells at every time point (P&amp;lt;0.0001). The increase in formation peaked at day 10, and a dose-dependent increase in COL VI levels was observed with PDGF-DD treatment. Interestingly, CTGF treatment did not enhance the synthesis of COL VI at any time point, and TGF-β treatment suppressed PRO-C6 levels compared to the untreated cells (not significant). The stimulation with 30 nM ETP caused an increase in PRO-C1 (P&amp;lt;0.0001) and PRO-C3 (P&amp;lt;0.0001) compared to the unstimulated cells on days 6, 10, and 13. The increase in collagen formation peaked at day 10 for both markers, with a 7.28-fold increase for COL I and a 4.13-fold increase for COL III. Conclusion The production of COL VI, an important mediator of fibrosis and inflammation through its bioactive fragment endotrophin, shows a differential expression after stimulation of kidney fibroblasts with different pro-fibrotic growth factors. Interestingly, members of the PDGF family induced COL VI production, whereas CTGF and TGF-β did not. Moreover, we confirmed that ETP itself could stimulate kidney fibroblasts to produce more ECM proteins; hence COL VI may self-perpetuate fibrosis. This SiaJ model, combined with ECM formation biomarkers, could be used to elucidate the mechanisms behind acute and sustained matrix production profiles in vivo.

Understanding the Impact of Uterine Fibroids on Human Endometrium Function.

Uterine fibroids (leiomyomas) are the most common benign gynecological tumors in women of reproductive age worldwide. They cause heavy menstrual bleeding, usually leading to severe anemia, pelvic pain/pressure, infertility, and other debilitating morbidities. Fibroids are believed to be monoclonal tumors arising from the myometrium, and recent studies have demonstrated that fibroids actively influence the endometrium globally. Studies suggest a direct relationship between the number of fibroids removed and fertility problems. In this review, our objective was to provide a complete overview of the origin of uterine fibroids and the molecular pathways and processes implicated in their development and growth, which can directly affect the function of a healthy endometrium. One of the most common characteristics of fibroids is the excessive production of extracellular matrix (ECM) components, which contributes to the stiffness and expansion of fibroids. ECM may serve as a reservoir of profibrotic growth factors such as the transforming growth factor β (TGF-β) and a modulator of their availability and actions. Fibroids also elicit mechanotransduction changes that result in decreased uterine wall contractility and increased myometrium rigidity, which affect normal biological uterine functions such as menstrual bleeding, receptivity, and implantation. Changes in the microRNA (miRNA) expression in fibroids and myometrial cells appear to modulate the TGF-β pathways and the expression of regulators of ECM production. Taken together, these findings demonstrate an interaction among the ECM components, TGF-β family signaling, miRNAs, and the endometrial vascular system. Targeting these components will be fundamental to developing novel pharmacotherapies that not only treat uterine fibroids but also restore normal endometrial function.

Losartan

Skeletal muscle injury is a common and debilitating orthopaedic condition. Unfortunately, current treatment protocols inadequately facilitate full muscular recovery. New research is investigating modalities that regulate postinjury muscle fibrosis or scar tissue, which in excess can increase muscle stiffness and risk of reinjury. Losartan, a well-known angiotensin II receptor blocker, reduces muscle fibrosis after acute muscle injury in animal models by indirectly inhibiting transforming growth factor-β1, a profibrotic growth factor released during muscle healing. Although promising results have been shown thus far, additional human clinical trials are needed to confirm these findings and provide guidance on treatment.

Biological Targets for Dupuytren Disease.

Dupuytren disease is a connective tissue disorder occurring on the palm, causing flexion contractures of fingers. There is a pressing need for therapeutic interventions that can slow, stop, or even incrementally reverse the progression of the disease. Numerous in vitro studies have shed light on cellular and molecular agents that contribute to contractures. This article comprehensively reviews various growth factors that can be targeted to prevent and limit the progression and recurrence of Dupuytren contracture (DC). Fibroblasts are the major cell population that has been reported for the contractures in DC, and they are also known to exacerbate the cytokine production. Limiting the fibroblast function by targeting the growth factor production will be of great benefit in treating DC. This review will focus on the studies that have shown to limit the exaggerated function of fibroblasts by reducing the expression of profibrotic growth factors by using antagonizing agents.

Potential effect of adrenomedullin on metabolic and endocrinal dysfunctions in the experimentally induced polycystic ovary: Targeting implication of endoplasmic reticulum stress.

This study investigated the potential effect of adrenomedullin (ADM) on metabolic and endocrinal dysfunctions in experimentally induced polycystic ovary. Twenty-four female Wistar rats were allocated into three groups: control;polycystic ovary syndrome (PCOS) in which PCOS was induced by letrozole, orally in a dose of 1 mg/kg once daily for 3 weeks;and ADM group in which ADM was injected intraperitonally in a dose of 3.5/μg/twice daily for 4 weeks. At the end of the experimental period, the serum sex hormone profile, ADM, fasting glucose, insulin, homeostatic model assessment of insulin resistance, and lipid parameters were determined. Ovarian tissue homogenates were used to determine malondialdehyde, total antioxidant capacity, glutathione peroxidase activity, tumor necrosis factor α, interleukin 6, B cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein. The profibrotic growth factors, including transforming growth factor β1 and connective tissue growth factor, were determined;and also, the relative gene expression of endoplasmic reticulum (ER) stress, including (Xbox-binding protein-1 [XBP-1], activating transcription factor 6 [ATF6], and homologous protein [CHOP]), serine/threonine kinase 1 (Akt1), phosphatidylinositol 3-kinase (PI3K), and peroxisome proliferator-activated receptor γ (PPAR-γ) were determined. Finally, histopathological analysis of the ovaries was evaluated. PCOS group exhibited increased ER stress, suppressing of PI3K/Akt1 and PPAR-γ pathways, imbalance of sex hormonal profile, hyperglycemia, insulin resistance, dyslipidemia, increased profibrotic factors, and abnormal ovarian histopathological picture, while ADM treatment alleviated these disturbances occurring in the PCOS model. We concluded that ADM mitigated PCOS via attenuating the ER stress, in addition to activation of PI3K/Akt1 and PPAR-γ pathways, its antioxidant, anti-inflammatory, antiapoptotic, and antifibrotic properties.

The Integrin Inhibitor Cilengitide and Bleomycin-Induced Pulmonary Fibrosis : Cilengitide and Bleomycin-Induced Pulmonary Fibrosis.

Fibroproliferation and excess deposition of extracellular matrix (ECM) are the pathologic hallmarks of idiopathic pulmonary fibrosis (IPF), a chronic progressive disorder with high mortality and suboptimal treatment options. Although the etiologic mechanisms responsible for the development and progression of IPF remain unclear, cell-ECM interactions and growth factors are considered important. Cilengitide is a cyclic RGD pentapeptide with anti-angiogenic activity that targets αvβ3, αvβ5 and α5β1, integrins known to mediate cell-ECM interactions and activate the pro-fibrotic growth factor Transforming Growth Factor beta (TGF-β). Cilengitide was studied in vitro with the use of NIH/3T3 cells and primary lung fibroblasts, and in vivo in the well-characterized bleomycin-induced lung injury model. The extent of ECM deposition was determined by RT-PCR, Western blot, histologic analysis and hydroxyproline assay of lung tissue. Bronchoalveolar lavage analysis was used to determine cell counts. Cilengitide treatment of cultured fibroblasts showed decreased adhesion to vitronectin and fibronectin, both integrin-dependent events. Cilengitide also inhibited TGF-β-induced fibronectin gene expression and reduced the accumulation of mRNAs and protein for fibronectin and collagen type I. Both preventive and treatment effects of daily injections of cilengitide (20mg/kg) failed to inhibit the development of pulmonary fibrosis as determined by histological analysis (Ashcroft scoring), bronchoalveolar lavage (BAL) fluid cell counts, and hydroxyproline content. Overall, our data suggest that, despite its in vitro activity in fibroblasts, daily injections of cilengitide (20mg/kg) did not inhibit the development of or ameliorate bleomycin-induced pulmonary fibrosis in mice.

Smooth muscle cell-specific SOCS3 deficiency promote pericardial fibrosis and diastolic dysfunction in aging mice

Abstract Background Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible negative regulator of signal transducer and activator of transcription-3 (STAT3) signaling pathway. We have previously shown that cardiac-specific SOCS3 deficiency spontaneously develop cardiac dysfunction with advanced age. However, the role of SOCS3 in smooth muscle cells in cardiovascular pathophysiology remains elusive. In this study, we determined whether STAT3 and SOCS3 in smooth muscle cells would play a role in cardiovascular pathophysiology. Methods and results To target inactivation of the SOCS3 gene to smooth muscle cells, SOCS3-flox mice were bred with transgenic mice expressing Cre recombinase under control of the mouse SM22-α promoter (sm-SOCS3-KO mice). Left ventricular weight to body weight ratio was significantly increased in sm-SOCS3-KO mice compared with wild-type mice at 12 months of age (p&amp;lt;0.05). Echocardiographic analyses of smSOCS3-KO mice showed significantly increased left ventricular diastolic dysfunction compared with wild-type from 12 months of age (p&amp;lt;0.05). Sirius-red staining revealed that thickness of pericardium and cardiac interstitial fibrosis in sm-SOCS3-KO mice were markedly greater compared with wild-type mice at 12 months of age (p&amp;lt;0.05). Western blot analyses showed that phosphorylated STAT3 was significantly increased in sm-SOCS3-KO hearts compared with wild-type mice at 12 months of age (p&amp;lt;0.05), whereas no significant differences were observed at 2 months of age. To investigate the mechanism that gave rise to promoted cardiac fibrosis and diastolic dysfunction during aging in sm-SOCS3-KO, we conducted a real-time PCR array analysis for fibrosis. The expression of pro-fibrotic CTGF (connective tissue growth factor), PDGFb (platelet growth factor-b), and TGF (transforming growth factor) family genes including TGFb1, TGFb2, and TGFb3, were significantly higher in sm-SOCS3-KO hearts than those in wild-type at 6 months of age. Conclusion Thus, smooth muscle cell-specific SOCS3 deletion induces increased pericardial fibrosis, cardiac interstitial fibrosis, and increased diastolic dysfunction in aging mice, possibly through the augmentation of pro-fibrotic growth factors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant JSPS KAKENHI

A combination of circulating chemokines as biomarkers of obesity-induced insulin resistance at puberty.

In obesity adipose tissue undergoes structural re-modelling leading to a chronic low-grade inflammatory state linked to insulin resistance (IR). We aimed to develop a clinically relevant biomarker model for stratifying IR in adolescents with obesity. Cytokines [tumour cell derived factor 1α, monocyte chemoattract protein (MCP) 1, eotaxin and fractalkine], growth factors [brain-derived neurotrophic factor, pro-fibrotic platelet-derived growth factor (PDGF-BB) and insulin-like growth factor 1] and biochemical/metabolic factors were analysed in serum of 143 pubertal patients with obesity (50% IR; 50% non-IR) and 33 controls. Factor analysis, correlation, binary logistic regression and receiver operating characteristic analysis were used to evaluate combinations of these biomarkers as possible diagnostic tools for IR. Two biomarker IR models combining levels of triglycerides (TG)/HDL, eotaxin, MCP-1 and PDGF-BB in pubertal patients with obesity of both sexes were defined. Altered levels of MCP-1, eotaxin, and PDGF-BB constitute a main component that determines 27.7% of the variance explaining IR. Growth and inflammatory factors comprise two other components linked to the first, together accounting for 59.2% of the variance determining IR. PDGF-BB, MCP-1, eotaxin, TG and cholesterol concentrations constitute a solid panel of biomarkers associated with IR in pubertal children with obesity that could be useful in their stratification in a clinical setting for stratification.

Nutraceuticals as potential therapeutics for vesicant-induced pulmonary fibrosis.

Exposure to vesicants, including sulfur mustard and nitrogen mustard, causes damage to the epithelia of the respiratory tract and the lung. With time, this progresses to chronic disease, most notably, pulmonary fibrosis. The pathogenic process involves persistent inflammation and the release of cytotoxic oxidants, cytokines, chemokines, and profibrotic growth factors, which leads to the collapse of lung architecture, with fibrotic involution of the lung parenchyma. At present, there are no effective treatments available to combat this pathological process. Recently, much interest has focused on nutraceuticals, substances derived from plants, herbs, and fruits, that exert pleiotropic effects on inflammatory cells and parenchymal cells that may be useful in reducing fibrogenesis. Some promising results have been obtained with nutraceuticals in experimental animal models of inflammation-driven fibrosis. This review summarizes the current knowledge on the putative preventive/therapeutic efficacy of nutraceuticals in progressive pulmonary fibrosis, with a focus on their activity against inflammatory reactions and profibrotic cell differentiation.

Epithelial Vasopressin Type-2 Receptors Regulate Myofibroblasts by a YAP-CCN2-Dependent Mechanism in Polycystic Kidney Disease.

Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.

Antepartum Aspirin Administration Reduces Activin A and Cardiac Global Longitudinal Strain in Preeclamptic Women.

BackgroundApproximately 60% of women have Stage B heart failure 1 year after a preeclamptic delivery. Emerging evidence suggests that the profibrotic growth factor activin A, which has been shown to induce cardiac fibrosis and hypertrophy, is elevated in preeclampsia and may be inhibited by aspirin therapy. We hypothesized that preeclamptic women receiving aspirin would have lower activin A levels and reduced global longitudinal strain (GLS), a sensitive measure of cardiac dysfunction, than women who do not receive aspirin. To test our hypothesis, we performed a cohort study of women with preeclampsia or superimposed preeclampsia and compared activin A levels and GLS in parturients who did or did not receive aspirin.Methods and ResultsNinety‐two parturients were enrolled, of whom 25 (27%) received aspirin (81 mg/day) therapy. GLS, plasma activin A, and follistatin, which inactivates activin A, were measured. Women receiving aspirin therapy had lower median (interquartile range) levels of activin A (8.17 [3.70, 10.36] versus 12.77 [8.37, 31.25] ng/mL; P=0.001) and lower activin/follistatin ratio (0.59 [0.31, 0.93] versus 1.01 [0.64, 2.60] P=0.002) than women who did not receive aspirin, which also remained significant after multivariable analysis. Furthermore, GLS was worse in patients who did not receive aspirin (−19.84±2.50 versus −17.77±2.60%; P=0.03) despite no differences in blood pressure between groups.ConclusionsOur study suggests that antepartum aspirin therapy reduced serum activin A levels and improved GLS in preeclamptic patients, suggesting that aspirin may mitigate the postpartum cardiac dysfunction seen in women with preeclampsia.