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1.
Biomolecules ; 12(11)2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36358925

RESUMO

Cardiac fibrosis is an important pathological process in many diseases. Wdr5 catalyzes the trimethylation of lysine K4 on histone H3. The effects of Wdr5 on the cardiac fibrosis phenotype and the activation or transformation of cardiac fibroblasts were investigated by Ang-II-infused mice by osmotic mini-pump and isolated primary neonatal rat cardiac fibroblasts. We found that the Wdr5 expression and histone H3K4me3 modification were significantly increased in Ang-II-infused mice. By stimulating primary neonatal rat cardiac fibroblasts with Ang II, we detected that the expression of Wdr5 and H3K4me3 modification were also significantly increased. Two Wdr5-specific inhibitors, and the lentivirus that transfected Sh-Wdr5, were used to treat primary mouse cardiac fibroblasts, which not only inhibited the histone methylation by Wdr5 but also significantly reduced the activation and migration ability of Ang-II-treated fibroblasts. To explore its mechanism, we found that the inhibition of Wdr5 increased the expression of P53, P21. Cut&Tag-qPCR showed that the inhibition of Wdr5 significantly reduced the enrichment of H3K4me3 in the Mdm2 promoter region. For in vivo experiments, we finally proved that the Wdr5 inhibitor OICR9429 significantly reduced Ang-II-induced cardiac fibrosis and increased the expression of P21 in cardiac fibroblasts. Inhibition of Wdr5 may mediate cardiac fibroblast cycle arrest through the Mdm2/P53/P21 pathway and alleviate cardiac fibrosis.


Assuntos
Histonas , Miofibroblastos , Ratos , Camundongos , Animais , Miofibroblastos/metabolismo , Histonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fibroblastos/metabolismo , Fibrose
2.
Sci Rep ; 12(1): 20003, 2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36411329

RESUMO

Adult tendons heal via fibrovascular scarring with inferior biomechanical properties. Mohawk (Mkx) emerged as a pivotal actor in tenolineage commitment. However, its precise function in tendinopathy remains poorly understood. This study investigates the cellular and molecular mechanisms underlying Mkx' role in fibrovascular healing. Human samples were collected to test fibrovascular markers. We then performed RNAseq on Mkx-/- mice compared to their wild type littermates to decipher Mkx regulome. We therefore sought to reproduce TSPCs transition to myofibroblasts in-vitro by over-expressing MyoD and followed by phenotypic and experimental cells' characterization using microscopy, qRT-PCR, flow cytometry sorting, presto-blue cell viability assay and immunofluorescence. Two different in vivo models were used to assess the effect of the MyoD-expressing myofibroblasts: transplantation in the dorsal area of immunodeficient mice and in an adult Achilles tendon injury model. To prevent angiofibrosis, we tested the molecule Xav939 and proceeded with histological stainings, q-RT PCR transcriptional quantification of angifibrotic markers, mechanical tests, and immunofluorescence. Tendinopathy samples showed fibrovascular healing with decreased tenolineage phenotype. Transcriptomic analysis of Mkx-/- tendons revealed myofibroblast-associated biological processes. Over-expression of MyoD in WT tendon stem progenitor cells (TSPCs) gave rise to myofibroblasts reprogramming in-vitro and fibrovascular scarring in-vivo. MKX directly binds to MyoD promoter and underlies global regulative processes related to angiogenesis and Wnt signaling pathway. Blocking Wnt signaling with the small molecule Xav393 resulted in higher histological and biomechanical properties. Taken together, our data provide the first in vivo and in-vitro evidence of tendon stem progenitor cells to myofibroblasts transition and show improved tendon healing via angiofibrosis modulation, thus opening potential therapeutic avenues to treat tendinopathy patients.


Assuntos
Proteínas de Homeodomínio , Miofibroblastos , Tendinopatia , Animais , Humanos , Camundongos , Cicatriz/patologia , Proteínas de Homeodomínio/metabolismo , Miofibroblastos/metabolismo , Células-Tronco/metabolismo , Tendinopatia/patologia , Tendões/metabolismo , Via de Sinalização Wnt , Diferenciação Celular
3.
Sci Rep ; 12(1): 16832, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207437

RESUMO

The spatiotemporal interaction and constant iterative feedback between fibroblasts, extracellular matrix, and environmental cues are central for investigating the fibroblast-induced musculoskeletal tissue regeneration and fibroblast-to-myofibroblast transition (FMT). In this study, we created a fibroblast-laden 3D tissue analogue to study (1) how mechanical loading exerted on three-dimensional (3D) tissues affected the residing fibroblast phenotype and (2) to identify the ideal mechanical strain amplitude for promoting tissue regeneration without initiating myofibroblast differentiation. We applied uniaxial tensile strain (0, 4, 8, and 12%) to the cell-laden 3D tissue analogues to understand the interrelation between the degree of applied mechanical loading amplitudes and FMT. Our data demonstrated that 4% mechanical strain created an anabolic effect toward tissue regeneration, but higher strain amplitudes over-stimulated the cells and initiated fibrotic tissue formation. Under increased mechanical strain amplitudes, fibroblasts were activated from a homeostatic state to a proto-myofibroblast state which resulted in increased cellularity accompanied by increased expressions of extracellular matrix (ECM) components, activation stressors (TGF-ß1 and TGF-ßR1), and profibrotic markers. This further transformed fibroblasts into α-smooth muscle actin expressing myofibroblasts. Understanding the interplay between the applied degree of mechanical loading exerted on 3D tissues and residing fibroblast phenotypic response is important to identify specific mechanomodulatory approaches for tissue regeneration and the informed mechanotherapy-guided tissue healing strategies.


Assuntos
Anabolizantes , Miofibroblastos , Actinas/metabolismo , Anabolizantes/farmacologia , Diferenciação Celular/fisiologia , Células Cultivadas , Fibroblastos/metabolismo , Fibrose , Humanos , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
4.
FASEB J ; 36(11): e22606, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36250931

RESUMO

Kinesin family member 26b (Kif26b) is essential for kidney development, and its deletion in mice leads to kidney agenesis. However, the roles of this gene in adult settings remain elusive. Thus, this study aims to investigate the role of Kif26b in the progression of renal fibrosis. A renal fibrosis model with adenine administration using Kif26b heterozygous mice and wild-type mice was established. Renal fibrosis and the underlying mechanism were investigated. The underlying pathways and functions of Kif26b were evaluated in an in vitro model using primary renal fibroblasts. Kif26b heterozygous mice were protected from renal fibrosis with adenine administration. Renal expressions of connective tissue growth factor (CTGF) and myofibroblast accumulation were reduced in Kif26b heterozygous mice. The expression of nonmuscle myosin heavy chain II (NMHCII), which binds to the C-terminus of Kif26b protein, was also suppressed in Kif26b heterozygous mice. The in vitro study revealed reduced expressions of CTGF, α-smooth muscle actin, and myosin heavy chain 9 (Myh9) via transfection with siRNAs targeting Kif26b in renal fibroblasts (RFB). RFBs, which were transfected by the expression vector of Kif26b, demonstrated higher expressions of these genes than non-transfected cells. Finally, Kif26b suppression and NMHCII blockage led to reduced abilities of migration and collagen gel contraction in renal fibroblasts. Taken together, Kif26b contributes to the progression of interstitial fibrosis via migration and myofibroblast differentiation through Myh9 in the renal fibrosis model. Blockage of this pathway at appropriate timing might be a therapeutic approach for renal fibrosis.


Assuntos
Rim , Cinesinas , Miofibroblastos , Animais , Camundongos , Actinas/genética , Actinas/metabolismo , Adenina/metabolismo , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Fibrose , Rim/metabolismo , Cinesinas/genética , Miofibroblastos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Diferenciação Celular , Movimento Celular
5.
Int J Mol Sci ; 23(19)2022 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-36233248

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, interstitial lung disease with a poor prognosis. Although specific anti-fibrotic medications are now available, the median survival time following diagnosis remains very low, and new therapies are urgently needed. To uncover novel therapeutic targets, we examined how biochemical properties of the fibrotic lung are different from the healthy lung. Previous work identified lactate as a metabolite that is upregulated in IPF lung tissue. Importantly, inhibition of the enzyme responsible for lactate production prevents fibrosis in vivo. Further studies revealed that fibrotic lesions of the lung experience a significant decline in tissue pH, likely due to the overproduction of lactate. It is not entirely clear how cells in the lung respond to changes in extracellular pH, but a family of proton sensing G-protein coupled receptors has been shown to be activated by reductions in extracellular pH. This work examines the expression profiles of proton sensing GPCRs in non-fibrotic and IPF-derived primary human lung fibroblasts. We identify TDAG8 as a proton sensing GPCR that is upregulated in IPF fibroblasts and that knockdown of TDAG8 dampens myofibroblast differentiation. To our surprise, BTB, a proposed positive allosteric modulator of TDAG8, inhibits myofibroblast differentiation. Our data suggest that BTB does not require TDAG8 to inhibit myofibroblast differentiation, but rather inhibits myofibroblast differentiation through suppression of RhoA mediated signaling. Our work highlights the therapeutic potential of BTB as an anti-fibrotic treatment and expands upon the importance of RhoA-mediated signaling pathways in the context of myofibroblast differentiation. Furthermore, this works also suggests that TDAG8 inhibition may have therapeutic relevance in the treatment of IPF.


Assuntos
Fibrose Pulmonar Idiopática , Proteína rhoA de Ligação ao GTP , Diferenciação Celular/fisiologia , Fibroblastos/metabolismo , Fibrose , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Lactatos/metabolismo , Pulmão/patologia , Miofibroblastos/metabolismo , Prótons , Proteína rhoA de Ligação ao GTP/metabolismo
6.
Exp Cell Res ; 421(1): 113374, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36206825

RESUMO

Renal fibrosis is a global health concern with limited curative treatment. Canonical transient receptor potential channel 6 (TRPC6), a nonselective cation channel, has been shown to regulate the renal fibrosis in murine models. However, the molecular mechanism is unclear. Fibroblast-myofibroblast transdifferentiation is one of the critical steps in the progression of renal fibrosis. In the present study, we demonstrate that transforming growth factor (TGF)-ß1 exposure significantly increases the TRPC6 expression in renal interstitial fibroblast NRK-49F cells. Pharmacological inhibition of TRPC6 and knockdown of Trpc6 by siRNA alleviate TGF-ß1-increased expression levels of α-smooth muscle actin (α-SMA) and collagen I, two key markers of myofibroblasts. Although direct activation of TRPC6 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) does not affect the expression of α-SMA and collagen I, OAG potentiates TGF-ß1-induced fibroblast-myofibroblast transdifferentiation. Further study demonstrates that TGF-ß1 exposure increases the phosphorylation level of p38 and Yes-associated protein (YAP) translocation into the nuclei. Inhibition of p38 and YAP decreases TGF-ß1-enhanced TRPC6 and α-SMA expression. In conclusion, we demonstrate that TRPC6 is a key regulator of TGF-ß1-induced fibroblast-myofibroblast transdifferentiation and provides the mechanism of how TGF-ß1 exposure regulates TRPC6 expression in NRK-49F fibroblasts.


Assuntos
Transdiferenciação Celular , Nefropatias , Canal de Cátion TRPC6 , Animais , Camundongos , Actinas/metabolismo , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/fisiologia , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibrose , Nefropatias/metabolismo , Miofibroblastos/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Fatores de Crescimento Transformadores/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/uso terapêutico , Canal de Cátion TRPC6/antagonistas & inibidores , Canal de Cátion TRPC6/genética , Proteínas de Sinalização YAP , Ratos , Modelos Animais de Doenças
7.
Biomolecules ; 12(10)2022 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-36291655

RESUMO

Corneal opacification due to fibrosis is a leading cause of blindness worldwide. Fibrosis occurs from many causes including trauma, photorefractive surgery, microbial keratitis (infection of the cornea), and chemical burns, yet there is a paucity of therapeutics to prevent or treat corneal fibrosis. This study aimed to determine if andrographolide, a labdane diterpenoid found in Andrographis paniculate, has anti-fibrotic properties. Furthermore, we evaluated if andrographolide could prevent the differentiation of fibroblasts to myofibroblasts in vitro, given that the transforming growth factor beta-1(TGF-ß1) stimulated persistence of myofibroblasts in the cornea is a primary component of fibrosis. We demonstrated that andrographolide inhibited the upregulation of alpha smooth muscle actin (αSMA) mRNA and protein in rabbit corneal fibroblasts (RCFs), thus, demonstrating a reduction in the transdifferentiation of myofibroblasts. Immunofluorescent staining of TGF-ß1-stimulated RCFs confirmed a dose-dependent decrease in αSMA expression when treated with andrographolide. Additionally, andrographolide was well tolerated in vivo and had no impact on corneal epithelialization in a rat debridement model. These data support future studies investigating the use of andrographolide as an anti-fibrotic in corneal wound healing.


Assuntos
Diterpenos , Fator de Crescimento Transformador beta1 , Coelhos , Ratos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Miofibroblastos/metabolismo , Actinas/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Diterpenos/farmacologia , Córnea/metabolismo , Fibrose , RNA Mensageiro/genética
8.
Cytokine ; 160: 156047, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36183616

RESUMO

BACKGROUND: Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced by exacerbated immune response in corneal stroma. Myofibroblasts play a key role in this process by producing a disorganized extracellular matrix and inflammatory mediators. However, the immune response profile of myofibroblasts during viral infections is still under study. The aim of this work was to analyze the cytokine profile of human limbal myfibroblasts (HLMs) stimulated with the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) and to identify their signaling pathways. METHODS: HLMs were isolated from cadaveric sclera-corneal rims and stimulated with poly I:C (10 µg/ml) for 12 h. The secretion of 36 cytokines was measured using the Human Cytokine Array Panel A. The secretion of IFN-ß was quantified by ELISA. The expression of pattern recognition receptors (PRRs) such as TLR3, RIG-1 and MDA5 were analyzed by western blot assays. Furthermore, translocation of the nuclear factors NF-κB, IRF3, and IRF7 was assessed by fluorescence staining. In addition, the differentially expressed cytokines were analyzed using the Core Analysis Tool of the Ingenuity Pathway Analysis IPA software. RESULTS: HLMs stimulated with poly I:C increased (fold change > 2) the secretion of G-CSF, sTREM-1, CXCL1, CCL1, CXCL8, CXCL10, CXCL11, CCL2, CCL5, IL-13, IL-6, IL-1ra, and IFN-ß compared with HLMs under basal conditions. Poly I:C stimulation also induced the expression of RIG-1 (p < 0.001), but the expression of TLR3 and MDA5 was unmodified. Finally, HLMs increased nuclear translocation of NF-κB, IRF3, and IRF7 after poly I:C stimulation. Bioinformatic analysis identified canonical signaling pathways associated with cell adhesion and diapedesis, chemokine signaling, and activation of IRFs by cytosolic pattern recognition receptors. CONCLUSIONS: These results demonstrate that HLMs secrete cytokines involved in immune cell activation and chemotaxis. The data suggest a key role for HLMs during viral infections in cornea and extend our knowledge about the signaling pathways they trigger.


Assuntos
NF-kappa B , Viroses , Antivirais/farmacologia , Córnea , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Interferon beta/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-13/genética , Interleucina-6/genética , Miofibroblastos/metabolismo , NF-kappa B/metabolismo , Poli I-C/farmacologia , RNA de Cadeia Dupla , Receptores de Reconhecimento de Padrão , Receptor 3 Toll-Like/metabolismo
9.
Biochem Soc Trans ; 50(5): 1247-1255, 2022 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-36281993

RESUMO

Cardiac fibroblasts play an essential role in maintaining the structural framework of the heart. Upon stress, fibroblasts undergo a cell state transition to activated fibroblasts (also referred to as myofibroblasts), a highly synthetic cell type that proliferates, migrates, and secrets both extracellular matrix as well as signaling factors that can modulate cellular crosstalk [J. Clin. Invest. 132, e148554]. Activated fibroblasts are critical regulators of cardiac wound healing after injury, but their excessive and persistent activation promote tissue fibrosis, a hallmark feature of the pathological remodeling of the heart. While much of the previous work in cardiac fibroblast biology has focused on the role of canonical signaling pathways or components of the extracellular matrix, recent efforts have been focused on deciphering the gene regulatory principles governing fibroblast activation. A better understanding of the molecular mechanisms that trigger and sustain the fibrotic process in heart disease has the potential to accelerate the development of therapies that specifically target the cardiac activated fibroblasts, which are at the moment unavailable. This concise review focuses on the mechanisms underlying the chromatin and transcriptional regulation of cardiac fibroblast activation. We discuss recent work from our group and others in this space, highlighting the application of single-cell genomics in the characterization of fibroblast function and diversity, and provide an overview on the prospects of targeting cardiac fibroblasts in heart disease and the associated challenges.


Assuntos
Fibroblastos , Cardiopatias , Humanos , Fibroblastos/metabolismo , Fibrose , Coração/fisiologia , Cardiopatias/genética , Cardiopatias/metabolismo , Miocárdio/metabolismo , Miofibroblastos/metabolismo
10.
Eur Heart J ; 43(45): 4739-4750, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36200607

RESUMO

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.


Assuntos
Insuficiência Cardíaca , Remodelação Ventricular , Camundongos , Animais , Fibrose , Miofibroblastos/metabolismo , Cardiomegalia/metabolismo , Fibroblastos/metabolismo , Insuficiência Cardíaca/patologia , Miocárdio/patologia , Camundongos Endogâmicos C57BL
11.
Circ Res ; 131(7): 620-636, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36052698

RESUMO

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.


Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Animais , Cardiomiopatias/metabolismo , Colágeno/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Fibroblastos/metabolismo , Fibrose , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Insuficiência Cardíaca/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Miofibroblastos/metabolismo , Tamoxifeno/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Quinases raf
12.
ACS Chem Biol ; 17(10): 2734-2743, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36076154

RESUMO

Celastrol (CEL), a pentacyclic triterpene compound, has been proven to have a definite antipulmonary fibrosis effect. However, its direct targets for antipulmonary fibrosis remain unknown. In this study, we designed and synthesized a series of celastrol-based probes to identify the direct targets in human pulmonary fibroblasts using an activity-based protein profiling strategy. Among many fished targets, we identified a key protein, cullin-associated and neddylation-dissociated 1 (CAND1), which was involved in fibroblast-myofibroblast transformation (FMT). More importantly, we found that the inhibitory effect of celastrol on FMT is dependent on CAND1, through improving the interactions between CAND1 and Cullin1 to promote the activity of Skp1/Cullin1/F-box ubiquitin ligases. In silico studies and cysteine mutation experiments further demonstrated that Cys264 of CAND1 is the site for conjugation of celastrol. This reveals a new mechanism of celastrol against pulmonary fibrosis and may provide a novel therapeutic option for antipulmonary fibrosis.


Assuntos
Proteínas Culina , Fibrose Pulmonar , Humanos , Proteínas Culina/genética , Proteínas Culina/metabolismo , Miofibroblastos/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Cisteína , Triterpenos Pentacíclicos , Ubiquitina/metabolismo , Ligases
13.
Front Immunol ; 13: 978262, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36159833

RESUMO

Renal fibrosis commonly occurs in the process of chronic kidney diseases. Here, we explored the role of Jumonji domain containing 3 (Jmjd3)/interferon regulatory factor 4 (IRF4) axis in activation of myeloid fibroblasts and transition of M2 macrophages into myofibroblasts transition (M2MMT) in kidney fibrosis. In mice, Jmjd3 and IRF4 were highly induced in interstitial cells of kidneys with folic acid or obstructive injury. Jmjd3 deletion in myeloid cells or Jmjd3 inhibitor reduced the levels of IRF4 in injured kidneys. Myeloid Jmjd3 depletion impaired bone marrow-derived fibroblasts activation and M2MMT in folic acid or obstructive nephropathy, resulting in reduction of extracellular matrix (ECM) proteins expression, myofibroblasts formation and renal fibrosis progression. Pharmacological inhibition of Jmjd3 also prevented myeloid fibroblasts activation, M2MMT, and kidney fibrosis development in folic acid nephropathy. Furthermore, IRF4 disruption inhibited myeloid myofibroblasts accumulation, M2MMT, ECM proteins accumulation, and showed milder fibrotic response in obstructed kidneys. Bone marrow transplantation experiment showed that wild-type mice received IRF4-/- bone marrow cells presented less myeloid fibroblasts activation in injured kidneys and exhibited much less kidney fibrosis after unilateral ureteral obstruction. Myeloid Jmjd3 deletion or Jmjd3 inhibitor attenuated expressions of IRF4, α-smooth muscle actin and fibronectin and impeded M2MMT in cultured monocytes exposed to IL-4. Conversely, overexpression IRF4 abrogated the effect of myeloid Jmjd3 deletion on M2MMT. Thus, Jmjd3/IRF4 signaling has a crucial role in myeloid fibroblasts activation, M2 macrophages to myofibroblasts transition, extracellular matrix protein deposition, and kidney fibrosis progression.


Assuntos
Miofibroblastos , Insuficiência Renal Crônica , Actinas/metabolismo , Animais , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibrose , Ácido Fólico/farmacologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interleucina-4/metabolismo , Histona Desmetilases com o Domínio Jumonji , Macrófagos/metabolismo , Camundongos , Miofibroblastos/metabolismo , Insuficiência Renal Crônica/patologia
14.
Int J Mol Sci ; 23(18)2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36142285

RESUMO

Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier integrity. The supernatant of HEMFs treated with acidic bile salts reflective of in vivo reflux increases squamous epithelial thickness. We aimed to identify the involved mechanisms using an unbiased approach. Acidic-bile-salt-treated primary HEMF cultures (n = 4) were submitted for RNA-Seq and analyzed with Partek Flow followed by Ingenuity Pathway Analysis (IPA). A total of 1165 molecules (579 downregulated, 586 upregulated) were differentially expressed, with most top regulated molecules either extracellular or in the plasma membrane. Increases in HEMF CXCL-8, IL-6, AREG, and EREG mRNA, and protein secretion were confirmed. Top identified canonical pathways were agranulocyte and granulocyte adhesion and diapedesis, PI3K/AKT signaling, CCR5 signaling in macrophages, and the STAT3 pathway. Top diseases and biological functions were cellular growth and development, hematopoiesis, immune cell trafficking, and cell-mediated response. The targets of the top upstream regulator ErbB2 included CXCL-8, IL-6, and AREG and the inhibition of CXCL-8 in the HEMF supernatant decreased squamous epithelial proliferation. Our work shows an inflammatory/immune cell and proliferative pathways activation in HEMFs in the GERD environment and identifies CXCL-8 as a HEMF-derived chemokine with paracrine proliferative effects on squamous epithelium.


Assuntos
Carcinoma de Células Escamosas , Refluxo Gastroesofágico , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/farmacologia , Carcinoma de Células Escamosas/metabolismo , Humanos , Interleucina-6/metabolismo , Miofibroblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética
15.
Open Biol ; 12(9): 210356, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36102060

RESUMO

Fibroblasts are widely distributed cells found in most tissues and upon tissue injury, they are able to differentiate into myofibroblasts, which express abundant extracellular matrix (ECM) proteins. Overexpression and unordered organization of ECM proteins cause tissue fibrosis in damaged tissue. Fibroblast growth factor (FGF) family proteins are well known to promote angiogenesis and tissue repair, but their activities in fibroblast differentiation and fibrosis have not been systematically reviewed. Here we summarize the effects of FGFs in fibroblast to myofibroblast differentiation and ECM protein expression and discuss the underlying potential regulatory mechanisms, to provide a basis for the clinical application of recombinant FGF protein drugs in treatment of tissue damage.


Assuntos
Proteínas da Matriz Extracelular , Miofibroblastos , Proteínas da Matriz Extracelular/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fibroblastos , Fibrose , Humanos , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
16.
Front Immunol ; 13: 948658, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36148244

RESUMO

Renal fibrosis is the inevitable pathway of the progression of chronic kidney disease to end-stage renal disease, which manifests as progressive glomerulosclerosis and renal interstitial fibrosis. In a previous study, we observed severe interstitial fibrosis in the contralateral kidneys of 6-month unilateral ureteral obstruction (UUO) rats, which was accompanied by increased macrophage infiltration and phenotypic transformation; after eplerenone administration, these effects were reduced. Therefore, we hypothesized that this effect was closely related to mineralocorticoid receptor (MR) activation induced by the increased aldosterone (ALD) level. In this study, we used uninephrectomy plus continuous aldosterone infusion in mice to observe whether aldosterone induced macrophage-to-myofibroblast transition (MMT) and renal fibrosis and investigated the signaling pathways. Notably, aldosterone induced predominantly M1 macrophage-to-myofibroblast transition by activating MR and upregulating TGF-ß1 expression, which promoted renal fibrosis. These effects were antagonized by the MR blocker esaxerenone. These findings suggest that targeting the MR/TGF-ß1 pathway may be an effective therapeutic strategy for renal fibrosis.


Assuntos
Insuficiência Renal Crônica , Fator de Crescimento Transformador beta1 , Aldosterona/farmacologia , Animais , Eplerenona/farmacologia , Fibrose , Macrófagos/metabolismo , Camundongos , Miofibroblastos/metabolismo , Pirróis , Ratos , Receptores de Mineralocorticoides , Sulfonas , Fator de Crescimento Transformador beta1/metabolismo
17.
Clin Sci (Lond) ; 136(19): 1405-1423, 2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36156078

RESUMO

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor ß1 (TGFß) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors. ß-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFß signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in ß-catenin-positive cells. In vitro, activation of Wnt-ß-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of ß-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFß increased ß-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFß up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a ß-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFß signalling pathways in CD intestinal fibrosis.


Assuntos
Doença de Crohn , beta Catenina , Colágeno Tipo I/metabolismo , Doença de Crohn/tratamento farmacológico , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Fibrose , Formaldeído/metabolismo , Humanos , Intestinos , Ligantes , Miofibroblastos/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
18.
Cells ; 11(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36078140

RESUMO

Overgrowths of dermal fibroblasts and myofibroblast phenoconversion in response to TGF-ß stimulation are the hallmarks of skin fibrosis. Constitutive activation of dermal fibroblasts by TGF-ß induces the excessive production of extracellular matrix as well as certain key intracellular proteins which form a complex interaction network. Current therapies include monoclonal anti-bodies against TGF-ß and surgery, but these treatments generally elicit a limited effect on certain kinds of skin fibrosis. In the current study, we investigated the effects of alpinetin (AP) on human primary dermal fibroblasts (HPDFs) stimulated with TGF-ß1. Results demonstrated that AP exhibited strong inhibitory effects on TGF-ß1-induced proliferation and migration of HPDFs. AP also inhibited TGF-ß1-induced morphological changes of fibroblasts to myofibroblasts, and these were found to be from its effects on blocking actin stress fiber formation and organization. The expression of major fibrotic molecules including α-SMA and type I collagen upon TGF-ß1 stimulation was also inhibited by AP. In addition, AP attenuated TGF-ß1-induced production and organization of vimentin, ß-catenin, and N-cadherin, important for the pathophysiology of skin fibrosis. In conclusion, we revealed that AP has an ability to reverse the fibrotic effects of TGF-ß1 at the cellular level, and this discovery suggests the therapeutic potential of AP for skin fibrosis.


Assuntos
Fibroblastos , Flavanonas , Fator de Crescimento Transformador beta1 , Biomarcadores , Fibroblastos/metabolismo , Fibrose/metabolismo , Flavanonas/farmacologia , Humanos , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
19.
Acta Biomater ; 152: 300-312, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36055606

RESUMO

Cardiac fibrosis is characterized by a maladaptive remodeling of the myocardium, which is controlled by various inflammatory pathways and cytokines. This remodeling is accompanied by a significant stiffening of the matrix, which may contribute to further activate collagen synthesis and scar formation. Evidence suggests that TGF-ß1 signaling, the main pro-fibrotic pathway in cardiac fibrosis, might cooperates with the Hippo transcriptional pathway by activating YAP. To directly test the cooperation of mechanical cues and paracrine signaling in cardiac fibrosis, we developed a 3D model of cardiac extracellular matrix remodeling by generating tissue blocks with Gelatin Methacrylate, a bioink with tunable stiffness, and human cardiosphere-derived stromal cells. Using this strategy, we assessed the cooperation of TGF-ß1 and YAP transcriptional factor to matrix compaction. Using mechanical compression tests, Masson's trichrome staining, immunofluorescence, and RT-qPCR, we demonstrate that pharmacological inhibition of YAP complex reverts almost completely the pro-compaction phenotype and the matrix-remodeling activity of cells treated with TGF-ß1. Our data show a direct connection between the classical pro-fibrotic signaling driven by TGF-ß1 and the mechanically activated pathways under the control of YAP in cardiac remodeling. Treatment with the elective drug targeting YAP is sufficient to override this cooperation with potential benefits for anti-fibrotic therapeutic applications. STATEMENT OF SIGNIFICANCE: Heart failure is a pathology in continuous growth worldwide, characterized by a progressive fibrosis, which decreases the pumping efficiency of the heart. Experimental evidences suggest that fibroblasts, normally responsible for the turnover of the cardiac matrix, are involved in myocardial fibrosis by differentiating into 'myofibroblasts'. These cells remodel extensively the cardiac extracellular matrix and deposit abundant collagen with a consequent increase in stiffness. In the present contribution, we propose a new 3D model of cell-mediated cardiac extracellular matrix stiffening to investigate the mechano-chemical mechanisms underlying the onset of the pathology. We also consolidate a pharmacological treatment able to prevent the pathological activation of fibroblasts with potential benefits for anti-fibrotic treatment of the failing heart.


Assuntos
Miocárdio , Miofibroblastos , Fator de Crescimento Transformador beta1 , Proteínas de Sinalização YAP , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibrose , Gelatina , Humanos , Metacrilatos/metabolismo , Miocárdio/patologia , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas de Sinalização YAP/metabolismo
20.
Nat Commun ; 13(1): 5324, 2022 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-36088459

RESUMO

Tissue injury triggers activation of mesenchymal lineage cells into wound-repairing myofibroblasts, whose unrestrained activity leads to fibrosis. Although this process is largely controlled at the transcriptional level, whether the main transcription factors involved have all been identified has remained elusive. Here, we report multi-omics analyses unraveling Basonuclin 2 (BNC2) as a myofibroblast identity transcription factor. Using liver fibrosis as a model for in-depth investigations, we first show that BNC2 expression is induced in both mouse and human fibrotic livers from different etiologies and decreases upon human liver fibrosis regression. Importantly, we found that BNC2 transcriptional induction is a specific feature of myofibroblastic activation in fibrotic tissues. Mechanistically, BNC2 expression and activities allow to integrate pro-fibrotic stimuli, including TGFß and Hippo/YAP1 signaling, towards induction of matrisome genes such as those encoding type I collagen. As a consequence, Bnc2 deficiency blunts collagen deposition in livers of mice fed a fibrogenic diet. Additionally, our work establishes BNC2 as potentially druggable since we identified the thalidomide derivative CC-885 as a BNC2 inhibitor. Altogether, we propose that BNC2 is a transcription factor involved in canonical pathways driving myofibroblastic activation in fibrosis.


Assuntos
Cirrose Hepática , Miofibroblastos , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genômica , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Miofibroblastos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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