Cardiac Fibrosis In Response Research Articles

MiR-24-3p secreted as extracellular vesicle cargo by cardiomyocytes inhibits fibrosis in human cardiac microtissues.

Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix-producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models. Gain- and loss-of-function experiments were performed using human induced pluripotent stem cell-derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia-simulating conditions. hCM-derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo-cultured human cardiac slices.hCF transfection with miR-24-3p mimic prevented TGFβ1-mediated induction of FURIN, CCND1 and SMAD4-miR-24-3p target genes participating in TGFβ1-dependent fibrinogenesis -, regulating hCF-to-myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM-derived EVs modulated hCF activation. Ischaemia-simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant-derived agent with miR-24-3p-stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues. These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis.

Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p>0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.

Ginsenoside Rg1 reduces cardiac inflammation against myocardial ischemia/reperfusion injury by inhibiting macrophage polarization

BackgroundMyocardial ischemia/reperfusion (MI/R) injury is the main cause of death worldwide and poses a significant threat to cardiac health. Ginsenoside Rg1 has been shown to have inhibitory effects on inflammatory activation, oxidative stress, and cardiac injury, suggesting that Rg1 may have therapeutic effects on MI/R injury. However, the mechanism remains to be further studied. Materials and methodsLeft anterior descending coronary artery ligation was performed in Sprague-Dawley rats to construct an MI/R model in vivo. Organ index, electrocardiogram, infarct size, histopathological changes, and detection of cardiac injury and inflammatory factors in the rats were used to evaluate myocarditis, macrophage polarization, and fibrosis. We also used rat bone marrow-derived macrophages (BMDMs) to further investigate the effects of Rg1 on absent in melanoma 2 (AIM2) activation and macrophage polarization in vitro. ResultsAdministration of Rg1 exhibited dose-dependent cardioprotective effects and effectively reduced MI/R injury. Rg1 significantly attenuated myocardial inflammation and inhibited M1 macrophage polarization during MI/R injury. Furthermore, Rg1 significantly reduced cardiac fibrosis in response to MI/R injury. This anti-fibrotic effect may contribute to the preservation of cardiac structure and function following an ischemic insult. Meanwhile, Rg1 effectively inhibited the activation of the AIM2 inflammasome in vitro, highlighting its potential as a key regulator of inflammatory pathways. ConclusionOur findings elucidate the multifaceted mechanisms underlying Rg1's cardioprotective effects, including its ability to mitigate inflammation, modulate macrophage polarization, and inhibit fibrosis.

Cardiomyocyte PAI-1 influences the cardiac transcriptome and limits the extent of cardiac fibrosis in response to left ventricular pressure overload

Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid-acting inhibitor of endogenous plasminogen activators (uPA and tPA). The global PAI-1 knockout mice (PAI-1KO) develop age-dependent cardiac-selective fibrosis, and young global PAI-1KO mice exhibit augmented susceptibility to developing cardiac fibrosis in response to hypertension. Here, we tested the hypothesis that cardiomyocyte PAI-1 is necessary to provide cardioprotective effects in a left ventricular pressure overload-induced murine model of cardiac hypertrophy and fibrosis using cardiomyocyte-specific PAI-1 knockout (cmPAI-1KO) mice. The results revealed that cmPAI-1KO mice display significantly worse cardiac fibrosis than controls. To investigate the molecular mechanisms responsible for these effects, genome-wide cardiac transcriptome analysis was performed. Loss of cardiomyocyte PAI-1 led to differential expression of 978 genes compared to controls in response to left ventricular pressure overload. Pathway enrichment analysis identified the inflammatory response, cell substrate adhesion, regulation of cytokine production, leukocyte migration, extracellular matrix organization, and cytokine-mediated signaling pathways as being significantly upregulated in cmPAI-1KO hearts. Conversely, specific epigenetic repressors, cation transmembrane transport, muscle system processes, and nitric oxide signaling were significantly downregulated in cmPAI-1KO hearts compared to control hearts in response to left ventricular pressure overload. Collectively, the present study provides strong evidence of the impact of cardiomyocyte PAI-1 in regulation of the transcriptome network involved in the cardiac stress response. In response to stress, the deregulatory impact of cardiomyocyte PAI-1 loss on the cardiac transcriptome may be the underlying cause of cardiac-selective accelerated fibrogenesis in global PAI-1-deficient mice.

Pharmacological blockade of αV integrin (CD51) reduces the development of pressure-overload-induced cardiac fibrosis

Myocardial fibrosis is involved in most forms of heart diseases, progressively leading to adverse cardiac remodeling and heart failure. We previously identified a population of cardiac stromal cells that contribute to fibrosis development through a CD51 (alpha v integrin) dependent activation of pro-fibrotic pathways in a murine model of myocardial infarction. The pharmacological blockade of CD51 was associated with a significant reduction of fibrosis, preservation of cardiac function and improved survival. Investigate the impact of CD51 pharmacological blockers on the development of cardiac fibrosis induced by neurohormonal pro-hypertrophic stressors. We used C57Bl/6J mice infused with Angiotensin II (1.44 mg/kg/day) or dual Angiotensin II (1.44 mg/kg/day) + PhenylEphrine (50 mg/kg/day) through an osmotic pump for 28 or 14 days, respectively. Animals were treated with daily with intra-peritoneal injection of either CD51 blocker cilengitide or vehicle for the duration of the study. We found that both AngII and AngII + PE stimulation led to a similar cardiac hypertrophic remodeling: heart weight on body weight ratio as well as cardiomyocyte size were similarly increased. However, AngII + PE mice developed a significantly higher amount of interstitial fibrosis as compared to AngII mice (10.2 ± 4.3% vs. 4.5 ± 1.3% of total area respectively, P < 0.0001). Importantly, AngII + PE but not AngII mice exhibited a significant increase in CD51 expression and in the number of CD51+ cells as found in cytometry analysis. CD51 blockade significantly decreased the development of fibrosis under AngII + PE stimulation (7.6% ± 1.7, P < 0.05), as well as heart weight on body weight ratio (5.4 ± 0.5 vs. 5.7 ± 0.4, P < 0.05), but did not change the cardiomyocyte sizes. CD51 blockade also partially rescued the progressive drop in LVEF observed in mice stimulated with AngII + PE (47.5% ± 4.0 vs. 40.7% ± 13.8). Further cytometry analyses on cardiac tissues demonstrated that CD51 is expressed by stromal cells and by CCR2+ macrophages. Preliminary results indicate that the anti-fibrotic effects of CD51 blockade could come from a dual reduction on the activation of stromal and the immune-inflammatory response. Inhibition of CD51 reduces the development of cardiac fibrosis in response to pressure-overload, through the targeting of both stromal and inflammatory cardiac cells.

Cardiac fibroblasts contribute to sexually dimorphic responses to cardiac injury.

A new study shows that male rats are more susceptible to cardiac fibrosis in response to chronic adrenergic stimulation than female rats, probably as a result of the increased myofibroblast activation in male hearts, and suggests that sex hormones do not to control the cardiac fibroblast response in this setting.

Cardiac Fibrosis: Key Role of Integrins in Cardiac Homeostasis and Remodeling.

Cardiac fibrosis is a common finding that is associated with the progression of heart failure (HF) and impacts all chambers of the heart. Despite intense research, the treatment of HF has primarily focused upon strategies to prevent cardiomyocyte remodeling, and there are no targeted antifibrotic strategies available to reverse cardiac fibrosis. Cardiac fibrosis is defined as an accumulation of extracellular matrix (ECM) proteins which stiffen the myocardium resulting in the deterioration cardiac function. This occurs in response to a wide range of mechanical and biochemical signals. Integrins are transmembrane cell adhesion receptors, that integrate signaling between cardiac fibroblasts and cardiomyocytes with the ECM by the communication of mechanical stress signals. Integrins play an important role in the development of pathological ECM deposition. This review will discuss the role of integrins in mechano-transduced cardiac fibrosis in response to disease throughout the myocardium. This review will also demonstrate the important role of integrins as both initiators of the fibrotic response, and modulators of fibrosis through their effect on cardiac fibroblast physiology across the various heart chambers.

Anti-integrin \u03b1v therapy improves cardiac fibrosis after myocardial infarction by blunting cardiac PW1+ stromal cells

There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1+ cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1+ cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1+ cells (93% ± 1%), predominantly as the αVβ1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFβ at the surface of cardiac PW1+ cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1+CD51+ cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

BMP10 preserves cardiac function through its dual activation of SMAD-mediated and STAT3-mediated pathways

Bone morphogenetic protein 10 (BMP10) is a cardiac peptide growth factor belonging to the transforming growth factor β superfamily that critically controls cardiovascular development, growth, and maturation. It has been shown that BMP10 elicits its intracellular signaling through a receptor complex of activin receptor-like kinase 1 with morphogenetic protein receptor type II or activin receptor type 2A. Previously, we generated and characterized a transgenic mouse line expressing BMP10 from the α-myosin heavy chain gene promoter and found that these mice have normal cardiac hypertrophic responses to both physiological and pathological stimuli. In this study, we report that these transgenic mice exhibit significantly reduced levels of cardiomyocyte apoptosis and cardiac fibrosis in response to a prolonged administration of the β-adrenoreceptor agonist isoproterenol. We further confirmed this cardioprotective function with a newly generated conditional Bmp10 transgenic mouse line, in which Bmp10 was activated in adult hearts by tamoxifen. Moreover, the intraperitoneal administration of recombinant human BMP10 was found to effectively protect hearts from injury, suggesting potential therapeutic utility of using BMP10 to prevent heart failure. Gene profiling and biochemical analyses indicated that BMP10 activates the SMAD-mediated canonical pathway and, unexpectedly, also the signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway both in vivo and in vitro Additional findings further supported the notion that BMP10's cardioprotective function likely is due to its dual activation of SMAD- and STAT3-regulated signaling pathways, promoting cardiomyocyte survival and suppressing cardiac fibrosis.

P5369Alpha-V integrin regulates the contribution of PW1+ cells to cardiac fibrosis

Abstract Background Activated cardiac fibroblasts produce extracellular matrix proteins that accumulate during cardiac fibrosis. We have recently shown that PW1 is expressed in a subset of cardiac stromal cells and that cardiac PW1+ cells represent a cellular source of fibroblasts in the ischemic hearts. Purpose We aimed to further identify new cell surface markers expressed by cardiac PW1+ cells and to investigate their role in the fibrogenic behavior of these cells. Methods and results We first performed transcriptomic and proteomic profiling of FACS-isolated cardiac PW1+ from normal and ischemic hearts. RNA-sequencing output files were processed with bioinformatics algorithms to identify 378 specific cell-surface markers for cardiac PW1+ cells. By comparing these candidates with the proteomic profile, we then cross-identified 9 cell surface proteins primarily involved in cell motility, adhesion to the matrix, inflammatory response and response to wounding. One of these candidates (i.e., aV-integrin or CD51) was expressed in almost all cardiac PW1+ cells (93±1%), and was predominantly found in cells expressing PW1 in the myocardium. Cardiac PW1+ cells showed a predominant expression of aVβ1 complex which is known to mediate fibrosis through TGF-beta activation in a number of tissues. The transfer of isolated cardiac PW1+CD51+ cells into ischemic hearts was associated with fibrosis development. We further demonstrated that inhibition of aV-integrin in cardiac PW1+ cells reduces their profibrotic gene expression profile and their ability to differentiate into fibroblasts. Lastly, a pharmacological blockade of aV-integrin improved cardiac function and animal survival following myocardial infarction coupled with a reduced infarct size and fibrotic lesion. Conclusions These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of aV-integrin leads to reduced pathological outcomes following cardiac ischemia. Acknowledgement/Funding Fondation Leducq (grant 13CVD01, CardioStemNet project), Fédération Française de Cardiologie and Era-CVD (ANR-16-ECVD-0011-03, Clarify project)

Abstract P180: Deficiency of Sirtuin 3 Sensitizes Angiotensin II-Induced Cardiac Pericyte-Fibroblast Transition and Diastolic Dysfunction via Iron Overloading

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of SIRT3 resulted in diastolic dysfunction in mice. In present study, we aim to explore the role of SIRT3 in pericyte recruitment and cardiac fibrosis in response to hypertension. NG2 + tracing reporter NG2DsREDBAC mouse was crossed with SIRT3KO mice. Mice were infused with Ang-II (1000ng/kg/min) for 28 days, blood pressure, coronary flow reserve (CFR) and diastolic function (IVRT and MPI) were measured pre- and post- Ang-II treatment. Cardiac fibrosis and iron content were measured by Masson and Prussian staining. Pericytes and fibroblasts were measured by co-immunostaining. The expression of heme oxygenase-1 (HO-1), ferroportin (FPN), p47 phox , and transforming growth factor-β1(TFG-β1) was analyzed by western blots. Infusion of Ang-II resulted in a significant increase in blood pressure compared to basal levels in WT mice (MAP 94± 2 mHg vs 130±1.7 mHg, n=7, p&lt;0.05); whereas knockout of SIRT3 exacerbated Ang-II-induced increase in blood pressure (MAP WT 130±1.7 mHg vs SIRT3KO 145 ± 2.1 mHg, n=7, p&lt;0.05). Furthermore, knockout of SIRT3 sensitized Ang-II-induced reduction of CFR and elevation of IVRT and MPI in mice. Using NG2 pericyte tracing reporter mice, we found that mice infusion with Ang-II resulted in a significant increase in NG2 + pericyte recruitment in the heart. Knockout of SIRT3 enhanced Ang-II-induced NG2 + pericyte recruitment. Knockout of SIRT3 further enhanced Ang-II-induced cardiac fibrosis as well as fibrotic markers FSP1 and a-SMA staining. To examine whether pericytes differentiate into fibroblast, NG2 + pericytes were co-stained with FSP-1 + . Ang-II infusion led to a significant increase in NG2 + /FSP-1 + ; while knockout of SIRT3 further increased numbers of NG2 + /FSP-1 + in the heart. Mechanistically, knockout of SIRT3 increased iron staining in the heart. This was accompanied by a significant increase in the expression of HO-1, FPN, p47 phox , ROS, and TGF-β as compared to Ang-II treatment. Our study suggests that deficiency of SIRT3 may promote Ang-II-induced cardiac fibrosis via pericyte-fibroblast transition and by Fe2 + -ROS-TGF-β pathway.

Abstract 268: Strain-specific Cardiac Fibroblast Response to Isoproterenol-induced Cardiac Fibrosis

Fibroblasts are a heterogeneous population of cells that function within the injury response mechanisms across various tissues. Despite their importance in pathophysiology, the effects of different genetic backgrounds on fibroblast contribution to the development of disease has yet to be addressed. It has previously been shown that mice in the Hybrid Mouse Diversity Panel, which consists of 110 inbred mouse strains, display a spectrum in severity of cardiac fibrosis in response to chronic treatment of isoproterenol (ISO). Here, we characterized cardiac fibroblasts (CFbs) from three different mouse strains (C57BL/6J, C3H/HeJ, and KK/HIJ) which exhibited varying degrees of fibrosis after ISO treatment. The select strains of mice underwent sham or ISO treatment via intraperitoneally-implanted osmotic pumps for 21 days. Masson’s Trichrome staining showed significant differences in fibrosis in response to ISO, with KK/HIJ mice demonstrating the highest levels, C3H/HeJ exhibiting milder levels, and C57BL/6J demonstrating little to no fibrosis. When CFbs were isolated and cultured from each strain, the cells demonstrated similar traits at the basal level but responded to ISO stimuli in a strain-specific manner. Likewise, CFbs demonstrated differential behavior and gene expression in vivo in response to ISO. ISO treatment caused CFbs to proliferate similarly across all strains, however, immunofluorescence staining showed differential levels of CFb activation. Additionally, RNA-sequencing analysis revealed unique gene expression profiles of all three strains upon ISO treatment. Our study depicts the phenotypic heterogeneity of CFbs across different strains of mice and our results suggest that ISO-induced cardiac fibrosis is a complex process that is independent of fibroblast proliferation and is mainly driven by the activation/inhibition of genes involved in pro-fibrotic pathways.

Abstract 424: Effector T Cells Induce Cardiac Fibroblasts Transition to Myofibroblasts &amp; Contribute to Pressure Overload Induced Cardiac Fibrosis

Background: Cardiac fibrogenesis is a major pathogenic factor that occurs in heart failure (HF) and results in contractile dysfunction and ventricular dilation. Recently, we showed that T cell deficient mice (TCRα -/- ) do not develop cardiac fibrosis (CF) and have preserved cardiac function in the thoracic aortic constriction (TAC) mouse model of pressure overload (PO). Specifically, CD4 + T cells are activated in the cardiac draining lymph nodes and infiltrate the LV, where the Th1 and Th17 effector T cell signature transcription factors are significantly upregulated as compared with control mice. However, the T cell subsets involved and the mechanisms by which they contribute to CF and pathogenesis of non-ischemic HF remains to be determined. Thus, we hypothesize that heart infiltrated effector T cells perpetuate the fibrotic response by regulating the differentiation and activation of extracellular matrix-producing cardiac myofibroblasts. Methods and Results: Naïve or effector T cells differentiated in vitro or isolated from mice undergoing TAC or Sham surgery were co-cultured with adult C57BL/6 cardiac fibroblasts (CFB). In contrast with naïve T cells, effector T cells and PO activated T cells strongly adhered to CFB and mediated fibroblast to myofibroblasts transition as depicted by immunofluorescence expression of SMAα. Effector T cell supernatants only slightly mediated this transition, indicating that effector T cells direct contact with CFB, rather than cytokine release is required to mediate CFB transformation. Adoptive transfer of effector, but not naïve T cells, into TCRα -/- recipient mice in the onset of TAC resulted in T cells infiltration into the left ventricle and increased CF. Conclusions: Our data indicate that CD4+ effector T cells directly interact with CFB to induce CF in response to PO induced CF. Future studies will determine the adhesion mechanisms regulating this crosstalk and evaluate the pro-fibrotic mechanisms induced and whether this is a T effector cell specific subset. These results will provide an attractive tool to counteract the inflammatory/fibrotic process as an alternative option for the treatment of CF in non- ischemic HF.

Cardiac fibrosis in myocardial infarction-from repair and remodeling to regeneration.

Ischemic cell death during a myocardial infarction leads to a multiphase reparative response in which the damaged tissue is replaced with a fibrotic scar produced by fibroblasts and myofibroblasts. This also induces geometrical, biomechanical, and biochemical changes in the uninjured ventricular wall eliciting a reactive remodeling process that includes interstitial and perivascular fibrosis. Although the initial reparative fibrosis is crucial for preventing rupture of the ventricular wall, an exaggerated fibrotic response and reactive fibrosis outside the injured area are detrimental as they lead to progressive impairment of cardiac function and eventually to heart failure. In this review, we summarize current knowledge of the mechanisms of both reparative and reactive cardiac fibrosis in response to myocardial infarction, discuss the potential of inducing cardiac regeneration through direct reprogramming of fibroblasts and myofibroblasts into cardiomyocytes, and review the currently available and potential future therapeutic strategies to inhibit cardiac fibrosis.Graphical abstractReparative response following a myocardial infarction. Hypoxia-induced cardiomyocyte death leads to the activation of myofibroblasts and a reparative fibrotic response in the injured area. Right top In adult mammals, the fibrotic scar formed at the infarcted area is permanent and promotes reactive fibrosis in the uninjured myocardium. Right bottom In teleost fish and newts and in embryonic and neonatal mammals, the initial formation of a fibrotic scar is followed by regeneration of the cardiac muscle tissue. Induction of post-infarction cardiac regeneration in adult mammals is currently the target of intensive research and drug discovery attempts

Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes.

Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts.

Tumor necrosis factor: a mechanistic link between angiotensin-II-induced cardiac inflammation and fibrosis.

Continuous angiotensin-II infusion induced the uptake of monocytic fibroblast precursors that initiated the development of cardiac fibrosis; these cells and concurrent fibrosis were absent in mice lacking tumor necrosis factor receptor 1 (TNFR1). We now investigated their cellular origin and temporal uptake and the involvement of TNFR1 in monocyte-to-fibroblast differentiation. Within a day, angiotensin-II induced a proinflammatory environment characterized by production of inflammatory chemokines, cytokines, and TH1-interleukins and uptake of bone marrow-derived M1 cells. After a week, the cardiac environment changed to profibrotic with growth factor and TH2-interleukin synthesis, uptake of bone marrow-derived M2 cells, and the presence of M2-related fibroblasts. TNFR1 signaling was not necessary for early M1 uptake, but its absence diminished the amount of M2 cells. TNFR1-knockout hearts also showed reduced levels of cytokine expression, but not of TH-related lymphokines. Reconstitution of wild-type bone marrow into TNFR1-knockout mice was sufficient to restore M2 uptake, upregulation of proinflammatory and profibrotic genes, and development of fibrosis in response to angiotensin-II. We also developed an in vitro mouse monocyte-to-fibroblast maturation assay that confirmed the essential role of TNFR1 in the sequential progression of monocyte activation and fibroblast formation. Development of cardiac fibrosis in response to angiotensin-II was mediated by myeloid precursors and consisted of 2 stages. A primary M1 inflammatory response was followed by a subsequent M2 fibrotic response. Although the first phase seemed to be independent of TNFR1 signaling, the later phase (and development of fibrosis) was abrogated by deletion of TNFR1.

Workshop on cardiovascular extracellular matrix in health and disease in Baeza, Spain

The Workshop on Cardiovascular Extracellular Matrix in Health and Disease, International University of Andalusia, Baeza, Spain, 6-8 October 2014 served to discuss the current knowledge on the mechanisms integral to extracellular matrix homeostasis that are fundamental to understanding the pathological basis of several cardiovascular diseases, including the development of cardiac fibrosis in response to cardiac hypertrophy and myocardial infarction, and the extracellular matrix alterations contributing to aortic stenosis or aneurysms.

Genistein alleviates pressure overload-induced cardiac dysfunction and interstitial fibrosis in mice.

Pressure overload-induced cardiac interstitial fibrosis is viewed as a major cause of heart failure in patients with hypertension or aorta atherosclerosis. The purpose of this study was to investigate the effects and the underlying mechanisms of genistein, a natural phytoestrogen found in soy bean extract, on pressure overload-induced cardiac fibrosis. Genisten was administered to mice with pressure overload induced by transverse aortic constriction. Eight weeks later, its effects on cardiac dysfunction, hypertrophy and fibrosis were determined. Its effects on proliferation, collagen production and myofibroblast transformation of cardiac fibroblasts (CFs) and the signalling pathways were also assessed in vitro. Pressure overload-induced cardiac dysfunction, hypertrophy and fibrosis were markedly attenuated by genistein. In cultured CFs, genistein inhibited TGFβ1-induced proliferation, collagen production and myofibroblast transformation. Genistein suppressed TGFβ-activated kinase 1 (TAK1) expression and produced anti-fibrotic effects by blocking the TAK1/MKK4/JNK pathway. Further analysis indicated that it up-regulated oestrogen-dependent expression of metastasis-associated gene 3 (MTA3), which was found to be a negative regulator of TAK1. Silencing MTA3 by siRNA, or inhibiting the activity of the MTA3-NuRD complex with trichostatin A, abolished genistein's anti-fibrotic effects. Genistein improved cardiac function and inhibited cardiac fibrosis in response to pressure overload. The underlying mechanism may involve regulation of the MTA3/TAK1/MKK4/JNK signalling pathway. Genistein may have potential as a novel agent for prevention and therapy of cardiac disorders associated with fibrosis.

CYP epoxygenase 2J2 prevents cardiac fibrosis by suppression of transmission of pro-inflammation from cardiomyocytes to macrophages

Cytochrome P450 epoxygenase (CYP450)-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; but the underlying mechanism remains unclear. The present study aimed to elucidate how EETs regulated cardiac fibrosis in response to isoprenaline (Iso) or angiotensin (Ang) II. Cardiac-specific human CYP2J2 transgenic mice (Tr) and wild-type (WT) C57BL/6 littermates were infused with Iso- or Ang II. Two weeks after infusion, Tr mice showed more alleviative cardiac fibrosis and inflammation compared with WT mice. In vitro, we found Iso or Ang II induced nuclear transfer of NF-κB p65 and inflammatory cytokines expression in cardiomyocytes. Furthermore, inflammation response emerged in macrophages cultured in cardiomyocytes-conditioned medium. When pretreatment with 14,15-EET in cardiomyocytes, the inflammatory response was markedly suppressed and the transmission of inflammation from cardiomyocytes to macrophages was reduced. In conclusion, CYP2J2 and EETs prevent cardiac fibrosis and cardiac dysfunction by suppressing transmission of pro-inflammation from cardiomyocytes to macrophages in heart, suggesting that elevation of EETs level could be a potential strategy to prevent cardiac fibrosis.