Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury(ALI), including acute respiratory distress syndrome(ARDS), in patients after cardiac surgery. We previously foun...Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury(ALI), including acute respiratory distress syndrome(ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles(eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of e EVs after cardiopulmonary bypass, remains unclear. Plasma plasminogenactivated inhibitor-1(PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice(C57BL/6,Toll-like receptor 4 knockout(TLR4^(-/-))) and inducible nitric oxide synthase knockout(iNOS^(-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3(JAK2/3)-signal transducer and activator of transcription 3(STAT3)-interferon regulatory factor 1(IRF-1)pathway, along with i NOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors(AG490 or S3I-201, respectively), and was relieved in TLR4-/-and iNOS-/-mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1(FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.展开更多
Background： Follistatin-like I （FSTL 1） is a novel profibrogenic factor that induces pulmonary fibrosis （PF） through the transforming growth factor-beta 1 （TGF-[B 1 ）/Smad signaling. Little is known about its e...Background： Follistatin-like I （FSTL 1） is a novel profibrogenic factor that induces pulmonary fibrosis （PF） through the transforming growth factor-beta 1 （TGF-[B 1 ）/Smad signaling. Little is known about its effects on PF through the non-Smad signaling, like the mitogen-activated protein kinase （MAPK） pathway. Therefore, this study aimed to investigate the role ofFSTL 1 in PF through the MAPK signaling pathway and its mechanisms in lung fibrogenesis. Methods： PF was induced in Fstll~ and wild-type （WT） C57BL/6 mice with bleomycin. After 14 days, the mice were sacrificed, and lung tissues were stained with hematoxylin and eosin; the hydroxyproline content was measured to confirm PF. The mRNA and protein level of FSTLI and the change of MAPK phosphorylation were measured by quantitative polymerase chain reaction and Western blotting. The effect of Fst11 deficiency on fibroblasts differentiation was measured by Western blotting and cell immunofluorescence. MAPK signaling activation was measured by Western blotting in Fst11＋/ and WT fibroblasts treated with recombinant human FSTLI protein. We pretreated mouse lung fibroblast cells with inhibitors of the extracellular signal-regulated kinase （ERK）, p38, and Jun N-terminal kinase （JNK） signaling and analyzed their differentiation, proliferation, migration, and invasion by Western blotting, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis, and transwell assays. The Student＇s t-test was used to compare the differences between two groups. Results： Fstll deficiency attenuated phosphorylation of the ERK, p38, and JNK signaling in bleomycin-induced fibrotic lung tissue 14 days after injury （0.67 ± 0.05 vs. 1.22 ± 0.03, t = 14.92, P = 0.0001; 0.41 ± 0.01 vs. 1.15 ± 0.07; t = 11.19; P = 0.0004; and 0.41 ± 0.01 vs. 1.07± 0.07, t = 8.92, P = 0.0009; respectively）, compared with WT lungs at the same time and in primary lung fibroblasts （0.82 ± 0.01 vs. 1.01 ±0.04, t = 4.06, P = 0.0150; 1.04 ±0.03 vs. 1.24 ± 0.03, t= 4.44, P = 0.0100： and 0.76 ±0.05 vs. 0.99± 0.05, t = 4.48, P = 0.0100; respectively）, compared with TGF-β1-stimulated WT group. Recombinant human FSTLI protein in lung fibroblasts enhanced TG F-β1 -mediated phosphorylation of the ERK （ 1.19± 0.08 vs, 0.55 ± 0.04, t = 6.99, P = 0.0020）, p38 （ 1.18 ±0.04 vs. 0.66 ± 0.03, t = 11.20, P = 0.0020）, and .INK （ 1,11± 0.01 vs. 0.84 ± 0.04, t = 6.53, P = 0.0030）, compared with the TGF-β1-stimulated WT group. Fstll-deficient fibroblasts showed reduced alpha-smooth muscle actin （α-SMA） expression （0.70 ± 0.06 vs. 1.28 ±0.11, t = 4.65, P = 0.0035, compared with the untreated WT group; 1.40 ± 0.05 vs. 1.76± 0.02, t = 6.31, P = 0.0007; compared with the TGF-β1-treated WT group）. Compared with the corresponding condition in the control group, the TGF-β1/FSTL 1-mediated α-SMA expression was significantly suppressed by pretreatment with an inhibitor of p38 （0.73± 0.01 vs. 1.13 ± 0.10, t = 3.92, P = 0.0078） and JNK （0.78 ± 0.03 vs. 1.08 ± 0.06, t = 4.40,P = 0.0046） signaling. The proliferation of mouse lung fibroblast cells （MLgs） significantly decreased after treatment of an inhibitor of p38 （0.30 ±0.01 vs. 0.46 ±0.03, t = 4.64, P = 0.0009）, JNK （0.30 ± 0.01 vs. 0.49 ± 0.01, t = 12.84, P = 0.0001）, and Smad2/3 （0.18 ± 0.02 vs. 0.46 ±0.02, t = 12.69, P = 0.0001） signaling compared with the dimethylsulibxide group. The migration and invasion cells of MLgs significantly decreased in medium pretreated with an inhibitor of p38 （70.17 ±3.28 vs. 116.30 ± 7.11, t = 5.89, P = 0.0042 for the migratory cells; 19.87 ± 0.84 vs. 32.70 i 0.95, t =10.14, P = 0.0005 for the invasive cells）, JNK （72.30 ±3.85 vs. 116.30 ± 7.11, t = 5.44, P = 0.0056 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 11.00, P = 0.0004 for the invasive cells）, and Smad2/3 （64.76 ± 1.41 vs. 116.30 ± 7.11, t = 7.11, P = 0.0021 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ±0.95, t = 13.29, P = 0.0002 for the invasive cells） signaling compared with the corresponding condition in the dimethylsulfoxide group. Conclusion： FSTL1 affects lung fibroblast differentiation, proliferation, migration, and invasion through p38 and JNK signaling, and in this way, it might influence the development of PF.展开更多
Background:We previously showed that the expression of follistatin-like protein 1(FSTL1)was significantly down-regulated in metastatic clear-cell renal cell carcinoma(ccRCC).In this study,we aimed to characterize the ...Background:We previously showed that the expression of follistatin-like protein 1(FSTL1)was significantly down-regulated in metastatic clear-cell renal cell carcinoma(ccRCC).In this study,we aimed to characterize the role of FSTL1 in the development of ccRCC.Methods:The effects of FSTL1 on cell activity and cell cycle were investigated in ccRCC cell lines with altered FSTL1 expression.Gene expression microarray assays were performed to identify the major signaling pathways affected by FSTL1 knockdown.The expression of FSTL1 in ccRCC and its effect on postoperative prognosis were estimated in a cohort with 89 patients.Results:FSTL1 knockdown promoted anchorage-independent growth,migration,invasion,and cell cycle of ccRCC cell lines,whereas FSTL1 overexpression attenuated cell migration.FSTL1 knockdown up-regulated nuclear factor-κB(NF-κB)and hypoxia-inducible factor(HIF)signaling pathways,increased epithelial-to-mesenchymal transition,up-regulated interleukin-6 expression,and promoted tumor necrosis factor-α-induced degradation of NF-κB inhibitor(IκBα)in ccRCC cell lines.FSTL1 immunostaining was selectively positive in epithelial cytoplasm in the loop of Henle,and positive rate of FSTL1 was significantly lower in ccRCC tissues than in adjacent renal tissues(P<0.001).The mul-tivariate Cox regression analysis showed that the intratumoral FSTL1 expression conferred a favorable independent prognosis with a hazard ratio of 0.325(95%confidence interval 0.118-0.894).HIF-2αexpression was negatively cor-related with FSTL1 expression in ccRCC specimens(r=−0.229,P=0.044).Intratumoral expression of HIF-2α,rather than HIF-1α,significantly predicted an unfavorable prognosis in ccRCC(log-rank,P=0.038).Conclusions:FSTL1 plays a tumor suppression role possibly via repressing the NF-κB and HIF-2αsignaling pathways.To increase FSTL1 expression might be a candidate therapeutic strategy for metastatic ccRCC.展开更多
基金supported by the National Key Research and Development Program of China(2021YFA0805100)the National Natural Science Foundation of China(81830013,81770241,81970363,82000362,92268202,81170271,81370370,81490531,81670392,81600382)+6 种基金the National Natural Science Foundation of China Distinguished Young Scholar Grant(81325001)“973 Project”from the Ministry of Science and Technology of China(2009CB522104)Guangdong Basic and Applied Basic Research Foundation,China(2019B1515120092)the Science and Technology Planning Project of Guangzhou,China(202103000016)the Changjiang Scholars Program from the Ministry of Education of Chinathe Sun Yat-sen University Clinical Research 5010 Programthe Program of National Key Clinical Specialties。
文摘Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury(ALI), including acute respiratory distress syndrome(ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles(eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of e EVs after cardiopulmonary bypass, remains unclear. Plasma plasminogenactivated inhibitor-1(PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice(C57BL/6,Toll-like receptor 4 knockout(TLR4^(-/-))) and inducible nitric oxide synthase knockout(iNOS^(-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3(JAK2/3)-signal transducer and activator of transcription 3(STAT3)-interferon regulatory factor 1(IRF-1)pathway, along with i NOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors(AG490 or S3I-201, respectively), and was relieved in TLR4-/-and iNOS-/-mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1(FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.
文摘Background： Follistatin-like I （FSTL 1） is a novel profibrogenic factor that induces pulmonary fibrosis （PF） through the transforming growth factor-beta 1 （TGF-[B 1 ）/Smad signaling. Little is known about its effects on PF through the non-Smad signaling, like the mitogen-activated protein kinase （MAPK） pathway. Therefore, this study aimed to investigate the role ofFSTL 1 in PF through the MAPK signaling pathway and its mechanisms in lung fibrogenesis. Methods： PF was induced in Fstll~ and wild-type （WT） C57BL/6 mice with bleomycin. After 14 days, the mice were sacrificed, and lung tissues were stained with hematoxylin and eosin; the hydroxyproline content was measured to confirm PF. The mRNA and protein level of FSTLI and the change of MAPK phosphorylation were measured by quantitative polymerase chain reaction and Western blotting. The effect of Fst11 deficiency on fibroblasts differentiation was measured by Western blotting and cell immunofluorescence. MAPK signaling activation was measured by Western blotting in Fst11＋/ and WT fibroblasts treated with recombinant human FSTLI protein. We pretreated mouse lung fibroblast cells with inhibitors of the extracellular signal-regulated kinase （ERK）, p38, and Jun N-terminal kinase （JNK） signaling and analyzed their differentiation, proliferation, migration, and invasion by Western blotting, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis, and transwell assays. The Student＇s t-test was used to compare the differences between two groups. Results： Fstll deficiency attenuated phosphorylation of the ERK, p38, and JNK signaling in bleomycin-induced fibrotic lung tissue 14 days after injury （0.67 ± 0.05 vs. 1.22 ± 0.03, t = 14.92, P = 0.0001; 0.41 ± 0.01 vs. 1.15 ± 0.07; t = 11.19; P = 0.0004; and 0.41 ± 0.01 vs. 1.07± 0.07, t = 8.92, P = 0.0009; respectively）, compared with WT lungs at the same time and in primary lung fibroblasts （0.82 ± 0.01 vs. 1.01 ±0.04, t = 4.06, P = 0.0150; 1.04 ±0.03 vs. 1.24 ± 0.03, t= 4.44, P = 0.0100： and 0.76 ±0.05 vs. 0.99± 0.05, t = 4.48, P = 0.0100; respectively）, compared with TGF-β1-stimulated WT group. Recombinant human FSTLI protein in lung fibroblasts enhanced TG F-β1 -mediated phosphorylation of the ERK （ 1.19± 0.08 vs, 0.55 ± 0.04, t = 6.99, P = 0.0020）, p38 （ 1.18 ±0.04 vs. 0.66 ± 0.03, t = 11.20, P = 0.0020）, and .INK （ 1,11± 0.01 vs. 0.84 ± 0.04, t = 6.53, P = 0.0030）, compared with the TGF-β1-stimulated WT group. Fstll-deficient fibroblasts showed reduced alpha-smooth muscle actin （α-SMA） expression （0.70 ± 0.06 vs. 1.28 ±0.11, t = 4.65, P = 0.0035, compared with the untreated WT group; 1.40 ± 0.05 vs. 1.76± 0.02, t = 6.31, P = 0.0007; compared with the TGF-β1-treated WT group）. Compared with the corresponding condition in the control group, the TGF-β1/FSTL 1-mediated α-SMA expression was significantly suppressed by pretreatment with an inhibitor of p38 （0.73± 0.01 vs. 1.13 ± 0.10, t = 3.92, P = 0.0078） and JNK （0.78 ± 0.03 vs. 1.08 ± 0.06, t = 4.40,P = 0.0046） signaling. The proliferation of mouse lung fibroblast cells （MLgs） significantly decreased after treatment of an inhibitor of p38 （0.30 ±0.01 vs. 0.46 ±0.03, t = 4.64, P = 0.0009）, JNK （0.30 ± 0.01 vs. 0.49 ± 0.01, t = 12.84, P = 0.0001）, and Smad2/3 （0.18 ± 0.02 vs. 0.46 ±0.02, t = 12.69, P = 0.0001） signaling compared with the dimethylsulibxide group. The migration and invasion cells of MLgs significantly decreased in medium pretreated with an inhibitor of p38 （70.17 ±3.28 vs. 116.30 ± 7.11, t = 5.89, P = 0.0042 for the migratory cells; 19.87 ± 0.84 vs. 32.70 i 0.95, t =10.14, P = 0.0005 for the invasive cells）, JNK （72.30 ±3.85 vs. 116.30 ± 7.11, t = 5.44, P = 0.0056 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 11.00, P = 0.0004 for the invasive cells）, and Smad2/3 （64.76 ± 1.41 vs. 116.30 ± 7.11, t = 7.11, P = 0.0021 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ±0.95, t = 13.29, P = 0.0002 for the invasive cells） signaling compared with the corresponding condition in the dimethylsulfoxide group. Conclusion： FSTL1 affects lung fibroblast differentiation, proliferation, migration, and invasion through p38 and JNK signaling, and in this way, it might influence the development of PF.
基金supported by the National Key Basic Research Program(973 program)(2015CB554000 to GC)the National Natural Science Foundation of China(81520108021 and 91529305 to G.C.,81672518 and 81101928 to X.T.)the Three-year Action Plan on Public Health,Phase IV,Shanghai,China(15GWZK0801 to GC).
文摘Background:We previously showed that the expression of follistatin-like protein 1(FSTL1)was significantly down-regulated in metastatic clear-cell renal cell carcinoma(ccRCC).In this study,we aimed to characterize the role of FSTL1 in the development of ccRCC.Methods:The effects of FSTL1 on cell activity and cell cycle were investigated in ccRCC cell lines with altered FSTL1 expression.Gene expression microarray assays were performed to identify the major signaling pathways affected by FSTL1 knockdown.The expression of FSTL1 in ccRCC and its effect on postoperative prognosis were estimated in a cohort with 89 patients.Results:FSTL1 knockdown promoted anchorage-independent growth,migration,invasion,and cell cycle of ccRCC cell lines,whereas FSTL1 overexpression attenuated cell migration.FSTL1 knockdown up-regulated nuclear factor-κB(NF-κB)and hypoxia-inducible factor(HIF)signaling pathways,increased epithelial-to-mesenchymal transition,up-regulated interleukin-6 expression,and promoted tumor necrosis factor-α-induced degradation of NF-κB inhibitor(IκBα)in ccRCC cell lines.FSTL1 immunostaining was selectively positive in epithelial cytoplasm in the loop of Henle,and positive rate of FSTL1 was significantly lower in ccRCC tissues than in adjacent renal tissues(P<0.001).The mul-tivariate Cox regression analysis showed that the intratumoral FSTL1 expression conferred a favorable independent prognosis with a hazard ratio of 0.325(95%confidence interval 0.118-0.894).HIF-2αexpression was negatively cor-related with FSTL1 expression in ccRCC specimens(r=−0.229,P=0.044).Intratumoral expression of HIF-2α,rather than HIF-1α,significantly predicted an unfavorable prognosis in ccRCC(log-rank,P=0.038).Conclusions:FSTL1 plays a tumor suppression role possibly via repressing the NF-κB and HIF-2αsignaling pathways.To increase FSTL1 expression might be a candidate therapeutic strategy for metastatic ccRCC.
