| BackgroundPathological cardiac remodeling which is mainly characterized by enlargement and apoptosis of myocyte and cardiac fibrosis, eventually lead to heart failure. Cardiac fibrosis is an accumulation of excessive extracellular matrix proteins (ECM) in the myocardium and is associated with a decreased extent of microvasculature and increased stiffness, initially associated with diastolic dysfunction that frequently progresses to systolic dysfunction.While many previous studies have found that except the resident fibroblasts, cardiac fibroblasts are a heterogeneous population in the development of cardiac disease, and are likely derive from various distinct tissue niches such as endothelial, epcardium, fibrocytes from the bone marrow, monocytes and pericytes. Since the molecular mechanisms have not been clearly elucidated, a better understanding of the factors that regulate cardiac fibrosis could reveal potential therapeutic targets for treating cardiac remodeling.TLR5 is a member of Toll like receptors family, it expressed not only in immune cells (monocytes/macrophages, immature dendritic cells) but also in nonimmune cells, including cardiomyocytes and vascular endothelial cells. Recent researches demonstrated that, in addition to sensing of pathogens, TLRs are now demonstrated to sense host ligands as part of a wider role in the monitoring of danger signals. Researches have indicated that TLR5 can trigger cardiac innate immune responses and caused acute contractile dysfunction when combined with its ligand flagellin, and TLR5 can promote Epithelial-mesenchymal transition (EMT) in alveolar epithelial cells during pulmonary fibrosis. However, the role of TLR5 in the pressure overload-induced cardiac remodeling is still not elaborated. MethodsPart one:We used a model of aortic banding (AB)-induced cardiac remodeling, and detect the protein and mRNAexpression of TLR5. After AB for indicated time points (0W?1W?4W?8W), compared the expression of TLR5 with the sham operation group;Part two:Myocardial cells of newborn SD rats and H9c2 cardiomyocytes were both stimulated by 1 ?M Angiotensin ? (Ang ?) for 24h, detect the TLR5 expression before and after the stimulus; Human Umbilical Vein Endothelial Cells (HUVEC-12) were stimulated by lOng/ml TGF-?1 for 72h to induce endothelial-to-mesenchymal transition (EndMT), also detect the TLR5 expression before and after the stimulus; Part three:TLR5 knockout (KO) mice and wild type (WT) mice were used in this part. The approach for mice modeling is the same as part one. Heart weight/body weight (HW/BW), lung weight/body weight (LW/BW) and heart weight/tibial length (HW/TL) were used to assess weight change of heart and lung of mice; the picture of whole heart was used to assess the volume of heart, HE and WGA staining was used to evaluate the cross-sectional area (CSA) of cardiomyocytes; echocardiography was used in the assessment of cardiac function; PSR staining was used to evaluate the collagen volume of LV. Real time-PCR was used to detect the mRNA expression of cardiac hypertrophy and fibrosis;Part four:TLR5 knockout (KO) mice and wild type (WT) mice were used in this part. The approach for mice modeling is the same as part one. After C57 mice received AB for the indicated time points (0W?1W?2W?4W?8W), compared the expression of inflammatory factors (IL-1? IL-6?TNF?) with the sham operation group; 8W later, Real time-PCR was used to detect the mRNA expression of inflammatory factors and markers which represented the cardiac inflammatory phenotype triggered and expanded; immunofluorescence staining was used to detect the macrophages infiltration;Part five:(1) TLR5 knockout (KO) mice and wild type (WT) mice were used in this part. The approach for mice modeling is the same as part one. After WT mice received AB for the indicated time points (0W?1W?2W?4W?8W), compared the mRNA expression CD31 and VEGF; Western Blot was used to detect CD31, a-SMA, vimentin and the phosphorylation of Smad2/Smad3; immunofluorescence staining was used to detect the change of CD31/a-SMA and CD31/Collagen ?; Real time-PCR was used to detect the exression of transcription factors which play the key role during EndMT; (2) HUVEC-12 with and without Ad-TLR5 transfection was used in this part, different groups were stimulated by TGF-?1 for 72h, Cell morphological changes were observed under light microscope; Western Blot was used to detect CD31, a-SMA and vimentin; immunofluorescence staining was used to detect the change of CD31/vimentin; the migration ability of different groups was obtained by an in vitro scratch wound assay.ResultsPart one:The level of TLR5 protein and mRNA were both highly increased 1 week after AB compared with the sham-operated group, increased expression was also found at 4 and 8 weeks after AB with particularly high expression at 1 week;Part two:TLR5 was expressed in all of the three cell lines, but there was no evident change of the TLR5 mRNA in the neonatal rat cardiac myocytes and TLR5 protein in the H9c2 cardiomyoblasts induced by Ang ? despite hypertrophic response was evident; TLR5 mRNA and protein expression were both upregulated when the HUVEC-12 were treated with lOng/ml TGF-?1 for 3 consecutive days;Part three:Results of the HW/BW, LW/BW and HW/TL ratios and the cardiomyocyte cross sectional area (CSA) were all strikingly decreased in the AB induced TLR5 KO mice compared to the WT mice, the gross hearts, H&E and WGA staining results also confirmed the role of TLR5 deficiency on cardiac remodeling; after 8 weeks of AB, TLR5 KO mice demonstrated significant attenuation of wall thickness, chamber dilation, myocardial compliance and hemodynamics compared with the WT group; the mRNA expression of cardiac remolding markers demonstrated lower levels of ANP, BNP and (3-MHC while higher levels of a-MHC in AB-induced TLR5 deficiency mice compared with the wild-type mice; AB-induced cardiac fibrosis (perivascular and interstitial) was significantly lower in global TLR5 KO mice versus WT, Our data demonstrated that these indicators of tissue mRNA expression [collagen la, collagen III?, fibronectin, connective tissue growth factor (CTGF), vimentin, a-SMA, TGF-?1, fibroblast specific protein (FSP1)] were all reduced in TLR5 KO mice;Part four:Sham-operated mice exhibited low level mRNA expression of IL-1, IL-6 and TNF-a, After 1 week of AB, mRNA expression of IL-1, IL-6 and TNF-a in WT myocardial were all upregulated, peaking at 2 weeks, and gradually reduced.8 weeks after AB, it was still statistically higher in the AB group compared with the Sham-operated group, while, in the TLR5 deficiency group subject to AB, pro-inflammatory cytokines and MIP-2 mRNA expression were significantly lower than WT, immunofluorescence assays also demonstrated that macrophages infiltration was lower in TLR5 deficiency group; there was an increasing mRNA expression of MPO, TREM-1and its ITAM-containing adaptor DAP12 in myocardium in the WT AB group, while in the AB-induced TLR5 KO group, this phenomenon was significantly reversal.Part five:After 1 week of AB, significant CD31 and vascular endothelial growth factor (VEGF) mRNA upregulation was noted in the pressure-overload hearts and gradually increased, peaking at 4 weeks, and reduced at 8 weeks, we further found that CD31 mRNA expression levels were positively correlated with VEGF mRNA expression levels in the pressure-overload hearts; 8 weeks after AB, WB demonstrated that myocardium of TLR5 deficiency had significantly higher levels of CD31 than WT while levels of a-SMA and vimentin and were significantly lower; It was also confirmed by co-localization of CD31/a-SMA and CD31/Collagen?; p-Smad2 and p-Smad3 were both decreased in the AB-induced TLR5 deficiency mice compared with the WT group; Our results also showed that some transcription factors which play an important role during EndMT like snail, snail2, TWIST1, TWIST2, N-cadherin were all reduced in TLR5 deficiency mice; In basal-medium cultures, the HUVEC-12 arrayed into a cobblestone-like structure, and gradually transformed into fusiform structure when stimulated by TGF-?1 for 3 days, the situation will get worse when treated the HUVEC-12 transfected with Ad-TLR5 by TGF-?1, while, there was no effect on cell structure when added Ad-TLR5 only; The results of WB demonstrated that, HUVEC-12 stimulated by TGF-?1 had significantly lower levels of CD31 than the control group while levels of a-SMA and vimentin were significantly higher. The situation will get worse when treated the HUVEC-12 transfected with Ad-TLR5 by TGF-?1, CD31 level was further reduced while a-SMA and vimentin were further higher compared with the HUVEC-12 treated by TGF-?1 group. There was no effect on the change of cell marker when added Ad-TLR5 only, It was also confirmed by co-localization of CD31/vimentin; when treated the HUVEC-12 transfected with Ad-TLR5 by TGF-?1, cells migrated even faster than the only TGF-?1 induced group. Conclusion 1. Cardiac remodeling increase the expression of TLR5;2. TLR5 knockout attenuates cardiac hypertrophy, interstitial fibrosis and dysfunction induced by pressure overload;3. TLR5 knockout attenuates inflammation and macrophage infiltration induced by pressure overload;4. TLR5 promotes cardiac fibrosis induced by pressure overload through promoting endothelial-mesenchymal transition... |
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