Fowl adenovirus serotype 4 (FAdV-4) infection induces inflammatory responses in chicken embryonic cardiac fibroblasts via PI3K/Akt and IκBα/NF-κB signaling pathways.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of the acute infectious disease hepatitis-hydropericardium syndrome (HHS). Previous studies have focused on the mechanisms of FAdV-4 caused liver injury, while studies revealing potential mechanisms of inflammatory injury in FAdV-4-infected chicken cardiac cells remain scare. Here we found that FAdV-4 successfully infected chicken embryonic cardiac fibroblasts (CECF) cells in vitro and significantly upregulated production of inflammatory cytokines including IL-1ß, IL-6, IL-8, and TNF-α, suggesting induction of a strong inflammatory response. Mechanistically, FAdV-4 infection increased expression of phosphorylated Akt in a time-dependent manner, while phosphorylation of Akt and production of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α were greatly reduced in FAdV-4-infected CECF cells after treatment with LY294002, a potent inhibitor of PI3K, indicating that the inflammatory response induced by FAdV-4 infection is mediated by the PI3K/Akt signaling pathway. Furthermore, FAdV-4 infection increased expression of phosphorylated IκBα, a recognized indicator of NF-κB activation, and treatment with the BAY11-7082, a selective IκBα phosphorylation and NF-κB inhibitor, significantly reduced IκBα phosphorylation and inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) production in FAdV-4-infected CECF cells, suggesting a critical role of IκBα/NF-κB signaling in FAdV-4-induced inflammatory responses in CECF cells. Taken together, our results suggest that FAdV-4 infection induces inflammatory responses through activation of PI3K/Akt and IκBα/NF-κB signaling pathways in CECF cells. These results reveal potential mechanisms of inflammatory damage in chicken cardiac cells caused by FAdV-4 infection, which sheds new insight into clarification of the pathogenic mechanism of FAdV-4 infection and development of new strategies for HHS prevention and control.

Fibroblasts activation by embryonic signal switching: A novel mechanism of placental growth factor-induced cardiac remodeling.

INTRODUCTION: Cardiac remodeling is defined as cellular interstitial changes that lead dysfunction of the heart after injury. Placental growth factor (PlGF), a member of the VEGF family, has been reported to regulate cardiac hypertrophy in hemodynamic state. We therefore analyze the function of PlGF during cardiac remodeling using cardiac cells and fibroblasts, under Angiotensin II (AngII) stimulation. METHODS: PlGF overexpressed mouse embryonic fibroblasts derived from C57BL/6 mice, were made by deficient retrovirus vector, designated as C57/PlGF. Only retrovirus vector introduced C57 cells (C57/EV) were used as control. After AngII stimulation, wound scratching assay and MTT proliferation assay with or without p38 MAPK inhibitor, SB205580 were performed in retrovirally-introduced C57 cells. Reactive oxygen species (ROS) production, NF-kB activation, IL-6 and TNF-α production were also measured. Then we assessed AngII-induced cell proliferation of mouse cardiac fibroblasts (CFs) and rat primary cardiomyocytes incubating with C57/PlGF conditioned-medium. RESULTS: The PlGF production in C57/PlGF were confirmed by ELISA (1093.48 ± 3.5 pg/ml, ±SE). AngII-induced cell migration, proliferation and H2O2 production were increased in C57/PlGF compared with C57/EV. SB205580 inhibited the AngII-induced cell proliferation in C57/PlGF. In C57/PlGF cells, NF-kB activation was higher, followed by up-regulation of IL-6 and TNF-α production. CFs and cardiomyocytes proliferation increased when stimulated with C57/PlGF conditioned-medium. DISCUSSION: The activation of fibroblast is stimulated by PlGF signaling via p38 MAPK/NF-kB pathway accompanied by elevation of ROS and inflammatory response. Furthermore, these signals stimulate the activation of CFs and cardiomyocytes, indicating that high circulating level of PlGF have a potential to regulate cardiac remodeling.

WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Periostin regulates lysyl oxidase through ERK1/2 MAPK-dependent serum response factor in activated cardiac fibroblasts.

Collagen crosslinking, mediated by lysyl oxidase, is an adaptive mechanism of the cardiac repair process initiated by cardiac fibroblasts postmyocardial injury. However, excessive crosslinking leads to cardiac wall stiffening, which impairs the contractile properties of the left ventricle and leads to heart failure. In this study, we investigated the role of periostin, a matricellular protein, in the regulation of lysyl oxidase in cardiac fibroblasts in response to angiotensin II and TGFß1. Our results indicated that periostin silencing abolished the angiotensin II and TGFß1-mediated upregulation of lysyl oxidase. Furthermore, the attenuation of periostin expression resulted in a notable reduction in the activity of lysyl oxidase. Downstream of periostin, ERK1/2 MAPK signaling was found to be activated, which in turn transcriptionally upregulates the serum response factor to facilitate the enhanced expression of lysyl oxidase. The periostin-lysyl oxidase association was also positively correlated in an in vivo rat model of myocardial infarction. The expression of periostin and lysyl oxidase was upregulated in the collagen-rich fibrotic scar tissue of the left ventricle. Remarkably, echocardiography data showed a reduction in the left ventricular wall movement, ejection fraction, and fractional shortening, indicative of enhanced stiffening of the cardiac wall. These findings shed light on the mechanistic role of periostin in the collagen crosslinking initiated by activated cardiac fibroblasts. Our findings signify periostin as a possible therapeutic target to reduce excessive collagen crosslinking that contributes to the structural remodeling associated with heart failure.

Engineering a robust and anisotropic cardiac-specific extracellular matrix scaffold for cardiac patch tissue engineering.

Extracellular matrix (ECM) fabricated using human induced pluripotent stem cells (hiPSCs)-derived cardiac fibroblasts (hiPSC-CFs) could serve as a completely biological scaffold for an engineered cardiac patch, leveraging the unlimited source and outstanding reproducibility of hiPSC-CFs. Additionally, hiPSC-CF-derived ECM (hiPSC-CF-ECM) holds the potential to enhance maturation of exogenous cardiomyocytes, such as hiPSC-derived cardiomyocytes (hiPSC-CMs), by providing a microenvironment rich in cardiac-specific biochemical and signaling cues. However, achieving sufficient robustness of hiPSC-CF-ECM is challenging. This study aims to achieve appropriate ECM deposition, scaffold thickness, and mechanical strength of an aligned hiPSC-CF-ECM by optimizing the culture period, ranging from 2 to 10 weeks, of hiPSC-CFs grown on micro-grated substrates, which can direct the alignment of both hiPSC-CFs and their secreted ECM. The hiPSC-CFs demonstrated a production rate of 13.5 µg ECM per day per 20,000 cells seeded. An anisotropic nanofibrous hiPSC-CF-ECM scaffold with a thickness of 20.0 ± 2.1 µm was achieved after 6 weeks of culture, followed by decellularization. Compositional analysis through liquid chromatography-mass spectrometry (LC-MS) revealed the presence of cardiac-specific fibrillar collagens, non-fibrillar collagens, and matricellular proteins. Uniaxial tensile stretching of the hiPSC-CF-ECM scaffold indicated robust tensile resilience. Finally, hiPSCs-CMs cultured on the hiPSC-CF-ECM exhibited alignment following the guidance of ECM nanofibers and demonstrated mature organization of key structural proteins. The culture duration of the anisotropic hiPSC-CF-ECM was successfully refined to achieve a robust scaffold containing structural proteins that resembles cardiac microenvironment. This completely biological, anisotropic, and cardiac-specific ECM holds great potential for cardiac patch engineering.

Boldine prevents the inflammatory response of cardiac fibroblasts induced by SGK1-NFκB signaling pathway activation.

Cardiac fibroblasts (CF) are mesenchymal-type cells responsible for maintaining the homeostasis of the heart's extracellular matrix (ECM). Their dysfunction leads to excessive secretion of ECM proteins, tissue stiffening, impaired nutrient and oxygen exchange, and electrical abnormalities in the heart. Additionally, CF act as sentinel cells in the cardiac tissue microenvironment, responding to various stimuli that may affect heart function. Deleterious stimuli induce an inflammatory response in CF, increasing the secretion of cytokines such as IL-1ß and TNF-α and the expression of cell adhesion molecules like ICAM1 and VCAM1, initially promoting damage resolution by recruiting immune cells. However, constant harmful stimuli lead to a chronic inflammatory process and heart dysfunction. Therefore, it is necessary to study the mechanisms that govern CF inflammation. NFκB is a key regulator of the cardiac inflammatory process, making the search for mechanisms of NFκB regulation and CF inflammatory response crucial for developing new treatment options for cardiovascular diseases. SGK1, a serine-threonine protein kinase, is one of the regulators of NFκB and is involved in the fibrotic effects of angiotensin II and aldosterone, as well as in CF differentiation. However, its role in the CF inflammatory response is unknown. On the other hand, many bioactive natural products have demonstrated anti-inflammatory effects, but their role in CF inflammation is unknown. One such molecule is boldine, an alkaloid obtained from Boldo (Peumus boldus), a Chilean endemic tree with proven cytoprotective effects. However, its involvement in the regulation of SGK1 and CF inflammation is unknown. In this study, we evaluated the role of SGK1 and boldine in the inflammatory response in CF isolated from neonatal Sprague-Dawley rats. The involvement of SGK1 was analyzed using GSK650394, a specific SGK1 inhibitor. Our results demonstrate that SGK1 is crucial for LPS- and IFN-γ-induced inflammatory responses in CF (cytokine expression, cell adhesion molecule expression, and leukocyte adhesion). Furthermore, a conditioned medium (intracellular content of CF subject to freeze/thaw cycles) was used to simulate a sterile inflammation condition. The conditioned medium induced a potent inflammatory response in CF, which was completely prevented by the SGK1 inhibitor. Finally, our results indicate that boldine inhibits both SGK1 activation and the CF inflammatory response induced by LPS, IFN-γ, and CF-conditioned medium. Taken together, our results position SGK1 as an important regulator of the CF inflammatory response and boldine as a promising anti-inflammatory drug in the context of cardiovascular diseases.

Cardiac Fibroblasts Enhance MMP2 Activity to Suppress Gap Junction Function in Cardiomyocytes.

Although it is crucial to promptly restore blood perfusion to revive the ischemic myocardium, reperfusion itself can paradoxically contribute to the electrical instability and arrhythmias of the myocardium. Several studies have revealed that cardiac fibroblasts can impact cardiac electrophysiology through various mechanisms including the deposition of extracellular matrix, release of chemical mediators, and direct electrical coupling with myocytes. Previously, we have shown that hypoxia/reoxygenation (H/R)-treated rat fibroblasts conditional medium (H/R-FCM) could decrease the spontaneous beating frequency of rat neonatal cardiomyocytes and downregulate the expression of gap junction proteins. However, the specific mechanism by which H/R-FCM affects the gap junctions requires further investigation. H/R-FCM was obtained by culturing confluent rat cardiac fibroblasts (RCF) for 4 h under hypoxic conditions. Gap junction function, hemichannel activity, and expression of Cx43 were examined upon treatment with H/R-FCM. Gelatin zymography was performed to detect matrix metalloproteinase (MMP) activity in the conditioned medium. The effect of H/R-FCM and MMP2 inhibitors on cardiac electrophysiology and arrhythmias was investigated with an isolated rat ischemia/reperfusion (I/R) model. H/R-FCM treatment impaired gap junction function, downregulated Cx43 expression, and increased hemichannel activity in rat cardiomyocytes (H9c2). The adverse effect of H/R-FCM on gap junction, which was confirmed by the cardiomyocyte H/R model, was involved in the activation of MMP2. MMP2 inhibition could partially attenuate the detrimental effects of I/R on myocardial electrophysiological indices and arrhythmia susceptibility. Our study indicates that inhibition of MMP2 may be a promising therapeutic target for the treatment of reperfusion arrhythmia.

Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms. METHODS: Clinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation. RESULTS: The expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3ß (GSK3ß)/ß-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3ß/ß-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3ß activity through ERK-mediated phosphorylation, thereby affecting ß-catenin degradation. CONCLUSION: ACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3ß/ß-catenin signaling pathway.

[A transcriptomic analysis of correlation between mitochondrial function and energy metabolism remodeling in mice with myocardial fibrosis following myocardial infarction].

OBJECTIVE: To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction (MI) and its correlation with the increase of glycolytic flux. METHODS: Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI. At 28 days after the operation, 5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing. FPKM method was used to calculate gene expression levels to identify the differentially expressed genes (DEGs) in MI mice, which were analyzed using GO and KEGG databases to determine the pathways affecting the disease process. Heat maps were drawn to show the differential expressions of the pathways and the related genes in the enrichment analysis. In primary cultures of neonatal mouse cardiac fibroblasts (CFs), the changes in mitochondrial respiration and glycolysis levels in response to treatment with the pro-fibrotic agonist TGF-ß1 were analyzed using Seahorse experiment. RESULTS: The mouse models of MI showed significantly increased diastolic and systolic left ventricular diameter (P < 0.05) and decreased left ventricular ejection fraction (P < 0.0001). A total of 124 up-regulated and 106 down-regulated DEGs were identified in the myocardial tissues of MI mice, and GO and KEGG enrichment analysis showed that these DEGs were significantly enriched in fatty acid metabolism, organelles and other metabolic pathways and in the mitochondria. Heat maps revealed fatty acid beta oxidation, mitochondrial dysfunction and increased glycolysis levels in MI mice. In the primary culture of CFs, treatment with TGF-ß1 significantly reduced the basal and maximum respiratory levels and increased the basal and maximum glycolysis levels (P < 0.0001). CONCLUSION: During myocardial fibrosis, energy metabolism remodeling occurs in the CFs, manifested by lowered mitochondrial function and increased energy generation through glycolysis.

Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFß-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

miR-486-5p diagnosed atrial fibrillation, predicted the risk of left atrial fibrosis, and regulated angiotensin II-induced cardiac fibrosis via modulating PI3K/Akt signaling through targeting FOXO1.

This study focused on miR-486-5p in atrial fibrillation (AF) evaluating its clinical significance and revealing its regulatory mechanism in cardiac fibroblasts, aiming to explore a novel biomarker for AF. The study enrolled 131 AF patients and 77 non-AF individuals. With the help of polymerase chain reaction (PCR), the expression of miR-486-5p was evaluated. The significance of miR-486-5p in the diagnosis of AF and the occurrence of left atrial fibrosis (LAF) was assessed by receiver operating curve (ROC) and logistic analyses. The regulatory effect and mechanism of miR-486-5p on cardiac fibrosis were investigated in human cardiac fibroblasts treated with angiotensin II. miR-486-5p was significantly upregulated in AF patients and discriminated AF patients from non-AF individuals. Increasing miR-486-5p showed a significant association with decreasing left ventricular ejection fraction (LVEF), increasing left atrial diameter (LAD) and left ventricular end-diastolic diameter (LVEDd), and the high incidence of LAF in AF patients. Moreover, miR-486-5p was identified as a risk factor for LAF and could distinguish AF patients with LAF and without LAF. In cardiac fibroblasts, angiotensin II induced the upregulation of miR-486-5p and promoted cell proliferation, migration, and collagen synthesis. miR-486-5p negatively regulated forkhead box O1 (FOXO1) and its knockdown could reverse the promoted effect of angiotensin II. FOXO1 alleviated the effect of miR-486-5p, and the miR-486-5p/FOXO1 could activate PI3K/Akt signaling. The activation of PI3K/Akt signaling alleviated the enhanced proliferation, migration, and collagen synthesis of cardiac fibroblasts induced by angiotensin II, and its inhibition showed opposite effects. Increased miR-486-5p served as a biomarker for the diagnosis and development prediction of AF. miR-486-5p regulated cardiac fibroblast viability and collagen synthesis via modulating the PI3K/Akt signaling through targeting FOXO1.

Steering cell orientation through light-based spatiotemporal modulation of the mechanical environment.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved,in vitrotechniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3Din vitromodel systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

Circulating soluble fibroblast activation protein (FAP) levels are independent of cardiac and extra-cardiac FAP expression determined by targeted molecular imaging in patients with myocardial FAP activation.

INTRODUCTION: Tissue Fibroblast Activation Protein alpha (FAP) is overexpressed in various types of acute and chronic cardiovascular disease. A soluble form of FAP has been detected in human plasma, and low circulating FAP concentrations are associated with increased risk of death in patients with acute coronary syndrome. However, little is known about the regulation and release of FAP from fibroblasts, and whether circulating FAP concentration is associated with tissue FAP expression. This study characterizes the release of FAP in human cardiac fibroblasts (CF) and analyzes the association of circulating FAP concentrations with in vivo tissue FAP expression in patients with acute (ST-segment elevation myocardial infarction, STEMI) and chronic (severe aortic stenosis, AS) myocardial FAP expression. METHODS AND RESULTS: FAP was released from CF in a time- and concentration-dependent manner. FAP concentration was higher in supernatant of TGFß-stimulated CF, and correlated with cellular FAP concentration. Inhibition of metallo- and serine-proteases diminished FAP release in vitro. Median FAP concentrations of patients with acute (77 ng/mL) and chronic (75 ng/mL, p = 0.50 vs. STEMI) myocardial FAP expression did not correlate with myocardial nor extra-myocardial nor total FAP volume (P ≥ 0.61 in all cases) measured by whole-body FAP-targeted positron emission tomography. CONCLUSION: We describe a time- and concentration dependent, protease-mediated release of FAP from cardiac fibroblasts. Circulating FAP concentrations were not associated with increased in vivo tissue FAP expression determined by molecular imaging in patients with both chronic and acute myocardial FAP expression. These data suggest that circulating FAP and tissue FAP expression provide complementary, non-interchangeable information.

3D bioprinted aged human post-infarct myocardium tissue model.

Background and Aims: Fibrotic tissue formed after myocardial infarction (MI) can be as detrimental as MI itself. However, current in vitro cardiac fibrosis models fail to recapitulate the complexities of post-MI tissue. Moreover, although MI and subsequent fibrosis is most prominent in the aged population, the field suffers from inadequate aged tissue models. Herein, an aged human post-MI tissue model, representing the native microenvironment weeks after initial infarction, is engineered using three-dimensional bioprinting via creation of individual bioinks to specifically mimic three distinct regions: remote, border, and scar. Methods: The aged post-MI tissue model is engineered through combination of gelatin methacryloyl, methacrylated hyaluronic acid, aged type I collagen, and photoinitiator at variable concentrations with different cell types, including aged human induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, cardiac fibroblasts, and cardiac myofibroblasts, by introducing a methodology which utilizes three printheads of the bioprinter to model aged myocardium. Then, using cell-specific proteins, the cell types that comprised each region are confirmed using immunofluorescence. Next, the beating characteristics are analyzed. Finally, the engineered aged post-MI tissue model is used as a benchtop platform to assess the therapeutic effects of stem cell-derived extracellular vesicles on the scar region. Results: As a result, high viability (>74%) was observed in each region of the printed model. Constructs demonstrated functional behavior, exhibiting a beating velocity of 6.7 µm/s and a frequency of 0.3 Hz. Finally, the effectiveness of hiPSC-EV and MSC-EV treatment was assessed. While hiPSC-EV treatment showed no significant changes, MSC-EV treatment notably increased cardiomyocyte beating velocity, frequency, and confluency, suggesting a regenerative potential. Conclusion: In conclusion, we envision that our approach of modeling post-MI aged myocardium utilizing three printheads of the bioprinter may be utilized for various applications in aged cardiac microenvironment modeling and testing novel therapeutics.

The Senescent Heart-"Age Doth Wither Its Infinite Variety".

Cardiovascular diseases are a leading cause of morbidity and mortality world-wide. While many factors like smoking, hypertension, diabetes, dyslipidaemia, a sedentary lifestyle, and genetic factors can predispose to cardiovascular diseases, the natural process of aging is by itself a major determinant of the risk. Cardiac aging is marked by a conglomerate of cellular and molecular changes, exacerbated by age-driven decline in cardiac regeneration capacity. Although the phenotypes of cardiac aging are well characterised, the underlying molecular mechanisms are far less explored. Recent advances unequivocally link cardiovascular aging to the dysregulation of critical signalling pathways in cardiac fibroblasts, which compromises the critical role of these cells in maintaining the structural and functional integrity of the myocardium. Clearly, the identification of cardiac fibroblast-specific factors and mechanisms that regulate cardiac fibroblast function in the senescent myocardium is of immense importance. In this regard, recent studies show that Discoidin domain receptor 2 (DDR2), a collagen-activated receptor tyrosine kinase predominantly located in cardiac fibroblasts, has an obligate role in cardiac fibroblast function and cardiovascular fibrosis. Incisive studies on the molecular basis of cardiovascular aging and dysregulated fibroblast function in the senescent heart would pave the way for effective strategies to mitigate cardiovascular diseases in a rapidly growing elderly population.

Roles of Integrin in Cardiovascular Diseases: From Basic Research to Clinical Implications.

Cardiovascular diseases (CVDs) pose a significant global health threat due to their complex pathogenesis and high incidence, imposing a substantial burden on global healthcare systems. Integrins, a group of heterodimers consisting of α and ß subunits that are located on the cell membrane, have emerged as key players in mediating the occurrence and progression of CVDs by regulating the physiological activities of endothelial cells, vascular smooth muscle cells, platelets, fibroblasts, cardiomyocytes, and various immune cells. The crucial role of integrins in the progression of CVDs has valuable implications for targeted therapies. In this context, the development and application of various integrin antibodies and antagonists have been explored for antiplatelet therapy and anti-inflammatory-mediated tissue damage. Additionally, the rise of nanomedicine has enhanced the specificity and bioavailability of precision therapy targeting integrins. Nevertheless, the complexity of the pathogenesis of CVDs presents tremendous challenges for monoclonal targeted treatment. This paper reviews the mechanisms of integrins in the development of atherosclerosis, cardiac fibrosis, hypertension, and arrhythmias, which may pave the way for future innovations in the diagnosis and treatment of CVDs.

Mechanism of sodium butyrate, a metabolite of gut microbiota, regulating cardiac fibroblast transdifferentiation via the NLRP3/Caspase-1 pyroptosis pathway.

BACKGROUND: Cardiac fibroblasts (CFs) are activated after initial injury, and then differentiate into myofibroblasts (MFs), which play a pivotal role as the primary mediator cells in pathological remodeling. Sodium butyrate (NaB), being a metabolite of gut microbiota, exhibits anti-inflammatory property in local therapies on sites other than the intestine. Thus, this study aimed to probe the mechanism by which NaB regulates CFs transdifferentiation through the NLRP3/Caspase-1 pyroptosis pathway. METHODS: CFs were cultured in vitro and induced into MFs by TGFß1. CFs were identified by immunofluorescence labelling technique of vimentin and α-SMA, followed by treatment with NaB or NLRP3 inflammasome inhibitor (CY-09) and its activator [nigericin sodium salt (NSS)]. The expression levels of α-SMA, GSDMD-N/NLRP3/cleaved Caspase-1 proteins, and inflammatory factors IL-1ß/IL-18/IL-6/IL-10 were determined using immunofluorescence, Western blot and ELISA. Cell proliferation and migration were evaluated using the CCK-8 assay and the cell scratch test, respectively. RESULTS: Following the induction of TGFß1, CFs exhibited increased expression levels of α-SMA proteins and IL-6/IL-10, as well as cell proliferative and migratory abilities. TGFß1 induced CFs to differentiate into MFs, while NaB inhibited this differentiation. NaB inactivated the NLRP3/Caspase-1 pyroptosis pathway. CY-09 demonstrated inhibitory effects on the NLRP3/Caspase-1 pyroptosis pathway, leading to a reduction in TGFß1-induced CFs transdifferentiation. NSS activated the NLRP3/Caspase-1 pyroptosis pathway, and thus partially counteracting the inhibitory effect of intestinal microbiota metabolite NaB on CFs transdifferentiation. CONCLUSION: NaB, a metabolite of the gut microbiota, inhibited the activation of the NLRP3/Caspase-1 pyroptosis pathway in TGFß1-induced CFs, repressed the transdifferentiation of CFs into MFs.

In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts.

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5 days later re-stimulated with Pam3CSK for 24 h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5 days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1ß, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.

M2 macrophage-derived paracrine factor TNFSF13 affects the fibrogenic alterations in endothelial cells and cardiac fibroblasts by mediating the NF-κB and Akt pathway.

Heart failure remains a global threaten to public health, cardiac fibrosis being a crucial event during the development and progression of heart failure. Reportedly, M2 macrophages might affect endothelial cell (ECs) and fibroblast proliferation and functions through paracrine signaling, participating in myocardial fibrosis. In this study, differentially expressed paracrine factors between M0/1 and M2 macrophages were analyzed and the expression of TNFSF13 was most significant in M2 macrophages. Culture medium (CM) of M2 (M2 CM) coculture to ECs and cardiac fibroblasts (CFbs) significantly promoted the cell proliferation of ECs and CFbs, respectively, and elevated α-smooth muscle actin (α-SMA), collagen I, and vimentin levels within both cell lines; moreover, M2 CM-induced changes in ECs and CFbs were partially abolished by TNFSF13 knockdown in M2 macrophages. Lastly, the NF-κB and Akt signaling pathways were proved to participate in TNFSF13-mediated M2 CM effects on ECs and CFbs. In conclusion, TNFSF13, a paracrine factor upregulated in M2 macrophages, could mediate the promotive effects of M2 CM on EC and CFb proliferation and fibrogenic alterations.