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1.
Anticancer Res ; 41(10): 4753-4759, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34593424

RESUMO

BACKGROUND/AIM: De-differentiation is a key step for the progression of cancer cells. This study investigated the anti-tumor effect of kartogenin (KGN), which has the ability to differentiate cells, on prostate cancer (PC) cells. MATERIALS AND METHODS: The effects of KGN on androgen receptor (AR) nuclear localization, prostate-specific antigen (PSA) expression, and Smad2 activation as well as the growth of PC cell lines (LNCaP, 22Rv1 and PC-3) were analyzed. RESULTS: KGN significantly inhibited growth of AR-expressing LNCaP and 22Rv1 cells but not of AR-lacking PC-3 cells. KGN decreased AR nuclear localization and PSA expression, but did not enhance the anti-tumor effect of bicalutamide in LNCaP cells. KGN activated Smad2 both in the absence and presence of TGF-ß1. KGN also inhibited growth of docetaxel-resistant PC cells, 22Rv1DR, and re-sensitized them to the agent. CONCLUSION: KGN has a potential as a novel therapeutic for PC patients after treatment failure.


Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , Ácidos Ftálicos/farmacologia , Receptores Androgênicos/metabolismo , Proteína Smad2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/patologia , Fator de Crescimento Transformador beta1/farmacologia
2.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639099

RESUMO

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-ß (TGF-ß) and TGF-ß inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-ß1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-ß-treated group and TGF-ß-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-ß-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-ß1, TGF-ß2, and TGF-ß3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.


Assuntos
Endotélio Vascular/patologia , Fibroblastos/patologia , Fibrose/patologia , Túbulos Renais Proximais/patologia , Impressão Tridimensional/instrumentação , Fator de Crescimento Transformador beta1/farmacologia , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/induzido quimicamente , Fibrose/metabolismo , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo
3.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638875

RESUMO

Pluripotent adult stem cells have potential applications in cell therapy and tissue engineering. Urine-derived stem cells (UDSCs) differentiate into various cell types. Here, we attempted to differentiate human UDSCs (hUDSCs) into smooth muscle cells (SMCs) using transforming growth factor-beta 1 (TGF-ß1) and/or PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Both quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that the expression of messenger ribonucleic acid (mRNA) and proteins for alpha-smooth muscle actin (α-SMA), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC), which are specific markers for SMCs, increased on day 9 after differentiation and again on day 14. The differentiated cells from human UDSCs (hUDSCs) with a combination of TGF-ß1 and PD98059 showed the highest expression of SMC marker proteins. Immunocytochemical staining performed to assess the molecular expression revealed CNN and α-SMA colocalizing in the cytoplasm. The cells that differentiated from hUDSCs with a combination of TGF-ß1 and PD98059 showed the strongest expression for CNN1, α-SMA, and SM-MHC. Functional testing of the differentiated cells revealed a stronger contractile capacity for the cells differentiated with a combination of PD98059 and TGF-ß1 than those differentiated with a single factor. These results suggest the combination of PD98059 and TGF-ß1 to be a more effective differentiation method and that differentiated SMCs could be used for restoring the functions of the sphincter muscle or bladder.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Células Musculares , Células-Tronco , Fator de Crescimento Transformador beta1/farmacologia , Urina/citologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células Musculares/citologia , Células Musculares/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
4.
Nat Commun ; 12(1): 6242, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34716325

RESUMO

Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigate mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we find significant differences in gene expression in most cell types present in scar tissue. Fibroblasts show the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), are further analyzed in functional assays, revealing a role in TGFß1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo shows anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.


Assuntos
Cicatriz/patologia , Dipeptidil Peptidase 4/genética , Proteínas de Membrana/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Cicatriz/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Feminino , Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/fisiologia , Análise de Célula Única , Fosfato de Sitagliptina/farmacologia , Fator de Crescimento Transformador beta1/farmacologia
5.
BMC Musculoskelet Disord ; 22(1): 680, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380439

RESUMO

BACKGROUND: Skeletal muscle atrophy and fibrosis are pathological conditions that contribute to morbidity in numerous conditions including aging, cachexia, and denervation. Muscle atrophy is characterized as reduction of muscle fiber size and loss of muscle mass while muscle fibrosis is due to fibroblasts activation and excessive production of extracellular matrix. Purinergic receptor P2Y2 has been implicated in fibrosis. This study aims to elucidate the roles of P2Y2 in sleketal muscle atrophy and fibrosis. METHODS: Primary muscle fibroblasts were isolated from wild type and P2Y2 knockout (KO) mice and their proliferating and migrating abilities were assessed by CCK-8 and Transwell migration assays respectively. Fibroblasts were activated with TGF-ß1 and assessed by western blot of myofibroblast markers including α-SMA, CTGF, and collagen I. Muscle atrophy and fibrosis were induced by transection of distal sciatic nerve and assessed using Masson staining. RESULTS: P2Y2 KO fibroblasts proliferated and migrated significantly slower than WT fibroblasts with or without TGF-ß1.The proliferation and ECM production were enhanced by P2Y2 agonist PSB-1114 and inhibited by antagonist AR-C118925. TGF-ß1 induced fibrotic activation was abolished by P2Y2 ablation and inhibited by AKT, ERK, and PKC inhibitors. Ablation of P2Y2 reduced denervation induced muscle atrophy and fibrosis. CONCLUSIONS: P2Y2 is a promoter of skeletal muscle atrophy and activation of fibroblasts after muscle injury, which signaling through AKT, ERK and PKC. P2Y2 could be a potential intervention target after muscle injury.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Músculo Esquelético/patologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores Purinérgicos P2Y2/metabolismo , Animais , Células Cultivadas , Fibroblastos/patologia , Fibrose , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
6.
Exp Eye Res ; 210: 108725, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34375589

RESUMO

Antiproliferative therapies are crucially important for improving the success rate of the glaucoma filtration surgeries. In this study, we investigated the potential efficacy of Forkhead Domain Inhibitory-6 (FDI-6) in inhibiting post-trabeculectomy subconjunctival fibrosis. In vitro, the effect of FDI-6 (10 µM) on fibrotic response and its underlying mechanism were investigated in rabbit tenon's fibroblasts (RTFs) treated with or without transforming growth factor-ß1 (TGF-ß1, 20 ng/mL). In vivo, FDI-6 (40 µM) was injected subconjunctivally to a rabbit trabeculectomy model. Intraocular pressure (IOP) changes were monitored within the 14-day period post-surgery. Bleb morphology and subepithelial fibrosis at the operating area were evaluated with slit lamp and confocal microscopic examinations and with histologic examinations. The results showed that, in cell culture studies, FDI-6 suppressed the proliferation, migration, collagen gel contraction and the expression levels of fibronectin (FN) and α-smooth muscle actin (α-SMA) in RTFs with TGF-ß treatment by down-regulating the TGF-ß1/Smad2/3 signaling pathway. In animal studies, the IOPs of the FDI-6-treated group were significantly lower than those of the saline-treated group after trabeculectomy. The FDI-6-treated eyes showed a better bleb appearance with fewer blood vessels compared to the saline-treated eyes. The analysis of confocal microscopy in vivo and histopathology revealed that subconjunctival fibrosis after trabeculectomy was significantly attenuated in the FDI-6-treated group compared to the controls. In conclusion, our studies indicate that FDI-6 exerts an inhibitory effect on subconjunctival fibrosis caused by trabeculectomy, holding potentials as a new antiproliferative agent used in anti-glaucoma filtration surgeries in the future.


Assuntos
Túnica Conjuntiva/patologia , Modelos Animais de Doenças , Glaucoma/cirurgia , Complicações Pós-Operatórias/prevenção & controle , Piridinas/uso terapêutico , Tiofenos/uso terapêutico , Trabeculectomia , Actinas/metabolismo , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibrose/prevenção & controle , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Marcação In Situ das Extremidades Cortadas , Injeções Intraoculares , Pressão Intraocular/efeitos dos fármacos , Masculino , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Cápsula de Tenon/efeitos dos fármacos , Cápsula de Tenon/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/efeitos dos fármacos
7.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360888

RESUMO

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.


Assuntos
Condrócitos/metabolismo , Condrócitos/patologia , Osteoartrite/metabolismo , Transdução de Sinais/genética , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad3/química , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Hipertrofia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/genética , Osteoartrite/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Domínios Proteicos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Membrana Sinovial/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia
8.
Exp Eye Res ; 211: 108747, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34450184

RESUMO

PURPOSE: Cornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. METHODS: Human corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-ß1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. RESULTS: Compared to the positive control TGF-ß1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). CONCLUSION: Our results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.


Assuntos
Cicatriz/prevenção & controle , Doenças da Córnea/prevenção & controle , Substância Própria/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Epitélio Corneano/efeitos dos fármacos , Actinas/metabolismo , Administração Oftálmica , Biomarcadores/metabolismo , Cicatriz/metabolismo , Doenças da Córnea/metabolismo , Ceratócitos da Córnea/efeitos dos fármacos , Ceratócitos da Córnea/metabolismo , Substância Própria/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Medicina Tradicional Chinesa , Soluções Oftálmicas , Fator de Crescimento Transformador beta1/farmacologia
9.
Cells ; 10(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34440821

RESUMO

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-ß1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-ß1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Escleroderma Sistêmico/patologia , Succinatos/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Elementos de Resposta Antioxidante/efeitos dos fármacos , Elementos de Resposta Antioxidante/genética , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/metabolismo , Escleroderma Sistêmico/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
10.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202024

RESUMO

Orbital fibrosis, a hallmark of tissue remodeling in Graves' ophthalmopathy (GO), is a chronic, progressive orbitopathy with few effective treatments. Orbital fibroblasts are effector cells, and transforming growth factor ß1 (TGF-ß1) acts as a critical inducer to promote myofibroblast differentiation and subsequent tissue fibrosis. Curcumin is a natural compound with anti-fibrotic activity. This study aims to investigate the effects of curcumin on TGF-ß1-induced myofibroblast differentiation and on the pro-angiogenic activities of orbital fibroblasts. Orbital fibroblasts from one healthy donor and three patients with GO were collected for primary cell culture and subjected to myofibroblast differentiation under the administration of 1 or 5 ng/mL TGF-ß1 for 24 h. The effects of curcumin on TGF-ß1-induced orbital fibroblasts were assessed by measuring the cellular viability and detecting the expression of myofibroblast differentiation markers, including connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA). The pro-angiogenic potential of curcumin-treated orbital fibroblasts was evaluated by examining the transwell migration and tube-forming capacities of fibroblast-conditioned EA.hy926 and HMEC-1 endothelial cells. Treatment of orbital fibroblasts with curcumin inhibited the TGF-ß1 signaling pathway and attenuated the expression of CTGF and α-SMA induced by TGF-ß1. Curcumin, at the concentration of 5 µg/mL, suppressed 5 ng/mL TGF-ß1-induced pro-angiogenic activities of orbital fibroblast-conditioned EA hy926 and HMEC-1 endothelial cells. Our findings suggest that curcumin reduces the TGF-ß1-induced myofibroblast differentiation and pro-angiogenic activity in orbital fibroblasts. The results support the potential application of curcumin for the treatment of GO.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Curcumina/farmacologia , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Oftalmopatia de Graves/etiologia , Oftalmopatia de Graves/metabolismo , Oftalmopatia de Graves/patologia , Humanos , Miofibroblastos/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
11.
Chem Biol Interact ; 346: 109570, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34217686

RESUMO

Verapamil is reported to prevent scar formation. However, whether verapamil is involved in the ureteral stricture scar and the underlying mechanism need further investigation. Fibroblasts were isolated from ureteral scar tissues. TGF-ß1 stimulation was used to induce fibrosis of fibroblasts. Inhibition of CaMK II was achieved by shRNA transfection. CCK-8 was performed to evaluate cell viability. qRT-PCR was applied to determine the level of mRNA while western blotting was used to determine the level of proteins. Immunofluorescence was used to detect the level of vimentin, collagen I and collagen III. Primary fibroblasts was successfully isolated from ureteral scar tissues. TGF-ß1 stimulation was capable to induce collagen production and fibrosis in primary fibroblasts while inhibition of CaMK II attenuate collagen production. Overexpression of wild type CaMK II lead to further increase of collagen production upon TGF-ß1 stimulation while the mutated CaMK II did not exert this promotion. Treatment of verapamil inhibits TGF-ß1 induced collagen production via inhibiting CaMK II. In present study, we revealed a vital role of Verapamil and CaMK II in the formation of ureteral scar. Verapamil inhibited TGF-ß1 induced collagen fiber formation by regulating CaMK II. Our finding might provide new insight into mechanism of prevention and treatment of ureteral scar.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Verapamil/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Cicatriz/tratamento farmacológico , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mutagênese , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Verapamil/uso terapêutico , Vimentina/metabolismo
12.
Environ Toxicol ; 36(11): 2225-2235, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34323359

RESUMO

Angiomotin-like 2 (AMOTL2) is a key modulator of signaling transduction and participates in the regulation of various cellular progresses under diverse physiological and pathological conditions. However, whether AMOTL2 participates in asthma pathogenesis has not been fully studied. In the present work, we studied the possible role and mechanism of AMOTL2 in regulating transforming growth factor-ß1 (TGF-ß1)-induced proliferation and extracellular matrix (ECM) deposition of airway smooth muscle (ASM) cells. Our results showed marked reductions in the abundance of AMOTL2 in TGF-ß1-stimulated ASM cells. Cellular functional investigations confirmed that the up-regulation of AMOTL2 dramatically decreased the proliferation and ECM deposition induced by TGF-ß1 in ASM cells. In contrast, the depletion of AMOTL2 exacerbated TGF-ß1-induced ASM cell proliferation and ECM deposition. Further research revealed that the overexpression of AMOTL2 restrained the activation of Yes-associated protein 1 (YAP1) in TGF-ß1-stimulated ASM cells. Moreover, the reactivation of YAP1 markedly reversed AMOTL2-mediated suppression of TGF-ß1-induced ASM cell proliferation and ECM deposition. Together, these findings suggest that AMOTL2 restrains TGF-ß1-induced proliferation and ECM deposition of ASM cells by down-regulating YAP1 activation.


Assuntos
Proteínas de Transporte/genética , Matriz Extracelular , Miócitos de Músculo Liso , Fator de Crescimento Transformador beta1 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Camundongos , Miócitos de Músculo Liso/citologia , Fator de Crescimento Transformador beta1/farmacologia
13.
Aging (Albany NY) ; 13(11): 14687-14708, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088884

RESUMO

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.


Assuntos
Envelhecimento/patologia , Autofagia , Células da Medula Óssea/metabolismo , Leucócitos Mononucleares/metabolismo , Comunicação Parácrina , Animais , Antígenos Ly/metabolismo , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/metabolismo , Meios de Cultivo Condicionados/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Comunicação Parácrina/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
14.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067060

RESUMO

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF ß1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3'-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF ß1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF ß1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.


Assuntos
Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Proteínas Relacionadas à Folistatina/metabolismo , MicroRNAs/metabolismo , Pericárdio/patologia , Animais , Biomarcadores/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Mesoderma/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Crescimento Transformador beta1/farmacologia
15.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069162

RESUMO

Therapeutic elevation of high-density lipoprotein (HDL) is thought to minimize atherogenesis in subjects with dyslipidemia. However, this is not the case in clinical practice. The function of HDL is not determined by its concentration in the plasma but by its specific structural components. We previously identified an index for the prediction of HDL functionality, relative HDL (rHDL) index, and preliminarily explored that dysfunctional HDL (rHDL index value > 2) failed to rescue the damage to endothelial progenitor cells (EPCs). To confirm the effectiveness of the rHDL index for predicting HDL functions, here we evaluated the effects of HDL from patients with different rHDL index values on the endothelial-mesenchymal transition (EndoMT) of EPCs. We also analyzed the lipid species in HDL with different rHDL index values and investigated the structural differences that affect HDL functions. The results indicate that HDL from healthy adults and subjects with an rHDL index value < 2 protected transforming growth factor (TGF)-ß1-stimulated EndoMT by modulating Smad2/3 and Snail activation. HDL from subjects with an rHDL index value > 2 failed to restore the functionality of TGF-ß1-treated EPCs. Lipidomic analysis demonstrated that HDL with different rHDL index values may differ in the composition of triglycerides, phosphatidylcholine, and phosphatidylinositol. In conclusion, we confirmed the applicability of the rHDL index value to predict HDL function and found structural differences that may affect the function of HDL, which warrants further in-depth studies.


Assuntos
Células Progenitoras Endoteliais/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Idoso , Dislipidemias/sangue , Células Progenitoras Endoteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipoproteínas HDL/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfatidilcolinas/sangue , Fosfatidilcolinas/química , Fosfatidilinositóis/sangue , Fosfatidilinositóis/química , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Triglicerídeos/sangue , Triglicerídeos/química , Adulto Jovem
16.
Cell Prolif ; 54(7): e13081, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34121240

RESUMO

OBJECTIVES: Idiopathic pulmonary fibrosis (IPF) is marked by the excessive accumulation of extracellular matrix, which participates in a variety of chronic diseases or injuries and seriously threatens human health. Due to the side effects of clinical drugs, there is still a need to develop novel and less toxic drugs to treat pulmonary fibrosis. MATERIALS AND METHODS: SKLB-YTH-60 was developed through computer-aided drug design, de novo synthesis and high-throughput screening. We employed the bleomycin (BLM)-induced lung fibrosis animal models and used TGF-ß1 to induce the epithelial-mesenchymal transition (EMT) of A549 cells in vitro. Meanwhile, the protein expression of collagen I and the α-smooth muscle actin (α-SMA), E-cadherin, p-FGFR1, p-PLCγ, p-Smad2/3 and p-Erk1/2 was detected by western blot. RESULTS: YTH-60 has obvious anti-proliferative activity on fibroblasts and A549 cells. Moreover, YTH-60 could impair the EMT of A549 cells and suppressed fibrosis by inhibiting FGFR and TGF-ß/Smad-dependent pathways. Intraperitoneal administration of preventive YTH-60 could significantly reduce the degree of fibrosis in mice and regulate the imbalance of the immune microenvironment. In addition, we observed that therapeutic YTH-60 treatment attenuated fibrotic changes in mice during the period of fibrosis. Importantly, YTH-60 has shown an acceptable oral bioavailability (F = 17.86%) and appropriate eliminated half-life time (T1/2  = 8.03 hours). CONCLUSIONS: Taken together, these preclinical evaluations suggested that YTH-60 could be a promising drug candidate for treating IPF.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/patologia , Células A549 , Animais , Sítios de Ligação , Bleomicina/toxicidade , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Desenho de Fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Meia-Vida , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controle , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/farmacologia
17.
Am J Sports Med ; 49(7): 1892-1903, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34081556

RESUMO

BACKGROUND: Tendons heal by fibrotic repair, increasing the likelihood of reinjury. Animal tendon injury and overuse models have identified transforming growth factor beta (TGF-ß) and bone morphogenetic proteins (BMPs) as growth factors actively involved in the development of fibrosis, by mediating extracellular matrix synthesis and cell differentiation. PURPOSE: To understand how TGF-ß and BMPs contribute to fibrotic processes using tendon-derived cells isolated from healthy and diseased human tendons. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon-derived cells were isolated from patients with a chronic rotator cuff tendon tear (large to massive, diseased) and healthy hamstring tendons of patients undergoing anterior cruciate ligament repair. Isolated cells were incubated with TGF-ß1 (10 ng/mL) or BMP-2 (100 ng/mL) for 3 days. Gene expression was measured by real-time quantitative polymerase chain reaction. Cell signaling pathway activation was determined by Western blotting. RESULTS: TGF-ß1 treatment induced ACAN mRNA expression in both cell types but less in the diseased compared with healthy cells (P < .05). BMP-2 treatment induced BGN mRNA expression in healthy but not diseased cells (P < .01). In the diseased cells, TGF-ß1 treatment induced increased ACTA2 mRNA expression (P < .01) and increased small mothers against decapentaplegic (SMAD) signaling (P < .05) compared with those of healthy cells. Moreover, BMP-2 treatment induced ACTA2 mRNA expression in the diseased cells only (P < .05). CONCLUSION: Diseased tendon-derived cells show reduced expression of the proteoglycans aggrecan and biglycan in response to TGF-ß1 and BMP-2 treatments. These same treatments induced enhanced fibrotic differentiation and canonical SMAD cell signaling in diseased compared with healthy cells. CLINICAL RELEVANCE: Findings from this study suggest that diseased tendon-derived cells respond differently than healthy cells in the presence of TGF-ß1 and BMP-2. The altered responses of diseased cells may influence fibrotic repair processes during tendon healing.


Assuntos
Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta , Animais , Proteínas Morfogenéticas Ósseas , Células Cultivadas , Humanos , Manguito Rotador , Tendões , Fator de Crescimento Transformador beta1/farmacologia
18.
Theranostics ; 11(14): 6905-6921, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093861

RESUMO

Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous. Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-ß1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches. Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-ß1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-ß1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells. Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-ß1-induced EMT of HCC cells: HGF or TGF-ß1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptor 1 de Folato/metabolismo , Ácido Fólico/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/patologia , Fucosiltransferases/metabolismo , Glicosilação , Células Hep G2 , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Espectrometria de Massas , Polissacarídeos/metabolismo , Proteoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
19.
Carbohydr Polym ; 268: 118256, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34127227

RESUMO

Inspired by the natural electrostatic interaction of cationic growth factors with anionic sulfated glycosaminoglycans in the extracellular matrix, we developed electrospun poly(hydroxybutyrate)/gelatin (PG) fibers conjugated with anionic sulfated carboxymethylcellulose (sCMC) to enable growth factor immobilization via electrostatic interaction for tissue engineering. The fibrous scaffold bound cationic molecules, was cytocompatible and exhibited a remarkable morphological and functional stability. Transforming growth factor-ß1 immobilized on the sCMC conjugated fibers was retained for at least 4 weeks with negligible release (3%). Immobilized fibroblast growth factor-2 and connective tissue growth factor were bioactive and induced proliferation and fibrogenic differentiation of infrapatellar fat pad derived mesenchymal stem cells respectively with efficiency similar to or better than free growth factors. Taken together, our studies demonstrate that sCMC conjugated PG fibers can immobilize and retain function of cationic growth factors and hence show potential for use in various tissue engineering applications.


Assuntos
Carboximetilcelulose Sódica/análogos & derivados , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Sistemas de Liberação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Tecidos Suporte/química , Fator de Crescimento Transformador beta1/farmacologia , Animais , Sequência de Carboidratos , Carboximetilcelulose Sódica/metabolismo , Carboximetilcelulose Sódica/toxicidade , Bovinos , Gelatina/química , Gelatina/metabolismo , Gelatina/toxicidade , Cabras , Proteínas Imobilizadas/farmacologia , Células-Tronco Mesenquimais , Muramidase/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Poliésteres/toxicidade , Soroalbumina Bovina/metabolismo , Eletricidade Estática , Engenharia Tecidual/métodos
20.
Front Immunol ; 12: 690697, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093596

RESUMO

Renal fibrosis is the final common pathway to chronic kidney diseases regardless of etiology. Parkinson disease protein 7 (PARK7) is a multifunctional protein involved in various cellular processes, but its pathophysiological role in kidneys remain largely unknown. Here, we have determined the role of PARK7 in renal fibrosis and have further elucidated the underlying mechanisms by using the in vivo mouse model of unilateral ureteric obstruction (UUO) and the in vitro model of transforming growth factor-b (TGFB1) treatment of cultured kidney proximal tubular cells. PARK7 decreased markedly in atrophic kidney tubules in UUO mice, and Park7 deficiency aggravated UUO-induced renal fibrosis, tubular cell apoptosis, ROS production and inflammation. In vitro, TGFB1 treatment induced fibrotic changes in renal tubular cells, which was accompanied by alterations of PARK7. Park7 knockdown exacerbated TGFB1-induced fibrotic changes, cell apoptosis and ROS production, whereas Park7 overexpression or treatment with ND-13 (a PARK7-derived peptide) attenuated these TGFB1-induced changes. Mechanistically, PARK7 translocated into the nucleus of renal tubular cells following TGFB1 treatment or UUO, where it induced the expression of SOD2, an antioxidant enzyme. Taken together, these results indicate that PARK7 protects against chronic kidney injury and renal fibrosis by inducing SOD2 to reduce oxidative stress in tubular cells.


Assuntos
Nefropatias/prevenção & controle , Túbulos Renais Proximais/enzimologia , Estresse Oxidativo , Proteína Desglicase DJ-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/biossíntese , Animais , Apoptose , Linhagem Celular , Modelos Animais de Doenças , Indução Enzimática , Fibrose , Nefropatias/enzimologia , Nefropatias/etiologia , Nefropatias/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Desglicase DJ-1/genética , Transdução de Sinais , Superóxido Dismutase/genética , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/complicações
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