Myofibroblast Transformation Research Articles

Genistein stimulates the viability and prevents myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-β

Primary open-angle glaucoma (POAG) is the most common type of glaucoma leading to blindness. The search for ways to prevent/treat this entity is one of the main challenges of today's ophthalmology. One of such solution seems to be biologically active substances of natural origin, such as genistein (GEN), which can affect the function of isolated trabecular meshwork by the inhibition of protein tyrosine kinase. However, the role of GEN in viability as well as myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-β is unknown. Using human trabecular meshwork cells (HTMCs) we investigated the effect of genistein on cell viability and myofibroblastic transformation stimulated by TGF-β1 and TGF-β2. Using Real-Time PCR, western blot and immunofluorescence we determined the effect on the expression changes of αSMA, TIMP1, collagen 1 and 3 at mRNA and protein level. We found that genistein increases the viability of HTMCs (1, 2, 3 μg/ml; P < 0.05 and 4, 5, 10, 15, 20 μg/ml; P < 0.01). Moreover, we found that addition of 10, 15 and 20 μg/ml is able to prevent myofibroblastic transformation of HTMCs by decreasing αSMA, TIMP1, collagen 1 and 3 mRNA and protein expression (P < 0.01). Based on the obtained results, we can conclude that genistein is a potential factor that can prevent the myofibroblastic transformation of HTMCs accompanying glaucoma. Describing GEN influence on myofibroblastic transformation processes in HTMC allows us to conclude that it can be considered a potential therapeutic agent or a substance supporting treatment in patients with glaucoma.

The SIRT1/Nrf2 signaling pathway mediates the anti-pulmonary fibrosis effect of liquiritigenin

BackgroundAt present, the treatment options available for idiopathic pulmonary fibrosis are both limited and often come with severe side effects, emphasizing the pressing requirement for innovative therapeutic alternatives. Myofibroblasts, which hold a central role in pulmonary fibrosis, have a close association with the Smad signaling pathway induced by transforming growth factor-β1 (TGF-β1) and the transformation of myofibroblasts driven by oxidative stress. Liquiritigenin, an active compound extracted from the traditional Chinese herb licorice, boasts a wide array of biomedical properties, such as anti-fibrosis and anti-oxidation. The primary objective of this study was to examine the impact of liquiritigenin on bleomycin-induced pulmonary fibrosis in mice and the underlying mechanisms.MethodsThe anti-pulmonary fibrosis and anti-oxidant effects of liquiritigenin in vivo were tested by HE staining, Masson staining, DHE staining and bio-chemical methods. In vitro, primary mouse lung fibroblasts were treated with TGF-β1 with or without liquiritigenin, the effects of liquiritigenin in inhibiting differentiation of myofibroblasts and facilitating the translocation of Nrf2 were valued using Quantitative real-time polymerase chain reaction (Q-PCR), western blotting and immunofluorescence. Nrf2 siRNA and SIRT1 siRNA were used to investigate the mechanism underlies liquiritigenin’s effect in inhibiting myofibroblast differentiation.ResultsLiquiritigenin displayed a dose-dependent reduction effect in bleomycin-induced fibrosis. In laboratory experiments, it was evident that liquiritigenin possessed the ability to enhance and activate sirtuin1 (SIRT1), thereby facilitating the nuclear translocation of Nrf2 and mitigating the oxidative stress-induced differentiation of primary mouse myofibroblasts. Moreover, our investigation unveiled that SIRT1 not only regulated myofibroblast differentiation via Nrf2-mediated antioxidant responses against oxidative stress but also revealed liquiritigenin's activation of SIRT1, enabling direct binding to Smad. This led to decreased phosphorylation of the Smad complex, constrained nuclear translocation, and suppressed acetylation of the Smad complex, ultimately curtailing the transcription of fibrotic factors. Validation in live subjects provided substantial evidence for the anti-fibrotic efficacy of liquiritigenin through the SIRT1/Nrf2 signaling pathway.ConclusionsOur findings imply that targeting myofibroblast differentiation via the SIRT1/Nrf2 signaling pathway may constitute a pivotal strategy for liquiritigenin-based therapy against pulmonary fibrosis.

PCSK9 regulates myofibroblast transformation through the JAK2/STAT3 pathway to regulate fibrosis after myocardial infarction

Cardiac fibrosis is pivotal in the progression of numerous cardiovascular diseases. This phenomenon is hallmarked by an excessive deposition of ECM protein secreted by myofibroblasts, leading to increased myocardial stiffness. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that belongs to the proprotein-converting enzyme family. It has emerged as a viable therapeutic target for reducing plasma low-density lipoprotein cholesterol. However, the exact mechanism via which PCSK9 impacts cardiac fibrosis remains unclear. In the present research, an increase in circulating PCSK9 protein levels was observed in individuals with myocardial infarction and rat models of myocardial infarction. Moreover, the inhibition of circulating PCSK9 in rats was found to reduce post-infarction fibrosis. In vitro experiments further demonstrated that overexpression of PCSK9 or stimulation by extracellular PCSK9 recombinant protein enhanced the transformation of cardiac fibroblasts to myofibroblasts. This process also elevated collagen Ⅰ, and Ⅲ, as well as α-SMA protein levels. However, these effects were countered when co-incubated with the STAT3 inhibitor S3I-201. This study suggests that PCSK9 may function as a novel regulator of myocardial fibrosis, primarily via the JAK2/STAT3 pathway.

Peritendinous adhesion: Therapeutic targets and progress of drug therapy

Peritendinous adhesion (PA) is one of the most common complications following hand surgery and characterized with abnormal hyperplasia of connective tissue and excessive deposition of extracellular matrix. Subsequently, various clinical symptoms such as chronic pain, limb dyskinesia and even joint stiffness occur and patients are always involved in the vicious cycle of “adhesion – release – re-adhesion”, which seriously compromise the quality of life. Until present, the underlying mechanism remains controversial and lack of specific treatment, with symptomatic treatment being the only option to relieve symptoms, but not contributing no more to the fundamentally rehabilitation of basic structure and function. Recently, novel strategies have been proposed to inhibit the formation of adhesion tissues including implantation of anti-adhesion barriers, anti-inflammation, restraint of myofibroblast transformation and regulation of collagen overproduction. Furthermore, gene therapy has also been considered as a promising anti-adhesion treatment. In this review, we provide an overview of anti-adhesion targets and relevant drugs to summarize the potential pharmacological roles and present subsequent challenges and prospects of anti-adhesion drugs.

Epidermal growth factor receptor activation is essential for kidney fibrosis development

Fibrosis is the progressive accumulation of excess extracellular matrix and can cause organ failure. Fibrosis can affect nearly every organ including kidney and there is no specific treatment currently. Although Epidermal Growth Factor Receptor (EGFR) signaling pathway has been implicated in development of kidney fibrosis, underlying mechanisms by which EGFR itself mediates kidney fibrosis have not been elucidated. We find that EGFR expression increases in interstitial myofibroblasts in human and mouse fibrotic kidneys. Selective EGFR deletion in the fibroblast/pericyte population inhibits interstitial fibrosis in response to unilateral ureteral obstruction, ischemia or nephrotoxins. In vivo and in vitro studies and single-nucleus RNA sequencing analysis demonstrate that EGFR activation does not induce myofibroblast transformation but is necessary for the initial pericyte/fibroblast migration and proliferation prior to subsequent myofibroblast transformation by TGF-ß or other profibrotic factors. These findings may also provide insight into development of fibrosis in other organs and in other conditions.

LAMB3 Promotes Myofibrogenesis and Cytoskeletal Reorganization in Endometrial Stromal Cells via the RhoA/ROCK1/MYL9 Pathway

LAMB3, a major extracellular matrix and basal membrane component, is involved in wound healing. We aimed to understand its role in Asherman’s syndrome (AS), which is associated with infertility, by using bioinformatics analysis and cultured endometrial stromal cells (ESCs). MRNAs extracted from tissues obtained from control subjects and patients with severe intrauterine adhesion were sequenced and subjected to bioinformatics analysis and the RhoA/ROCK1/MYL9 pathway was implicated and this subsequently studied using cultured primary ESCs. The effects of overexpression and knockdown and activation and inhibition of LAMB3 on the mesenchymal to myofibroblastic phenotypic transformation of ECCs were assessed using PCR and western blot analysis. Phalloidin was used to localize the actin cytoskeletal proteins. Silencing of LAMB3 reversed the TGF-β-induced ESC myofibroblast phenotype conversion, whereas overexpression of LAMB3 promoted this process. Activation and silencing of LAMB3 led to remodeling of the ESC cytoskeleton. Overexpression and silencing of LAMB3 caused activation and inhibition of ESCs, respectively. Y-27632 and LPA reversed the activation and inhibition of the RhoA/ROCK1/MYL9 pathway after overexpression and silencing, respectively. These results suggest that LAMB3 can regulate ESC fibrosis transformation and cytoskeleton remodeling via the RhoA/ROCK1/MYL9 pathway. This study provides a potential new target for gene therapy and drug intervention of AS.

Omentin-1 induces mechanically activated fibroblasts lipogenic differentiation through pkm2/yap/pparγ pathway to promote lung fibrosis resolution.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by extensive extracellular matrix (ECM) deposition by activated myofibroblasts, which are specialized hyper-contractile cells that promote ECM remodeling and matrix stiffening. New insights on therapeutic strategies aimed at reversing fibrosis by targeting myofibroblast fate are showing promise in promoting fibrosis resolution. Previously, we showed that a novel adipocytokine, omentin-1, attenuated bleomycin (BLM)-induced lung fibrosis by reducing the number of myofibroblasts. Apoptosis, deactivation, and reprogramming of myofibroblasts are important processes in the resolution of fibrosis. Here we report that omentin-1 reverses established lung fibrosis by promoting mechanically activated myofibroblasts dedifferentiation into lipofibroblasts. Omentin-1 promotes myofibroblasts lipogenic differentiation by inhibiting dimerization and nuclear translocation of glycolytic enzymes pyruvate kinase isoform M2 (PKM2) and activation of the downstream Yes-associated protein (YAP) by increasing the cofactor fructose-1,6-bisphosphate (F1, 6BP, FBP). Moreover, omentin-1 activates proliferator-activated receptor gamma (PPARγ) signaling, the master regulator of lipogenesis, and promotes the upregulation of the lipogenic differentiation-related protein perilipin 2 (PLIN2) by suppressing the PKM2-YAP pathway. Ultimately, omentin-1 facilitates myofibroblasts transformation into the lipofibroblast phenotype, with reduced collagen synthesis and enhanced degradation properties, which are crucial mechanisms to clear the ECM deposition in fibrotic tissue, leading to fibrosis resolution. Our results indicate that omentin-1 targets mechanical signal accelerates fibrosis resolution and reverses established lung fibrosis by promoting myofibroblasts lipogenic differentiation, which is closely associated with ECM clearance in fibrotic tissue. These findings suggest that targeting mechanical force to promote myofibroblast lipogenic differentiation is a promising therapeutic strategy against persistent lung fibrosis.

Integrative analysis links autophagy to intrauterine adhesion and establishes autophagy-related circRNA-miRNA-mRNA regulatory network.

Intrauterine adhesion (IUA) is a troublesome complication characterized with endometrial fibrosis after endometrial trauma. Increasing number of investigations focused on autophagy and non-coding RNA in the pathogenesis of uterine adhesion, but the underlying mechanism needs to be further studied. mRNA expression profile and miRNA expression profile were obtained from Gene Expression Omnibus database. The autophagy related genes were low. Venn diagram was used to set the intersection of autophagy genes and DEGs to obtain ARDEGs. Circbank was used to select hub autophagy-related circRNAs based on ARDEMs. Then, the differentially expressed autophagy-related genes, miRNAs and circRNAs were analyzed by functional enrichment analysis, and protein-protein interaction network analysis. Finally, the expression levels of hub circRNAs and hub miRNAs were validated through RT-PCR of clinical intrauterine adhesion samples. In vitro experiments were investigated to explore the effect of hub ARCs on cell autophagy, myofibroblast transformation and collagen deposition. 11 autophagy-related differentially expressed genes (ARDEGs) and 41 differentially expressed miRNA (ARDEMs) compared between normal tissues and IUA were identified. Subsequently, the autophagy-related miRNA-mRNA network was constructed and hub ARDEMs were selected. Furthermore, the autophagy-related circRNA-miRNA-mRNA network was established. According to the ranking of number of regulated ARDEMs, hsa-circ-0047959, hsa-circ-0032438, hsa-circ-0047301 were regarded as the hub ARCs. In comparison of normal endometrial tissue, all three hub ARCs were upregulated in IUA tissue. All hub ARDEMs were downregulated except has-miR-320c. In the current study, we firstly constructed autophagy-related circRNA-miRNA-mRNA regulatory network and identified hub ARCs and ARDEMs had not been reported in IUA.

Glycyrrhetinic Acid Mitigates Radiation-Induced Pulmonary Fibrosis via Inhibiting the Secretion of TGF-β1 by Treg Cells

Radiation-induced pulmonary fibrosis (RIPF) is a common side effect of radiation therapy for thoracic tumors without effective prevention and treatment methods at present. The aim of this study was to explore whether glycyrrhetinic acid (GA) has a protective effect on RIPF and the underlying mechanism. A RIPF mouse model administered GA was used to determine the effect of GA on RIPF. The cocultivation of regulatory T (Treg) cells with mouse lung epithelial-12 cells or mouse embryonic fibroblasts and intervention with GA or transforming growth factor-β1 (TGF-β1) inhibitor to block TGF-β1 was conducted to study the mechanism by which GA alleviates RIPF. Furthermore, injection of Treg cells into GA-treated RIPF mice to upregulate TGF-β1 levels was performed to verify the roles of TGF-β1 and Treg cells. GA intervention improved the damage to lung tissue structure and collagen deposition and inhibited Treg cell infiltration, TGF-β1 levels, epithelial mesenchymal transition (EMT), and myofibroblast (MFB) transformation in mice after irradiation. Treg cell-induced EMT and MFB transformation in vitro were prevented by GA, as well as a TGF-β1 inhibitor, by decreasing TGF-β1. Furthermore, reinfusion of Treg cells upregulated TGF-β1 levels and exacerbated RIPF in GA-treated RIPF mice. GA can improve RIPF in mice, and the corresponding mechanisms may be related to the inhibition of TGF-β1 secreted by Treg cells to induce EMT and MFB transformation. Therefore, GA may be a promising therapeutic candidate for the clinical treatment of RIPF.

Abstract P3159: Exosomes Derived From Podoplanin Positive Cells Induce Endo-Mt And Myofibroblast Transformation In Healthy Mouse Heart And After Ischemic Injury

Specific understanding of the differences in cell communications between physiologicaland pathological conditions harbor the understanding of mechanisms to recover theequilibrium in the injured tissue and restore the organ function. Neurohumoral factorsreleased after myocardial infarction (MI) induce the expression of platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN) in mesenchymalstromal cells, which pivot their structural support function and actively participate in theremodeling of the ischemic tissue releasing an abundance of extracellular vesicles calledexosomes. Exosomes (exo) derived from activated PDPN+ cells conditioned media wereused in vivo for the treatment of healthy mouse hearts, and in vitro for the treatment ofmouse cardiac endothelial cells (mCECs), NHI-3T3 and adult cardiac fibroblast. Ourongoing experiments showed that in vivo, PDPN-exo could independently alter healthyheart physiology and structure; mice hearts treated with PDPN-exo developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increaseof the end diastolic and systolic volumes. The fibrotic area was characterized by ECMdeposition, increased lymphatic vessels and infiltrating CD45+ cells. In vitro, PDPN-exoreprogramed mCECs, NHI-3T3 and adult cardiac fibroblast, upregulating the expressionof fibrotic markers suggesting an Endothelial-Mesenchymal Transition (EndoMT) andmyofibroblast transformation. Proteomic analysis of PDPN-exo suggests these transitionsmay depend upon the delivery of NOTCH1 via exosomes and the cleavage of it throughγ-Secretase. Experiments of gain and loss of NOTCH1 function showed thatoverexpression of NOTCH1 intracellular domain (NICD1) in vitro was sufficient to derivea lineage switching in mCECs and fibroblasts on the contrary in vivo delivery and in vitrotreatment with exosomes derived from PDPN+ cells silenced for NOTCH1 or treatmentwith γ-Secretase inhibitor did not alter the heart structure and function or the endothelialcells and fibroblast lineage. Thus, NOTCH1 delivery via PDPN-exo after MI play a majorrole in scar remodeling and its biology can be pharmacologically modulated.

Cytokine receptor-like factor 1 (CRLF1) promotes cardiac fibrosis via ERK1/2 signaling pathway.

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

Fibroblast-specific knockout of METTL1 attenuates myocardial infarction-induced cardiac fibrosis

Cardiac fibrosis, a common pathology in inherited and acquired heart diseases, necessitates the identification of diagnostic and therapeutic targets. Methyltransferase Like 1 (METTL1), an enzyme responsible for RNA modification by methylating guanosine to form m7G, is an emerging area of research in understanding cellular processes and disease pathogenesis. Dysregulation of m7G modification has been implicated in various diseases. However, the role of METTL1 in cardiac fibrosis remains unclear. This study aimed to investigate the role of METTL1 in myocardial infarction-induced heart failure and cardiac fibrosis. Our findings demonstrate that elevated METTL1-mediated RNA m7G methylation is observed in cardiac fibrosis tissues and TGF-β1-induced cardiac fibroblast proliferation and myofibroblast transformation. Furthermore, fibroblast-specific knockout of METTL1 attenuated myocardial infarction-induced heart failure and cardiac fibrosis. Additionally, METTL1 knockout decreased m7G methylated fibrotic genes and impaired their translation efficiency. These results suggest a novel pro-fibrosis role of METTL1-mediated RNA m7G methylation, highlighting its potential as a therapeutic target in cardiac fibrosis.

Substrate-dependent interaction of SPOP and RACK1 aggravates cardiac fibrosis following myocardial infarction

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition adaptor of cullin-3 (CUL3)/RING-type E3 ligase complex, is investigated for its role in cardiac fibrosis in our study. Cardiac fibroblasts (CFs) activation was achieved with TGF-β1 (20ng/mL) and MI mouse model was established by ligation of the left anterior descending coronary, and lentivirus was employed to mediate interference of SPOP expression. SPOP was increased both in fibrotic post-MI mouse hearts and TGF-β1-treated CFs. The gain-of-function of SPOP promoted myofibroblast transformation in CFs, and exacerbated cardiac fibrosis and cardiac dysfunction in MI mice, while the loss-of-function of SPOP exhibited the opposite effects. Mechanistically, SPOP bound to the receptor of activated protein C kinase 1 (RACK1) and induced its ubiquitination and degradation by recognizing Ser/Thr-rich motifs on RACK1, leading to Smad3-mediated activation of CFs. Forced RACK1 expression canceled the effects of SPOP on cardiac fibrosis. The study reveals therapeutic targets for fibrosis-related cardiac diseases.

The impact of UV cross-linking on corneal stromal cell migration, differentiation and patterning

Previous studies have demonstrated that UV cross-linking (CXL) increases stromal stiffness and produces alterations in extracellular matrix (ECM) microstructure. In order to investigate how CXL impacts both keratocyte differentiation and patterning within the stroma, and fibroblast migration and myofibroblast differentiation on top of the stroma, we combined CXL with superficial phototherapeutic keratectomy (PTK) in a rabbit model. Twenty-six rabbits underwent a 6 mm diameter, 70 μm deep phototherapeutic keratectomy (PTK) with an excimer laser to remove the epithelium and anterior basement membrane. In 14 rabbits, standard CXL was performed in the same eye immediately after PTK. Contralateral eyes served as controls. In vivo confocal microscopy through focusing (CMTF) was used to analyze corneal epithelial and stromal thickness, as well as stromal keratocyte activation and corneal haze. CMTF scans were collected pre-operatively, and from 7 to 120 days after the procedure. A subset of rabbits was sacrificed at each time point, and corneas were fixed and labeled in situ for multiphoton fluorescence microscopy and second harmonic generation imaging. In vivo and in situ imaging demonstrated that haze after PTK was primarily derived from a layer of myofibroblasts that formed on top of the native stroma. Over time, this fibrotic layer was remodeled into more transparent stromal lamellae, and quiescent cells replaced myofibroblasts. Migrating cells within the native stroma underneath the photoablated area were elongated, co-aligned with collagen, and lacked stress fibers. In contrast, following PTK + CXL, haze was derived primarily from highly reflective necrotic “ghost cells” in the anterior stroma, and fibrosis on top of the photoablated stroma was not observed at any time point evaluated. Cells formed clusters as they migrated into the cross-linked stromal tissue and expressed stress fibers; some cells at the edge of the CXL area also expressed α-SM actin, suggesting myofibroblast transformation. Stromal thickness increased significantly between 21 and 90 days after PTK + CXL (P < 0.001) and was over 35 μm higher than baseline at Day 90 (P < 0.05). Overall, these data suggest that cross-linking inhibits interlamellar cell movement, and that these changes lead to a disruption of normal keratocyte patterning and increased activation during stromal repopulation. Interestingly, CXL also prevents PTK-induced fibrosis on top of the stroma, and results in long term increases in stromal thickness in the rabbit model.

(375) Can Tamoxifen and a PDE5 inhibitor slow the progression of Peyronie’s Disease?

Abstract Introduction The available treatments for Peyronie’s Disease as recommended by international guidelines are either surgery or intralesional injection therapy. In recently cell and rat studies it has been shown that using a phosophdiesterase type 5 inhibitor (PDE5i) and a selective oestrogen receptor modulator (Tamoxifen) inhibited myofibroblast transformation and extracellular matrix production. Objective The aim of this study is to compare the combination of tamoxifen and a PDE5i versus no treatment or other oral medications in the progression of early Peyronie’s Disease. Methods This is a retrospective study of all patients with early Peyronie’s Disease, defined as less than 6 months duration or persistent pain presenting over a 3-year period. Patients were given Tamoxifen 20mg twice daily + Tadalafil 5mg daily for 2 months. The control group either received no treatment or simple oral medication such as Vitamin E. Data retrieved included demographics, duration of disease, change in curvature and pain, as well as further treatment modalities. Assessment of curvature was with photos or an artificial erection test. Data was analysed using the Mann-Whitney Test. Results A total of 147 patients were recruited with early Peyronie’s Disease, 102 patients given the active drug combination (median age 49, range 24-71) and 45 controls (median age 55, range 27-76). The results in each group are presented in Table 1. Overall, the patients on Tamoxifen and Tadalafil showed more improvement and less deterioration which was statistically significant (p=0.009) Conclusions The combination of Tamoxifen and Tadalafil has been shown to prevent the progression of Peyronie’s Disease in vitro and in this study in patients with early disease. Future prospective studies are warranted to confirm this finding. Disclosure No

Statins synergize with phosphodiesterase type 5 inhibitors but not with selective estrogen receptor modulators to prevent myofibroblast transformation in an in vitro model of Peyronie's disease.

Peyronie's disease (PD) is a fibrotic disorder characterized by plaque formation in the tunica albuginea (TA) of the penis, and we have previously shown that inhibition of transformation of TA-derived fibroblasts to myofibroblasts using a combination phosphodiesterase type 5 (PDE5) inhibitors and selective estrogen receptor modulators (SERMs) is effective in slowing the progression of early PD. The study sought to investigate whether combinations of statins with PDE5 inhibitors or SERMs would affect myofibroblast transformation in vitro. Primary fibroblasts were isolated from TA of patients with PD and stimulated with transforming growth factor β1 in the absence and presence of a range of concentrations of statins, PDE5 inhibitors, SERMs, and their combinations for 72hours before quantifying α-smooth muscle actin using in-cell enzyme-linked immunosorbent assay. The prevention of transforming growth factor β1-induced transformation of TA-derived fibroblasts to myofibroblasts was measured in vitro. Statins (simvastatin, lovastatin) inhibited myofibroblast transformation in a concentration-dependent manner with half maximal inhibitory concentration values of 0.77 ± 0.07μM and 0.8 ± 0.13μM, respectively. Simvastatin inhibited myofibroblast transformation in a synergistic fashion when combined with vardenafil (a PDE5 inhibitor; log alpha >0). Combination of tamoxifen (a SERM) and simvastatin did not show synergy (log alpha <0). When 3 drugs (simvastatin, vardenafil, and tamoxifen) were combined, the effect was not synergistic, but rather was additive. A combination of a statin with a PDE5 inhibitor might be useful in the clinic to slow the progression of the disease in patients with early PD; however, caution should be taken with such a combination because of the reported myopathy as a side effect. The use of primary human cells from patients with PD is a strength of this study. The mechanisms by which these drug classes exert synergy when used in combination was not investigated. This is the first demonstration of an antifibrotic synergy between statins and PDE5 inhibitors.

A Subset of Breg Cells, B10, Contributes to the Development of Radiation-Induced Pulmonary Fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiotherapy, but the underlying mechanisms are unknown. B10 cells, as negative B regulatory cells, play important roles in regulating inflammation and autoimmunity. However, the role of B10 cells in RIPF progression is unclear. The aim of this study was to determine the role of B10 cells in aggravating RIPF and the underlying mechanism. The role of B10 cells in RIPF was studied by constructing mouse models of RIPF and depleting B10 cells with an anti-CD22 antibody. The mechanism of B10 cells in RIPF was further explored through cocultivation of B10 cells and MLE-12 or NIH3T3 cells and administration of an IL-10 antibody to block IL-10. B10 cell numbers increased significantly during the early stage in the RIPF mouse models compared with the controls. In addition, depleting B10 cells with the anti-CD22 antibody attenuated the development of lung fibrosis in mice. Subsequently, we confirmed that B10 cells induced EMT and the transformation of myofibroblasts via activation of STAT3 signaling in vitro. After blockade of IL-10, it was verified that IL-10 secreted by B10 cells mediates the EMT of myofibroblasts, thereby promoting RIPF. Our study uncovers a novel role for IL-10-secreting B10 cells that could be a new target of research for relieving RIPF.

LncRNA GAS5 suppresses TGF-β1-induced transformation of pulmonary pericytes into myofibroblasts by recruiting KDM5B and promoting H3K4me2/3 demethylation of the PDGFRα/β promoter

BackgroundIdiopathic pulmonary fibrosis (IPF) is a condition that may cause persistent pulmonary damage. The transformation of pericytes into myofibroblasts has been recognized as a key player during IPF progression. This study aimed to investigate the functions of lncRNA growth arrest-specific transcript 5 (GAS5) in myofibroblast transformation during IPF progression.MethodsWe created a mouse model of pulmonary fibrosis (PF) via intratracheal administration of bleomycin. Pericytes were challenged with exogenous transforming growth factor-β1 (TGF-β1). To determine the expression of target molecules, we employed quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemical and immunofluorescence staining. The pathological changes in the lungs were evaluated via H&E and Masson staining. Furthermore, the subcellular distribution of GAS5 was examined using FISH. Dual-luciferase reporter assay, ChIP, RNA pull-down, and RIP experiments were conducted to determine the molecular interaction.ResultsGAS5 expression decreased whereas PDGFRα/β expression increased in the lungs of IPF patients and mice with bleomycin-induced PF. The in vitro overexpression of GAS5 or silencing of PDGFRα/β inhibited the TGF-β1-induced differentiation of pericytes to myofibroblasts, as evidenced by the upregulation of pericyte markers NG2 and desmin as well as downregulation of myofibroblast markers α-SMA and collagen I. Further mechanistic analysis revealed that GAS5 recruited KDM5B to promote H3K4me2/3 demethylation, thereby suppressing PDGFRα/β expression. In addition, KDM5B overexpression inhibited pericyte–myofibroblast transformation and counteracted the promotional effect of GAS5 knockdown on pericyte–myofibroblast transformation. Lung fibrosis in mice was attenuated by GAS5 overexpression but promoted by GAS5 deficiency.ConclusionGAS5 represses pericyte–myofibroblast transformation by inhibiting PDGFRα/β expression via KDM5B-mediated H3K4me2/3 demethylation in IPF, identifying GAS5 as an intervention target for IPF.

Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie's disease.

Drug repurposing has been shown to bring safe medications to new patient populations, as recently evidenced by the COVID-19 pandemic. We investigated whether we could use phenotypic screening to repurpose drugs for the treatment of Peyronie's disease (PD). PD is a fibrotic disease characterised by continued myofibroblast presence and activity leading to formation of a plaque in the penile tunica albuginea (TA) that can cause pain during erection, erectile dysfunction, and penile deformity. PD affects 3-9% of men with treatment options limited to surgery or injection of collagenase which can only be utilised at late stages after the plaque is formed. Currently there are no approved medications that can be offered to patients presenting with early disease before the formation of the plaque. Drug repurposing may therefore be the ideal strategy to identify medical treatments to address this unmet medical need in early PD. We used primary human fibroblasts from PD patients in a phenotypic screening assay that measures TGF-β1-induced myofibroblast transformation which is the main cellular phenotype that drives the pathology in early PD. A library of FDA-approved 1,953 drugs was screened in duplicate wells at a single concentration (10 μM) in presence of TGF-β1. The myofibroblast marker α-SMA was quantified after 72h incubation. A positive control of SB-505124 (TGF-β1 receptor antagonist) was included on each plate. Hits were defined as showing >80% inhibition, whilst retaining >80% cell viability. 26 hits (1.3%) were identified which were divided into the following main groups: anti-cancer drugs, anti-inflammation, neurology, endocrinology, and imaging agents. Five of the top-ten drugs that increase myofibroblast-transformation appear to act on VEGFR. This is the first phenotypic screening of FDA-approved drugs for PD and our results suggest that it is a viable method to predict drugs with potential for repurposing to treat early PD.

616 Investigating if hydroxypyridone anti-fungals can target already established myofibroblasts in an in vitro model of hypertrophic scarring

Hypertrophic scars are a common complication of burn injury, yet there are no medications to prevent their formation. Myofibroblasts, a key cell involved in scar formation, are resistant to apoptosis. We have previously shown that hydroxypyridone anti-fungals can inhibit myofibroblast transformation in burn scar-derived fibroblasts. This study aimed to investigate if these drugs can target established myofibroblasts. Primary human dermal fibroblasts, derived from burn patient scars, were exposed to transforming growth factor beta-1 (TGF-β1) for 72 h to induce myofibroblast transformation.