Calcitriol Suppressed Isoproterenol-induced Proliferation of Cardiac Fibroblasts via Integrinβ3/FAK/Akt Pathway

Objective Cardiac fibroblasts(CFs)proliferation and extracellular matrix deposition are important features of cardiac fibrosis.Various studies have indicated that vitamin D displays an anti-fibrotic property in chronic heart diseases.This study explored the role of vitamin D in the growth of CFs via an integrin signaling pathway.Methods MTT and 5-ethynyl-2′-deoxyuridine assays were performed to determine cell viability.Western blotting was performed to detect the expression of proliferating cell nuclear antigen(PCNA)and integrin signaling pathway.The fibronectin was observed by ELISA.Immunohistochemical staining was employed to evaluate the expression of integrinβ3.Results The PCNA expression in the CFs was enhanced after isoproterenol(ISO)stimulation accompanied by an elevated expression of integrin beta-3(β3).The blockade of the integrinβ3 with a specific integrinβ3 antibody reduced the PCNA expression induced by the ISO.Decreasing the integrinβ3 by siRNA reduced the ISO-triggered phosphorylation of FAK and Akt.Both the FAK inhibitor and Akt inhibitor suppressed the PCNA expression induced by the ISO in the CFs.Calcitriol(CAL),an active form of vitamin D,attenuated the ISO-induced CFs proliferation by downregulating the integrinβ3 expression,and phosphorylation of FAK and Akt.Moreover,CAL reduced the increased levels of fibronectin and hydroxyproline in the CFs culture medium triggered by the ISO.The administration of calcitriol decreased the integrinβ3 expression in the ISO-induced myocardial injury model.Conclusion These findings revealed a novel role for CAL in suppressing the CFs growth by the downregulation of the integrinβ3/FAK/Akt pathway.

Effect of aldosterone on the proliferation of rat cardiac fibroblasts

Objective:A cell model of cardiac fibroblasts proliferation induced by aldosterone was established to observe the effect of aldosterone on the proliferation of rat cardiac fibroblasts.Methods:Primary cardiac fibroblasts were cultured by trypsin digestion method and differential adhesion method,primary cardiac fibroblasts were sub-cultured by conventional digestion method,and the immunocytochemical assay was used to identify cardiac fibroblasts.The second-generation cardiac fibroblasts were randomly divided into five groups:standard control group,10-9 mol/L aldosterone(ALD1)group,10-8 mol/L aldosterone(ALD2)group,10-7 mol/L aldosterone(ALD3)group,and 10-6 mol/L aldosterone(ALD4)group.The viability of fibroblast cells in each group was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.Results:Vimentin staining assay showed that the cultured cells staining positive,and the purity of cultured mouse cardiac fibroblasts was 95%.The results of methyl thiazolyl tetrazolium showed that compared with the control group,the low concentration of aldosterone(10-9 mol/L)had no significant effect on the proliferation of normal cardiac fibroblasts.With the increase in the intensity of(10-8–10-6)mol/L,aldosterone could significantly promote the proliferation of cardiac fibroblasts.Moreover,there was no significant difference in absorbance value between the aldosterone group(10-6 mol/L)and the aldosterone group(10-7 mol/L)(P>0.05).The highest concentration of aldosterone group 10-7 mol/L promoted the proliferation of cardiac the optimum concentration was 10-7 mol/L.Conclusion:Aldosterone can promote the spread of cardiac fibroblasts in a specific concentration range.

Sodium tanshinone ⅡA sulfonate prevents radiation-induced damage in primary rat cardiac fibroblasts

This study investigated the effects of X-ray irradiation on primary rat cardiac fibroblasts(CFs) and its potential mechanism, as well as whether sodium tanshinone ⅡA sulfonate(STS) has protective effect on CFs and its possible mechanism. Our data demonstrated that X-rays inhibited cell growth and increased oxidative stress in CFs, and STS mitigated X-ray-induced injury. Enzyme-linked immuno-sorbent assay showed that X-rays increased the levels of secreted angiotensin Ⅱ(Ang Ⅱ) and brain natriuretic peptide(BNP). STS inhibited the X-ray-induced increases in Ang Ⅱ and BNP release. Apoptosis and cell cycle of CFs were analyzed using flow cytometry. X-rays induced apoptosis in CFs, whereas STS inhibited apoptosis in CFs after X-ray irradiation. X-rays induced S-phase cell cycle arrest in CFs, which could be reversed by STS. X-rays increased the expression of phosphorylated-P38/P38,cleaved caspase-3 and caspase-3 as well as decreased the expression of phosphorylated extracellular signal-regulated kinase 1/2(ERK1/2)/ERK 1/2 and B cell lymphoma 2(Bcl-2)/Bcl-2 associated X protein(BAX) in CFs, as shown by Western blotting. STS mitigated the X-ray radiation-induced expression changes of these proteins. In conclusion, our results demonstrated that STS may potentially be developed as a medical countermeasure to mitigate radiation-induced cardiac damage.

Transplantation of 5-azacytidine treated cardiac fibroblasts improves cardiac function of infarct hearts in rats

Background Cellular cardiomyoplasty by transplantation of various cell types has been investigated as potential treatments for the improvement of cardiac function after myocardial injury. A major barrier for the clinical application of cell transplantation is obtaining sufficiently large quantities of suitable cells. AIIogeneic cellular cardiomyoplasty may provide an alternative source of abundant, transplantable, myogenic cells by in vitro manipulation of cardiac fibroblasts using chemicals including 5-azacytidine. This study evaluated cardiomyogenic differentiation of cardiac fibroblasts, their survival in myocardial scar tissue, and the effect of the implanted cells on heart function. Methods Primary cardiac fibroblasts from neonatal rats were treated with 5-azacytidine （10 pmol/L） or control. Treatment of 5-azacytidine caused myogenic differentiation of cultured cardiac fibroblasts, as defined by elongation and fusion into multinucleated myotubes with sarcomeric structures as identified by electron microscopy, and positive immunostaining for cardiac specific proteins, troponin I and 13-myosin heavy chain （13-MHC） and the gap junction protein connexin 43. The myogenic cells （1.0x106） were transplanted into the infarcted myocardium 2 weeks after coronary artery occlusion. Results By 1 month after transplantation, the converted fibroblasts gave rise to a cluster of cardiac-like muscle cells that in the hearts occupied a large part of the scar with positive immunostaining for the myogenic proteins troponin I and 13-MHC. Engrafted cells also expressed the gap junction protein connexin 43 in a disorganized manner. There was no positive staining in the control hearts treated with injections of culture medium. Heart function was evaluated at 6 weeks after myocardial injury with echocardiographic and hemodynamic measurements. Improvement in cardiac function was seen in the hearts transplanted with the 5-azacytidine-treated cardiac fibroblasts which was absent in the hearts treated with control. Conclusion The 5-azacytidine has a unique capacity to induce myogenesis in cardiac fibroblasts in vitro and transplantation of cardiac-like muscle cells into ventricular scar tissue improves myocardial function.

Effects of Tetramethylpyrazine and Radix Salviae Miltiorrhizae on Collagen Synthesis and Proliferation of Cardiac Fibroblasts

Objective: To explore the effects of Tetramethylpyrazine (TMP) and Radix Salviae Miltiorrhizae (RSM) on collagen synthesis and proliferation of cardiac fibroblasts. Methods: Using collagenase and pancreatin digested rat cardiac tissue assay to isolate cardiac fibroblasts (FB). Different dosage of TMP, RSM and norepinephrine were used to study their effects on the collagen synthesis and proliferation of cultured cardiac FB. Results: Compared with the control group, moderate or high dosage TMP and RSM could significantly inhibit the collagen synthesis and the proliferation of cultured cardiac FB. Moreover, low-dose TMP (50 mg/L) and low-dose RSM (3 g/L) could antagonize the collagen synthesis and the proliferation of cultured cardiac FB stimulated by NE (500μg/L). Conclusion: Both TMP and RSM can inhibit the collagen synthesis and proliferation of cultured cardiac FB processes.The mechanisms of these effects might be correlated to their Ca++ antagonistic action. Original article on CJIM(Chin) 1998; 18(7): 423

Macrophage migration inhibitory factor suppresses angiotensinⅡ-induced expression of fibrosis-associated genes by inactivating Smad3 and activating Nrf2 in cardiac fibroblasts

Background Macrophage migration inhibitory factor （MIF） is a key pro-inflammatory cytokine, exhibiting antioxidant properties. However, the role of MIF in cardiac fibrosis is not well known. In the present study, the effects of MIF on Smad3 and Nrf2 signalings in cardiac fibroblasts were investigated. Methods Cardiac fibroblasts were isolated from 1-3 days old C57BL/6 mice, and the cardiac fibroblasts from passage 2 to 4 were used in this study. Expression of fibrosis-associated Collal, Col3al and oL-SMA in mouse cardiac fibroblasts was de- tected by immunofluorescence staining and Western-blot assay, respectively. Intracellular oxidants in mouse car- diac fibroblasts were measured by using the probe dichlorofluoroscindiacetate （DCFH-DA） under confocal mi- croscopy. Western-blot assay was also used to detect Smad3 and Nrf2, antioxidant proteins, MLL and HCF-1 in mouse cardiac fibroblasts. Results Immunofluorescence staining and Western- blot assay showed that MIF could markedly inhibit fibrosis-associated Collal, Col3al and oL-SMA expression in cardiac fibroblasts. DCFH- DA staining revealed that MIF can efficiently decrease reactive oxygen species （ROS） level in Ang-II-induced cardiac fibroblasts. Additionally, Smad3 activation was inhibited, but transcription factor Nrf2 and the downstream antioxidant genes, including HO-1, SOD-l, SOD2, Trx-2 and e-NOS, were increased in MIF-treated cardiac fibroblasts. MLL and HCF-lwere up-regulated by MIF, and either MLL knockdown or HCF-1 knock- down could consistently suppress Nrf2 expression in cardiac fibroblasts. Conclusions MIF possesses anti-fibro- sis effect by inactivating Smad3 and activating Nrf2 in cardiac fibroblasts.

Long non-coding RNAs in cardiac fibrosis:Regulation of cardiac fibroblasts

Background Cardiac fibrosis,characterized by excessive extracellular matrix(ECM)deposition and increased cardiac fibroblasts(CFs)activity,is a common pathology of various cardiovascular diseases.Cardiac fibrosis decreases ventricular compliance,increases diastolic filling pressure,decreases cardiac oxygen supply,and ultimately impairs the cardiac output.CFs are the main effecter cell type in regulating ECM and predominantly drive the fibrosis process.Despite the critical importance of CFs,our limited understanding of CFs impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression.Recently,long non-coding RNAs(lncRNAs)are emerging as important pathological and physiological regulators of cardiac fibrosis,shedding light on novel molecular mechanisms and potential therapeutic targets.This review discussed the current knowledge regarding the lnc RNAs involved in cardiac fibrosis and summarized their possible molecular mechanisms with special focus on the regulation of CFs.

Regulations of Thyroid Hormone on Cardiac Protein Kinase C Signal Pathway in vitro

The experiments were conducted to assess the influences of thyroid hormone on cardiac protein kinase C(PKC) signal pathway with cultured cardiac myocytes and fibroblasts as the models. Cells were pretreated with 1% newborn calf serum (NCS) or angiotensin II (Ang II), and then following by a triiodothyronine (T3) treatment. The PKC activity, PKCα and PKCε expressions were analyzed and compared. In 1% NCS pretreatment, T3 could inhibit PKC activity and PKCε expression in cardiac myocytes. The AngII pretreatment led to an increase of PKC activity and PKCε expression in cardiac myocytes, and an increase of PKC activity in cardiac fibroblasts. Following by T3 treatment, the increased PKC activity and PKCε expression in cardiac myocytes were markedly decreased. In conclusion, whether in 1% NCS or in Ang II pretreatment, T3 could inhibit PKC activity and PKCε expression in cardiac myocytes. Key words thyroid hormone - cardiac myocytes - cardiac fibroblasts - protein kinase C CLC number Q 572 Foundation item: Supported by the Natural Science Foundation of Hubei Province (98091)Biography: WANG Bao-hua (1974-), female, Ph. D, research direction: cardiovascular pathophysiology.

Cardiac stromal cells on stage:From dull filler to specialized actors

Cardiac stromal cells have faced through the years a significant evolution in their definitions concerning their phenotypes,markers,and functions.They are surging to key roles in physiopathology,becoming important targets to be exploited for cardiac repair.In this perspective,we briefly discuss their role in novel therapeutic strategies for enhancing cardiac repair and regeneration.

Distinct mononuclear diploid cardiac subpopulation with minimal cell-cell communications persists in embryonic and adult mammalian heart

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,12645 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.

Lipopolysaccharide induced activin A-follistatin imbalance affects cardiac fibrosis

Background Inflammation plays a pivotal role in cardiac remodeling, especially in myocardial fibrosis. Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Previous study has demonstrated that many inflammation stimulating factors trigger transforming growth factor-β （TGF-β） induction and reactive myocardial fibrosis. Activin A （ACT A） is a member of TGF-β superfamily, and follistatin （FS） is an activin-binding protein, i.e. an antagonist of ACT A. Our previous studies have shown that ACT A-FS imbalance occurs in rats with heart failure （HF）, and overexpression of ACT A can lead to ventricular remodeling, and resultant HF. Low expression of FS after myocardial infarction further exacerbated HF. The pathogenic change resulting from overexpression of ACT A is consistent with that of overexpression of angiotensin II （Angll）. Ventricular remodeling includes cardiocyte remodeling and myocardial interstitial collagen deposition and fibrosis, Therefore, the present study was designed to investigate the effects of inflammatory factors on the ACT A-FS and the secretions of cardiac fibroblasts in order to explore in depth the mechanism of myocardial fibrosis. Methods A rat model with HF was established, and the results showed that there was a greater degree of cardiac fibrosis in HF rats. In addition, we found that there was an imbalance of the ACT A/FS system in HF rats, which was characterized by increased levels of ACT A. Further, primary rat cardiac fibroblasts were cultured and the MTT assay was performed to determine the effect of the inflammatory factor-bacterial endotoxin lipopolysaccharide （LPS） on cardiac fibroblast proliferation. Results The results showed that LPS can stimulate the cardiac fibroblasts to proliferate in a dose-dependent manner. Cellular immunohistochemical staining showed that the rat cardiac fibroblasts themselves could express ACT A and FS proteins, and stimulation by LPS could apparently promote the cultured primary rat cardiac fibroblasts to secrete ACT A, but inhibit the secretion of FS. The results also showed that ACT A promoted, in a dose-dependent manner, the proliferation of the cultured primary rat cardiac fibroblasts, and the expression of collagen types I and Ill. Moreover, ACT A promoted, in a dose dependent manner, the cardiac fibroblasts to secrete nitric oxide （NO）, and unregulated the expression of inducible nitric oxide synthase （iNOS） mRNA. Conclusions These results suggest that the inflammatory mediator LPS can promote ACT A-FS imbalance in cardiac fibroblasts, mainly overexpression of ACT A. Overexpression of ACT A promotes the proliferation and the secretion of collagens in cardiac fibroblasts through autocrine/paracrine stimulation of NO, and is involved in the pathological process of myocardial fibrosis.

Cardiac fibrosis: Histological features, molecular pathways and therapeutic targets

Cardiac fibrosis is defined as the unbalanced production and degradation of cardiac interstitial extracellular matrix(ECM),leading to systolic and diastolic dysfunction,arrhythmias,and adverse outcomes of many cardiac pathophysiological conditions.The accumulation of myocardial ECM increases the risk of arrhythmias and impairs cardiac function,ultimately leading to the development of heart failure.Although slowing or reversing the development of excessive accumulation of ECM and cardiac fibrosis is important for maintaining cardiac function,there is currently no approved treatment for them.Activated cardiac fibroblasts are the main effector cells of cardiac fibrosis.Their expansion after pathophysiologic stimuli such as pressure overload,volume overload,metabolic dysfunction,wound healing,and aging is primarily driven by activating resident interstitial populations.While cardiac fibroblasts are the primary effector cells in the fibrotic heart,monocytes/macrophages,lymphocytes,mast cells,vascular cells,and cardiomyocytes may also contribute to the fibrotic response,by secreting critical fibrotic factors and matricellular proteins.This review discusses histological features,molecular pathways involved in the pathogenesis of cardiac fibrosis and possible therapeutic targets.Understanding the occurrence,development and diffusion mechanisms of cardiac fibrosis has important clinical implications for the discovery of drugs to prevent the progression of cardiac fibrosis.

Compensatory function of bradykinin B1 receptor in the inhibitory effect of captopril on cardiomyocyte hypertrophy and cardiac fibroblast proliferation in neonatal rats

Background Bradykinin （BK） acts mainly on two receptor subtypes： B1 and B2, and activation of B2 receptor mediates the most well-known cardioprotective effects of angiotensin converting enzyme inhibitors （ACEi）, however, the role that B1 receptor plays in ACEi has not been fully defined. We examined the role of B1 receptor in the inhibitory effect of ACE inhibitor captopril on rat cardiomyocyte hypertrophy and cardiac fibroblast proliferation induced by angiotensin Ⅱ （Ang Ⅱ） and explored its possible mechanism. Methods Neonatal cardiomyocytes and cardiac fibroblasts （CFs） were randomly treated with Ang Ⅱ, captopdl, B2 receptor antagonist （HOE-140） and B1 receptor antagonist （des-Arg^10, Leu^9-kallidin） alone or in combination. Flow cytometry was used to evaluate cell cycle, size and protein content. Nitric oxide （NO） and intracellular cyclic guanosine monophosphate （cGMP） level were measured by colorimetry and radioimmunoassay. Results After the CFs and cardiomyocytes were incubated with 0.1 μmol/L Ang Ⅱ for 48 hours, the percentage of CFs in the S stage, cardiomyocytes size and protein content significantly increased （both P 〈0.01 vs control）, and these increases were inhibited by 10 μmol/L captopril. However, NO and cGMP levels were significantly higher than that with Ang Ⅱ alone （both P 〈0.01）. 1 μmol/L HOE-140 or 0.1 pmol/L des-Arg^10, Leu^9-kallidin attenuated the effects of captopril, which was blunted further by blockade of both B1 and B2 receptors. Conclusions Acting via B2 receptor, BK contributes to the antihypertrophic and antiproliferative effects of captopril on cardiomyocytes and CFs. In the absence of B2 receptor, B1 receptor may act a compensatory mechanism for the B2 receptor and contribute to the inhibition of cardiomyocyte hypertrophy and CFs proliferation by captopril. NO and cGMP play an important role in the effect of B1 receptor.

Cardiac Fibroblast-Specific Activating Transcription Factor 3 Promotes Myocardial Repair after Myocardial Infarction

Background：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.Methods：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/flColla2-Cre＋ （CF-specific ATF3 overexpression group,n =5） and ATF3TGfl/flColla2-Cre-male mice （without CF-specific ATF3 overexpression group,n =5）.In addition,five mice of ATF3TGfl/flCol1a2-Cre＋ and ATF3TGfl/flCol 1 a2-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 28th day after MI surgery in ATF3TGfl/flColla2-Cre＋ and ATF3TGfl/flColla2-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.Results：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 semm stimulation.Notably,ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.Conclusions：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.

Atorvastatin reduces myocardial fibrosis in a rat model with post- myocardial infarction heart failure by increasing the matrix metaHoproteinase-2/tissue matrix metalloproteinase inhibitor-2 ratio

Background The cholesterol-lowering statin drugs have some non-lipid-lowering effects, such as inhibiting myocardial remodeling. However, the underlying mechanism is still unclear. Methods The left anterior descending coronary artery was ligated to establish a rat model of heart failure, and the rats were divided into a sham operation （SO） group, myocardial infarction model （MI） group, and MI-atorvastatin group. Changes in hemodynamic parameters were recorded after the final drug administration. Histological diagnosis was made by reviewing hematoxylin and eosin （HE） stained tissue. Real-time quantitative polymerase chain reaction （PCR） was performed to determine the expressions of type I and type III collagen, matrix metalloproteinase-2 （MMP-2）, and tissue matrix metalloproteinase inhibitor-2 （TIMP-2）. Further, primary rat cardiac fibroblasts were cultured and the MTT assay was performed to determine the effect of atorvastatin on cardiac fibroblast proliferation. Results The model of heart failure was established and the results of HE staining and Masson＇s trichrome staining revealed that the rats in the heart failure group showed obvious hyperplasia of fibrotic tissue, which was significantly reduced in the atorvastatin group. Real-time quantitative PCR showed that the MI group showed a significantly increased expression of type I and type III coltagen, MMP-2, and TIMP-2, but a significantly reduced MMP-2/T＇IMP- 2 ratio. Compared with the MI group, the atorvastatin group showed significantly reduced expression of type I and III collagen, unchanged expression of MMP-2, significantly reduced expression of TIMP-2, and an increased MMP-2/ TIMP-2 ratio. We further found that atorvastatin significantly inhibited the Ang II-induced fibroblast proliferation and the expression of type I and type III collagen in cardiac fibroblasts while increasing the MMP-2/TIMP-2 ratio. Conclusions These data suggest that atorvastatin can inhibit cardiac fibroblast proliferation and enhance collagen degradation by increasing the MMP-2/TIMP-2 ratio, thereby inhibiting the formation of myocardial fibrosis in rats with heart failure after myocardial infarction.

Cell-derived extracellular matrix-coated silk fibroin scaffold for cardiogenesis of brown adipose stem cells through modulation of TGF-β pathway

The cell-derived extracellular matrix(ECM)-modified scaffolds have advantages of mimic tissue specificity and been thought to better mimic the native cellular microenvironment in vitro.ECM derived from cardiac fibroblasts(CFs)are considered as key elements that provide a natural cell growth microenvironment and change the fate of cardiomyocytes(CMs).Here,a new hybrid scaffold is designed based on silk fibroin(SF)scaffold and CFs-derived ECM.CFs were seeded on the SF scaffold for 10days culturing and decellularized to produce CFs-derived ECM-coated SF scaffold.The results showed that the cell-derived ECM-modified silk fibroin scaffold material contained collagen,laminin,fibronectin and other ECM components with myocardial-like properties.Further to explore its effects on brown adipose stem cells(BASCs)differentiation into CMs.We found that the CFderived ECM-coated scaffold also increased the expression of CM-specific proteins(e.g.cardiac troponin T and α-actinin)of BASCs.Notably,the b1-integrin-dependent transforming growth factor-β1 signaling pathway was also involved in the regulation of CF-derived ECM by promoting the differentiation of BASCs into CMs.Overall,these findings provide insights into the bionic manufacturing of engineered cardiac tissues(ECTs)and establish a theoretical basis for the construction of ECTs.

Upregulation of miR-29b contributes to the anti-fibrotic effect of macrophage migration inhibitory factor in diabetic myocardial fibrosis

Background Macrophage migration inhibitory factor （MIF） possesses proinflammatory function when secreted from the cells, and it also exhibits antioxidant properties based on its intrinsic oxidoreductase activity. However, the role of MIF in cardiac fibrosis is not well known. In the present study, the effect of MIF on fibrosis-associated gene expression and the underlying mechanism were examined. Methods The collagen content in mouse myocardium was detected by Masson staining. Expressions of MIF and fibrosis-associated Collal, Col3al and α-SMA in mouse myocardium or mouse cardiac fibroblasts were detected by quantitative real-time PCR and Western blot assay, respectively. Mature miR-29b expressions in mouse myocardium and cardiac fibroblasts were determined by real-time PCR. Smad3 activation in MIF-treated cardiac fibroblasts was also detected by Western blot assay. Results Compared with the db/m control mice, the collagen content was significantly increased in the myocardium of diabetic db/db mice. MIF was up-regulated, but miR-29b was down-regulated in the diabetic myocardium. Quantitative real-time PCR and Western blot assay showed that MIF could inhibit fibrosis-associated Collal, Col3al and α-SMA expressions in mouse cardiac fibroblasts. Smad3 activation was inhibited, but miR-29b was up-regulated in MIF-treated cardiac fibroblasts. Enforced expression of miR-29b significantly suppressed Collal, Col3al, and oL-SMA mRNA and lprotein expressions in cardiac fibroblasts. Conclusions MIF possesses the anti-fibrosis activity through inhibiting Smad3 activation and through up-regulating miR-29b expression, and miR-29b can inhibit fibrosis-associated Collal, Col3al and α-SMA expressions in cardiac fibroblasts.