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ObjectiveTo investigate the regulatory effect of circular RNA circ_0120051 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe expression of circ_0120051 and its host gene of solute carrier family 8 member A1（SLC8A1） mRNA in the myocardium of healthy organ donors （n=24） and heart failure （HF） patients （n=21） were assessed by real-time quantitative polymerase chain reaction （RT-qPCR） assay. RNA stability of circ_0120051 was identified by RNase R exonuclease digestion assay. The cytoplasmic and nuclear distribution of circ_0120051 in human cardiomyocyte AC16 was detected by RT-qPCR assay. The expression of fibrosis-related genes in mouse cardiac fibroblasts （mCFs） with adenovirus-mediated overexpression of circ_0120051 was detected by RT-qPCR and Western blot assay， respectively. The effect of overexpression of circ_0120051 on the migration activity of mCFs was evaluated by wound-healing assay. RNA co-immunoprecipitation （RIP） was conducted to detect the interaction between circ_0120051 and miR-144-3p. The binding site of miR-144-3p in the 3'-UTR of isocitrate dehydrogenase 2 （Idh2） mRNA was identified by the dual luciferase reporter gene assay. ResultsCirc_0120051 was significantly up-regulated in the myocardium of HF patients， while the mRNA expression of its host gene SLC8A1 was not changed. Circ_0120051 was mainly located in the cytoplasm of human AC16 cells. Results of RNase R exonuclease digestion revealed that circ_0120051 possesses the characteristic stability of circular RNA compared to the linear SLC8A1 mRNA. Overexpression of circ_0120051 could inhibit the expression of fibrosis-related gene in mCFs and mCFs migration. RIP assay confirmed the specific interaction between circ_0120051 and miR-144-3p. Transfection of miR-144-3p mimic could efficiently promote the expression of fibrosis-related genes in mCFs and reverse the inhibitory effect of circ_0120051 on the fibrotic phenotype of mCFs. Results of the dual luciferase reporter gene assay confirmed the interaction between miR-144-3p and the 3'-UTR of Idh2. Transfection of miR-144-3p transcriptionally inhibited Idh2 expression， and overexpression of circ_0120051 enhanced IDH2 expression in mCFs. MiR-144-3p mimic and Idh2 small interfering RNA （siRNA） could consistently reverse the inhibitory effects of circ_0120051 on fibrosis-related genes expression in mCFs and mCFs migration. ConclusionsCirc_0120051 inhibits the fibrotic phenotype of cardiac fibroblasts via sponging miR-144-3p to enhance the target gene of IDH2 expression.
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ObjectiveTo investigate the effect of circSLC8A1_005 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe effect of adenovirus-mediated overexpression of circSLC8A1_005 on the expression of fibrosis-related genes， collagen type I alpha 1 chain （Col1a1）， collagen type Ⅲ alpha 1 chain （Col3a1） and smooth muscle actin alpha 2 （Acta2）， in mouse cardiac fibroblasts （mCFs） were detected. The proliferation and migration activities of mCFs were detected by EdU and wound-healing assay， respectively. Dual luciferase reporter gene assay was performed to detect the activity of potential internal ribozyme entry site （IRES） in circSLC8A1_005. CircSLC8A1_005-translated protein， SLC8A1-605aa， and its intracellular distribution was identified by Western blot assay. The effect of SLC8A1-605aa protein on transcription activity of Sod2 gene was detected by the dual luciferase reporter gene assay. RNA binding protein immunoprecipitation （RIP） was utilized to verify the interaction between SLC8A1-605aa and superoxide dismutase 2 （Sod2） mRNA. Actinomycin D treatment was used to detect the effect of SLC8A1-605aa on Sod2 mRNA stability in mCFs. ResultsAn efficient adenovirus-mediated overexpression of circSLC8A1_005 was achieved in mCFs. The enforced expression of circSLC8A1_005 suppressed proliferation and migration of mCFs， and inhibited the expression of fibrosis-related genes in mCFs. The dual luciferase reporter gene assay revealed the activities of 2 IRES in circSLC8A1_005. Results of Western blot assay showed that circSLC8A1_005 could translate protein SLC8A1-605aa with the prospected molecular weight of 70 ku， which is predominantly distributed in the nucleus. Overexpression of the circSLC8A1_005 and SLC8A1-605aa could consistently inhibit the fibrotic phenotype of mCFs. SLC8A1-605aa could up-regulate superoxide dismutase 2 （Sod2） expression， but not at the transcriptional level. RIP assay indicated that SLC8A1-605aa could specifically interact with Sod2 mRNA， and the results of actinomycin D assay showed that SLC8A1-605aa could enhance the stability of Sod2 mRNA in mCFs. ConclusionCircSLC8A1_005 inhibits the fibrotic phenotype of cardiac fibroblasts via translating SLC8A1-605aa protein， and SLC8A1-605aa may be a potential target for the treatment of myocardial fibrosis.
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ObjectiveTo investigate the effect of myosin heavy chain 7 gene-derived miRNA-208b-3p on the fibrotic phenotype of cardiac fibroblasts. MethodsmiRNA chip array was performed to detect the dysregulated miRNAs in the myocardium of diabetic db/db mice and db/m control mice. Neonatal mouse ventricular cardiomyocytes （NMVCs） and cardiac fibroblasts （CFs） were isolated from C57BL/6 mice and cultured. Real-time quantitative PCR （RT-qPCR） was conducted to determine the expression of miR-208b-3p in mouse CFs and NMVCs subjected to angiotensinⅡ（AngⅡ） and high glucose plus glucose oxidase （G/Go） treatment， respectively. Cell counting kit 8（CCk8） assay， flow cytometry and determination of fibrosis-related protein， including COL1A1， COL3A1and α-SMA， were performed in mCFs transfected with miR-208b-3p. Dual luciferase reporter assay was performed to confirm the interaction between miR-208b-3p and the 3'-UTR of metal response element binding transcription factor 2 （Mtf2） and progesterone receptor membrane component 1（Pgrmc1）， respectively. The expressions of Mtf2 and Pgrmc1 at the mRNA and protein levels in mCFs after miR-208b-3p mimic transfection were determined using RT-qPCR and Western blot assay， respectively. The small interfering RNA （siRNA） was used to inhibit Mtf2 and Pgrmc1 expression in mCFs， and the effects of Mtf2 siRNA， Pgrmc1 siRNA and miR-208b-3p on fibrosis-related protein expression in mCFs were investigated. ResultsResults of miRNA chip array and RT-qPCR assay showed that miR-208b-3p was up-regulated in the myocardium of the diabetic db/db mice. miR-208b precursor and the host gene of Myh7 were consistently increased in db/db mice. miR-208b-3p and Myh7 mRNA were expressed in mCFs and NMVCs， but the levels of miR-208b-3p and Myh7 mRNA in NMVCs were much higher than those in mCFs. miR-208b-3p was up-regulated in mCFs and NMVCs subjected to Ang Ⅱ and G/Go treatment， respectively. miR-208b-3p could significantly enhance fibrosis-related protein， including COL1A1， COL3A1 and α-SMA， in mCFs， without affecting the proliferation activity and cell cycle distribution of mCFs. Dual luciferase reporter assay revealed the interactions of miR-208b-3p with the 3'-UTR of Mtf2 and Pgrmc1. The results of RT-qPCR and Western blotting confirmed that miR-208b-3p inhibited Mtf2 and Pgrmc1 expression at the post- transcriptional level. Transfection with miR-208b-3p mimic， Mtf2 siRNA and Pgrmc1 siRNA could consistently enhance the fibrosis-related protein expression in the cardiac fibroblasts. ConclusionsmiR-208b-3p enhances fibrosis-related gene expression by targeting Mtf2 and Pgrmc1in mCFs.
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A small proportion of mononuclear diploid cardiomyocytes (MNDCMs), with regeneration potential, could persist in adult mammalian heart. However, the heterogeneity of MNDCMs and changes during development remains to be illuminated. To this end, 12 645 cardiac cells were generated from embryonic day 17.5 and postnatal days 2 and 8 mice by single-cell RNA sequencing. Three cardiac developmental paths were identified: two switching to cardiomyocytes (CM) maturation with close CM-fibroblast (FB) communications and one maintaining MNDCM status with least CM-FB communications. Proliferative MNDCMs having interactions with macrophages and non-proliferative MNDCMs (non-pMNDCMs) with minimal cell-cell communications were identified in the third path. The non-pMNDCMs possessed distinct properties: the lowest mitochondrial metabolisms, the highest glycolysis, and high expression of Myl4 and Tnni1. Single-nucleus RNA sequencing and immunohistochemical staining further proved that the Myl4+Tnni1+ MNDCMs persisted in embryonic and adult hearts. These MNDCMs were mapped to the heart by integrating the spatial and single-cell transcriptomic data. In conclusion, a novel non-pMNDCM subpopulation with minimal cell-cell communications was unveiled, highlighting the importance of microenvironment contribution to CM fate during maturation. These findings could improve the understanding of MNDCM heterogeneity and cardiac development, thus providing new clues for approaches to effective cardiac regeneration.
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Animals , Mice , Diploidy , Heart , Myocytes, Cardiac/metabolism , Cell Communication , Gene Expression Profiling , Mitochondria , Regeneration , Mammals/geneticsABSTRACT
Objective To investigate the effects of Smad7 knock down by lentivirus on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen secretion in vitro. Methods The primary cardiac fibroblasts were separated from the hearts of ten SD rats and identified by immunohistochemical method. The lentivirus transfection knocked down the expresson of Smad7 in cardiac fibroblasts, Western blotting was used to detect the efficiency of Smad7 knock down by lentivirus. The proliferation of cardiac fibroblasts was quantified by real-time unlabeled cell analyzer. Cell migration was evaluted by cell wound scratch assay. Western blotting was used to detect expression of α-smooth muscle actin（α-SMA） and collagen Ⅰ（Col Ⅰ）. Results Myocardial fibroblasts were successfully cultured and identified by immunocytochemical methods. The multiplicity of infection（MOI） that lentivirus transduction of myocardial fibroblasts was 100. After lentivirus transduction, 88.33% myocardial fibroblasts expressed green fluorescent protein, showed that the lentivirus could significantly reduce the protein expression of Smad7. Smad7 deficiency decreased the proliferation and migration of cardiac fibroblasts, increased the protein expression of α-SMA and decreased collagen secretion. The results indicated that Smad7 deficiency significantly down-regulated the proliferation and migration of cardiac fibroblasts, increased α-SMA protein expression and reduced ColⅠ protein expression. Conclusion Smad7 deficiency can significantly change the cardiac fibroblasts function, that is related to the pathological mechanism that lead to myocardial fibrosis
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Objective:To investigate the implication of micro RNA-21(miR-21) in Endostar combined with X-ray irradiation of cardiac fibroblasts (CF).Methods:Rat CFs were used in this experiment and been divided into the blank control group, 10 Gy X-ray irradiation group, Endostar group, 10 Gy X-ray+ Endostar group, 10 Gy X-ray+ Endostar+ NC mimic group (negative control 1), 10 Gy X-ray+ Endostar+ miR-21 mimic group, 10 Gy X-ray+ Endostar+ NC inhibitor group (negative control 2) and 10 Gy X-ray+ Endostar+ miR-21 inhibitor group. The proliferation of CF was determined by Methyl thiazolyl tetrazolium (MTT) assay. The expression level of Collagen Ⅰ protein was analyzed by Western blot. The expression levels of Collagen Ⅰ and miR-21 mRNA were assayed by real-time quantitative polymerase chain reaction (q-PCR).Results:In the 10 Gy X-ray+ Endostar+ miR-21 mimic group, the CF proliferation, Collagen Ⅰ and miR-21 mRNA were increased significantly compared with those in the blank control group, 10 Gy X-ray+ Endostar group, and negative control group 1 (all P<0.05). In the 10 Gy X-ray+ Endostar+ miR-21 inhibitor group, the CF proliferation and expression levels of Collagen Ⅰ mRNA were decreased significantly compared with those in the blank control group, 10 Gy X-ray+ Endostar group and negative control group 2(all P<0.05). Conclusions:The CF proliferation and Collagen Ⅰ expression are increased when the expression level of miR-21 gene is simulated. When inhibiting the expression of miR-21 gene, the CF proliferation and Collagen Ⅰ expression are reduced.
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Objective To investigate the effect of fibroblast growth factor (FGF) on the proliferation and transdifferentiation of cardiac fibroblasts ( CFs ) into myofibroblasts ( MFs ). Methods Rat CFs were isolated and cultured, and then induced by FGF. CCK-8 was used to detect the cell activity and proliferation. Immunofluorescence and Western blotting were used to detect the expression of a smooth muscle actin ( α-SMA ) and collagen I ( Col I ). Results The expression and activation of α-SMA and Col I increased with the increase of CFs culture generation. The number of CFs induced by FGF did not increased significantly; the expression of α-SMA in CFs induced by FGF1 and FGF2 decreased, and the number of activated MFs decreased. Conclusion FGF family has no effect on the proliferation of CFs, but FGF1 and FGF2 can inhibit the activation of CFs and reduce the differentiation into MFs.
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Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the
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Humans , Fibroblasts , Fibrosis , Myocardium/pathology , RNA, Long Noncoding , Signal Transduction , Transforming Growth Factor beta1ABSTRACT
Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca homeostasis, and Ca signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts , and the cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.
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Excess activation of cardiac fibroblasts inevitably induces cardiac fibrosis. Emodin has been used as a natural medicine against several chronic diseases. The objective of this study is to determine the effects of emodin on cardiac fibrosis and the underlying molecular mechanisms. Intragastric administration of emodin markedly decreased left ventricular wall thickness in a mouse model of pathological cardiac hypertrophy with excess fibrosis induced by transaortic constriction (TAC) and suppressed activation of cardiac fibroblasts induced by angiotensin II (AngII). Emodin upregulated expression of metastasis associated protein 3 (MTA3) and restored the MTA3 expression in the setting of cardiac fibrosis. Moreover, overexpression of MTA3 promoted cardiac fibrosis; in contrast, silence of MTA3 abrogated the inhibitory effect of emodin on fibroblast activation. Our findings unraveled the potential of emodin to alleviate cardiac fibrosis upregulating MTA3 and highlight the regulatory role of MTA3 in the development of cardiac fibrosis.
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<p><b>Background</b>Myocardial ischemia injury is one of the leading causes of death and disability worldwide. Cardiac fibroblasts (CFs) have central roles in modulating cardiac function under pathophysiological conditions. Activating transcription factor 3 (ATF3) plays a self-protective role in counteracting CF dysfunction. However, the precise function of CF-specific ATF3 during myocardial infarction (MI) injury/repair remains incompletely understood. The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI.</p><p><b>Methods</b>Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0, 0.5, 1.0, 3.0, and 7.0 days postoperation. Model for MI was constructed in ATF3TGfl/flCol1a2-Cre+ (CF-specific ATF3 overexpression group, n = 5) and ATF3TGfl/flCol1a2-Cre- male mice (without CF-specific ATF3 overexpression group, n = 5). In addition, five mice of ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- were subjected to sham MI operation. Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining (myocardial fibrosis area was detected by blue collagen deposition area) at the 28 day after MI surgery in ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- mice received sham or MI operation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue. BrdU staining was used to detect fibroblast proliferation.</p><p><b>Results</b>After establishment of an MI model, we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs. Genetic overexpression of ATF3 in CFs (ATF3TGfl/flCol1a2-Cre+ group) resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction (41.0% vs. 30.5%, t = 8.610, P = 0.001) and increased fractional shortening (26.8% vs. 18.1%, t = 7.173, P = 0.002), which was accompanied by a decrease in cardiac scar area (23.1% vs. 11.0%, t = 8.610, P = 0.001). qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs, displaying pro-proliferation properties. BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin II stimuli (11.5% vs. 6.8%, t = 31.599, P = 0.001) or serum stimuli (31.6% vs. 20.1%, t = 31.599, P = 0.001). The 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h (P2: 91.6% vs. 71.8%, t = 8.465, P = 0.015) and 48 h (P3: 81.6% vs. 51.1%, t = 9.029, P = 0.012) after serum stimulation. Notably, ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.</p><p><b>Conclusions</b>We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.</p>
Subject(s)
Animals , Male , Mice , Activating Transcription Factor 3 , Physiology , Disease Models, Animal , Fibroblasts , Physiology , Fibrosis , Mice, Inbred C57BL , Myocardial Infarction , Myocardium , Ventricular RemodelingABSTRACT
Objective To investigate the effect of lentivirus-stromal cell-derived factor-1α-green fluorescent protein(LV-SDF-1α-GFP) on the cardiac fibroblasts, the optimum conditions of infection, the expression and secretion of the target protein. Methods The cardiac fibroblasts of neonatal rats were primarily isolated and cultured by differential adherence methods, and were observed and identifi with immunofluorescence. LV-SDF-1α-GFP with different titers and conditions was transfected into cardiac fibroblasts. The expression of fluorescence and the optimal transfection conditions were observed. LV-SDF-1α-GFP target gene virus and negative control C0N145 virus were transfected into cardiac fibroblasts. The growth curve was drawn, and the effect of transfection on the proliferation of cardiac fibroblasts was explored. The cardiac fibroblasts were transfected with the optimum transfection dose, and the expression of SDF-1α was detected by Dot-blotting. The measurement data underwent statistical analysis. Results There was no statistical difference between the cardiac fibroblasts with SDF-1α transfected lentivirus and without no-transfected SDF-1α lentivirus. The peak of the expression of SDF-1α appeared in culture day 4 and statistical analysis showed significantly difference (P<0.05). Conclusion The LV-SDF-1α-GFP vector is of higher transfection efficiency to cardiac fibroblasts with the both low cytotoxicity and ability of secreting SDF-la protein.
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Objective To investigate the effect of microRNA-1 (miR-1) on the fibrosis of cardiac fibroblasts induced by high glucose. Methods The primary cultured fibroblasts from 1-3 days old Sprague-Dawley (SD) rats, were randomly divided into 4 groups (n = 3): normal glucose + lentivector-vehicle (CON+Lv-Vehicle group), normal glucose + lentivector-miR-1 (CON+Lv-miR1 group), high glucose + lentivector-vehicle (HG+Lv-Vehicle group), high glucose + lentivector-miR-1 (HG+Lv-miR1 group). Fibroblasts were cultured in glucose concentration 5.5 mmol/L and 25 mmol/L DMEM culture, and were injected lentiviral vector carrying miR-1 silencer sequence or the same volume of lentiviral vector. After 12 hours, the medium was replaced with fresh complete medium. After 3 days when transfection efficiency was up to 90%, the cellular miR-1 content was detected by real-time reverse transcription-polymerase chain reaction (qRT-PCR). The secretion of collagen Ⅰ and Ⅲ were measured by enzyme linked immunosorbent assay (ELISA). Expression of collagen Ⅰ and Ⅲ, matrix metalloproteinase 2 and 9 (MMP-2, MMP-9), autophagy related protein LC3B-Ⅱ, P62/SQSTM1 and Cathepsin D were assessed by Western Blot. Results Compared with the CON+Lv-Vehicle group, the content of miRNA in the CON+Lv-miR1 group had no statistical significance. Compared with the CON+Lv-Vehicle group, high glucose increased the amount of miR-1 (2-ΔΔCt: 1.82±0.17 vs. 1.00±0.04), collagen Ⅰ and Ⅲ secretion (mg/L: 14.55±0.33 vs. 7.28±0.22, 157.50±13.22 vs. 61.25±8.54) and expression (gray value: 432.35±56.00 vs. 100.00±15.00, 320.35±47.00 vs. 100.00±15.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 249.0±21.0 vs. 100.0±15.0, 142.3±20.0 vs. 100.0±16.0, 178±19 vs. 100±14, 378.3±20.0 vs. 100.0±15.0), decreased the expression of lysosomal associated protein Cathepsin D (gray value: 60±14 vs. 100±10), and the differences were statistically significant (all P < 0.01). Compared with the HG+Lv-Vehicle group, the amount of miR-1 in the HG+Lv-miR1 group was significantly decreased (2-ΔΔCt: 1.21±0.10 vs. 1.82±0.17), collagen Ⅰ and Ⅲ secretion (mg/L: 10.68±0.54 vs. 14.55±0.33, 87.25±13.55 vs. 157.50±13.22) and expression (gray value: 179.41±45.00 vs. 432.35±56.00, 173.41±50.00 vs. 320.35±47.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 172.0±23.0 vs. 249.0±21.0, 90.0±17.0 vs. 142.3±20.0, 138±15 vs. 178±19, 265.0±17.0 vs. 378.3±20.0) in the HG+Lv-miR1 group were decreased and the expression of lysosomal associated protein Cathepsin D was higher (gray value: 110±17 vs. 60±14), and the differences were statistically significant (all P < 0.05). Conclusions The expression of miRNA-1 was up-regulated in cardiac fibroblasts cultured in high glucose, and miRNA-1 silencing inhibited cardiac fibroblast induced fibrosis in high glucose. The mechanism may be related to the recovery of autophagy flux, up-regulation of Cathepsin D expression and inhibition of collagen production.
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Objective To investigate collagen metabolism modulation of Intermedin 1-53 ( IMD1-53 ) on angiotensin Ⅱ( AngⅡ)-induced rat cardiac fibroblasts .Methods SD neonatal rat cardiac fibroblasts were cultured and divided them into control group, AngⅡ +different concentrations IMD1-53 groups.ⅠandⅢcollagen expression in cardiac fibroblasts were measured by Westem blot , IMD1-53 receptor-like receptor ( calcitonin receptor-like receptor , CRLR) and transforming growth factor-β( TGF-β) mRNA expression were exammed by real-time PCR.Results IMD1-53 significantly inhibited AngⅡ-induced collagen synthesis in cardiac fibroblasts , and this effect was more ob-vious with the increase of IMD 1-53 ( P<0.01 ,P<0.05 ) .Similar phenomenon was recorded in TGF-βexpression in cardiac fibroblasts ( P<0.05 ) .Conclusions IMD1-53 inhibited collagen synthetic in cardiac fibrosis induced by AngⅡ, down-regulated TGF-βexpression.These may relate to IMD1-53 anti myocardial fibrosis.
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Notch signaling is an evolutionarily ancient, highly conserved pathway important for deciding cell fate, cellular development, differentiation, proliferation, apoptosis, adhesion, and epithelial-to-mesenchymal transition. Notch signaling is also critical in mammalian cardiogenesis, as mutations in this signaling pathway are linked to human congenital heart disease. Furthermore, Notch signaling can repair myocardial injury by promoting myocardial regeneration, protecting ischemic myocardium, inducing angiogenesis, and negatively regulating cardiac fibroblast-myofibroblast transformation. This review provides an update on the known roles of Notch signaling in the mammalian heart. The goal is to assist in developing strategies to influence Notch signaling and optimize myocardial injury repair.
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Animals , Humans , Heart Diseases/metabolism , Myocardium/metabolism , Receptors, Notch/metabolism , Signal Transduction/physiology , Apoptosis/physiology , Cell Differentiation/physiology , Heart Diseases/physiopathology , Ischemic Preconditioning, Myocardial , Mammals , Regeneration/physiologyABSTRACT
Cardiac fibroblasts play a pivotal role in cardiac remodeling.In response to various pro-fibrotic stimuli,such as pro-inflammatory cytokines,anoxia-reoxygenation and pressure overload as well as aging,cardiac fibroblasts undergo proliferation,migration and activation,leading to the accumulation of extracellular matrix components and increased thickness and stiffness of heart.The ligands for peroxisome proliferator-activated receptor γ,especially thiazolidinediones,modulate the function of cardiac fibroblasts and the progression of cardiac remodeling,especially under pathological conditions.Unfortunately these agents have not been found to be consistently beneficial in heart failure.Although the precise intracellular signaling pathways are not fully understood,existing evidence strongly supports the involvement of oxidative stress and related signaling pathways.Further,peroxisome proliferator-activated receptor γ and lectin-like oxidized low-density lipoprotein receptor-1 together play critical roles in thiazolidinediones-modulated cardiac fibroblast dysfunction.
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Objective To explore the effects of the β3 adrenergic receptor (β3-AR) agonist BRL37344 on cardiac fibroblast proliferation and collagen fiber hyperplasia in Wistar rats by promoting the phosphaticlylinositol 3 kinase-protein kinase B(PI3K-Akt) signal transduction pathway. Method Cardiac fibroblasts(CFbs) were isolated from 1 - 3 day-old Wistar rats under the sterile environment in the laboratory of the First Affiliated Hospital of Hasbin Medical University, by using enzyme digestion and an modified technique of differential anchoring velocity.The cultured myocardial cells were randomly (random number) divided into five groups. ① In blank group, rats were not treated with drug; ②in BRL group, rats were treated with BRL37344; ③in LY group, rats were treated with LY294002(PI3K antagonist) for one hour before treated with BRL37344;④in Akt-Ⅰ group,rats were treated with Akt-Ⅰ (Akt antagonist) for one hour before treated with BRL37344;⑤in L-A group, rats were treated with LY294002 and Akt-Ⅰ for one hour before treated with BRL37344. MTT colorimetric method and RT-PCR were used to observe the role of β3-AR agonist with or without PI3K antagonist and/or Akt antagonist in cardiac fibroblast proliferation (CFP) and collagen fiber hyperplasia (CFH). Comparisons between groups were made by one-way ANOVA and Tukey test. Results ①β3-AR was present in the CFbs. ②Compared with BRL group, the CFP and CFH in LY and Akt-Ⅰ groups were lower (P ＜0.01) and those in L-A group were lowest (P ＜ 0.01). ③Compared with blank group,the expressions of type Ⅰ and type Ⅲ fiber mRNA obliviously increased in BRL group (P ＜ 0.01),and at 48 hours,the expressionrs reached peak. At 48 hours,compared with BRL group,the expressions in LY and Akt-Ⅰ groups were lower, and were lowest in L-A group ( P ＜0.01). Conclusions BRL37344 promotes cardiac fibroblast proliferation and expressions of type Ⅰ and Ⅲ collagen fiber mRNA by activating the PI3K-Akt signal transduction pathway.
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Objective To observe the effects of ACE inhibitor enalaprilat(Ena) and protein kinase C(PKC) inhibitor chelerythrine(Chele)on proliferation,collagen Ⅰ,cell cycle,PKC and cyclinD1 protein expression of neonatal cardiac fibroblasts(CFb) and to probe its molecular mechanism.Methods CFb of neonatal Wistar rats were divided into control group,AngⅡ group,Chele+AngⅡ group,Chele+AngⅡ+Ena group and AngⅡ+Ena group.CFb were isolated by trypsin digestion method.MTT colorimetric assay was adopted to evaluate cell proliferation,immunocytochemical staining(IC) was used to measure collagen Ⅰ content,Western blotting and flow cytometry were used to detect PKC,cyclinD1 and cell cycle respectively.Results Compared with AngⅡ group,the MTT value decreased dramatically(P
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Objective To investigate the effects of activator protein-1(AP-1)decoy oligodeoxynucleotides (ODNs)on the myocardial fibrosis induced by angiotension Ⅱ(Ang Ⅱ)in vitro.Methods CFs of neonatal Spra- gue-Dawley(SD)rats were isolated by trypsin digestion method.CFs were co-cultured with 10~(-7)mol/L Ang Ⅱ in the presence of different concentration of activator protein-1(AP-1)decoy ODNs or mutational AP-1 decoy ODNs for 24 h.Collagen synthesis was assessed by hydroxyproline and the mRNA expression of collagen Ⅰ,collagen Ⅲ.Results The concentration of hydroxyproline increased significantly after treated by 10~(-7)mol/L Ang Ⅱ;decoy ODNs on the range of 10-200 nmol/L dose dependently decrease synthesis of collagen;Ang Ⅱ stimulates mRNA expression of collagen Ⅲ(1.04?0.07 vs 1.63?0.071,n=3,P
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Objective: To study the influence of Centipede Acidic Protein(CAP) on the proliferation and collagen synthesis of cultured neonatal rat cardiac fibroblasts(CFb) induced by angiotensinⅡ(AngⅡ),and to explore the mechanisms of CAP on cardiac fibrosis.Methods: Neonatal rat cardiac fibroblasts were treated with AngⅡ to produce fibrosis model.The effects of CAP on proliferation of CFb were observed by MTT colorimetric assay,synthesis of collagen was observed by the hydroxyproline concentration.The NO contents were measured by Nitric acid reductase method.The c-myc expression was examined by semi-quantitative RT-PCR analysis.Results: Compared with that of control group,the proliferation,collagen synthesis and the levels of c-myc mRNA expression of CFb in the model group increased,while the NO contents decreased obviously(P