| Background:Flavin containing monooxygenase 2(FM02)is a member of the flavin monooxygenase(FMO)family,which uses NADP+ and FAD as co-enzymes and prosthetic groups to metabolize xenobiotics.It has characteristics that are different from those of other members of the family,for it is expressed in the lungs which is easily associated with fibrosis,but not in the liver,an organ which is responsible for drug metabolism.Its role apart from metabolic-related function and hidden mechanisms need further elucidation.In our previous study,gene array was done on three SD rat groups(sham,MI,and MI+ human endometrium stem cell transplantation).We observed that FMO2 were significantly downregulated after MI,and will be compensated after transplantation of endometrium stem cells.What is more,we validated that FM02 is majorly expressed in cardiac fibroblasts rather than in cardiomyocytes.To summarize,we consider that FM02 might regulate fibrosis procedure subsequent to myocardial infarction.Methods and results:Here we overexpressed FM02 in SD rats after myocardial infarction and observed that cardiac function was significantly rescued at 14 and 28 days after myocardial infarction than in the control group.As to pathological analysis,rat heart sections were stained by Sirius Red and MASSON trichrome staining,which showed significant reduction in scar circumference in FMO2-overexpression group than that in controls,as accompanied by decreased fibrotic area within non-infarcted zone.In addition,cardiomyocytes surviving is better within MI segment in FM02-overexpression group than in control group.To further confirm that FM02 only exerts its effect in fibroblasts after MI,we generated a FMO2 overexpression lentivirus specificly targeting fibroblast(LV-miR133a/la TS FMO2),and we were surprisingly that the heart function of SD rats injected with LV-miR133a/la TS FM02 showed significant improvement compared to rat received negative control lentivirus.Also,SD rats injected with LV-miR133a/1a TS FM02 exhibited less scar circumference ratio,lowered fibrosis area in non-infarcted zone,and augmented cardiomyocyte within MI segment.Interestingly,we found that the heart function of FM02-/-rats(generated by CRISPR-cas9 technology)was also significantly worsened than that of normal SD rats of the same age and sex.In in vitro experiments,we isolated cardiac fibroblasts(NRCF)from neonatal rats and stimulated by TGF-beta for 24 hours to mimic fibrotic progress in vivo after myocardial infarction.FM02-overexpressed group represents a significantly lower expression of fibrotic-activated proteins,such as Periostin.Collagenl than controls.Further experiments showed that in many signaling pathways that promote fibrosis,overexpression of FM02 mainly inhibits the phosphorylation of SMAD2/3 in their pro-fibrotic sites.In order to further study the medium that mediate this signaling pathway,we used NRCF with FM02 overexpression and 24 hours of TGF-? treating for mass spectrometry detection and found that a member of the cytochrome p450 superfamily-CYP2J3 can form a complex with FMO2,and the result is also confirmed by subsequent Co-immunoprecipitation experiments.What's more,we found that overexpression of CYP2J3 alone could also inhibit the expression of target genes and signaling pathways related to fibrosis.On the other hand,even if FM02 is overexpressed,when CYP2J3 is interfered,the phenotype of inhibition towards downstream fibrogenic gene expressions is reversed,and inhibition of the phosphorylation of SMAD2/3 is reversed as well.To summarize,we demonstrated that CYP2J3 is an essential factor mediating the inhibition of fibrosis induced by FM02 compensation when TGF-beta is stimulated.Conclusion:The compensation of FM02 after MI could significantly ameliorates remodeling process while binding with CYP2J3,which is able to dephosphorylate SMAD2/3 downstreaming of TGF-beta signaling pathway.This investigation provided a brand new therapeutic target towards fibrosis procedure subsequent to myocardial infarction. |
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