Primary Mouse Cardiac Fibroblasts Research Articles

IL-1β-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling

The source and roles of fibroblasts and T-cells during maladaptive remodeling and myocardial fibrosis in the setting of pulmonary arterial hypertension (PAH) have been long debated. We demonstrate, using single-cell mass cytometry, a subpopulation of endogenous human cardiac fibroblasts expressing increased levels of CD4, a helper T-cell marker, in addition to myofibroblast markers distributed in human fibrotic RV tissue, interstitial and perivascular lesions in SUGEN/Hypoxia (SuHx) rats, and fibroblasts labeled with pdgfrα CreERt2/+ in R26R-tdTomato mice. Recombinant IL-1β increases IL-1R, CCR2 receptor expression, modifies the secretome, and differentiates cardiac fibroblasts to form CD68-positive cell clusters. IL-1β also activates stemness markers, such as NANOG and SOX2, and genes involved in dedifferentiation, lymphoid cell function and metabolic reprogramming. IL-1β induction of lineage traced primary mouse cardiac fibroblasts causes these cells to lose their fibroblast identity and acquire an immune phenotype. Our results identify IL-1β induced immune-competency in human cardiac fibroblasts and suggest that fibroblast secretome modulation may constitute a therapeutic approach to PAH and other diseases typified by inflammation and fibrotic remodeling.

Abstract 14400: Tipifarnib Mediated Reduction of Circulating Exosomes Inhibit Pressure Overload-Induced Cardiac Remodeling and Hypertrophy

Circulating extracellular vesicles, including exosomes, and their content have been implicated in cardiac tissue remodeling during cardiovascular disease progression. However, their role in cardiac hypertrophy and heart failure is very limited. Therefore, we investigated the role of circulating exosomes in cardiac dysfunction and remodeling in a transverse aortic constriction (TAC) model of heart failure using a recently identified exosome biogenesis inhibitor, Tipifarnib. Ten-week-old C57BL6J male mice were randomized into three groups i.e., Sham, TAC and Tipifarnib treated (10 mg/kg) TAC (TAC-Tip). The untreated TAC mice showed a significant increase in circulating exosomes along with gradual development of cardiac hypertrophy accompanied with reduced cardiac functions and increased fibrosis. On the contrary, TAC-Tip mice showed a significant reduction in circulating exosomes and remarkably improved cardiac left ventricular functions, reduced cardiac hypertrophy, fibrosis, and systemic inflammation. Strikingly, when primary mouse cardiac fibroblast cells were treated with exosomes derived from serum of TAC mice, the fibrosis related genes expression was significantly amplified compared to those treated with exosomes from TAC-Tip mice serum. Further, systemic injections of TAC-serum exosomes in normal, uninjured mice for 8 weeks, reduced cardiac function and increased cardiomyocyte hypertrophy. Small RNA sequencing analysis of serum exosomes from TAC and TAC-Tip mice displayed an extensively differential exosomal miRNAs profile. To evaluate the crucial role of miRNA in the circulating exosomes, we selected miR 331-5p based on the most differentially expressed miR and validated its expression in heart tissue and serum exosomes of Sham, TAC and TAC-Tip mice. Functional studies showed miR 331-5p inhibited HOXC8, a critical regulator of fibrosis. Overall, our studies reveal a mechanism by which treatment of Tipifarnib in TAC mice, alters not just the exosome numbers but also circulating exosomes content including miR 331-5p that directly inhibits HOXC8 and reduces fibrosis. Thus, Tipifarnib mediated inhibition of circulating exosomes may serve as a viable strategy to prevent progressive cardiac remodeling to heart failure.

Insulin-Like Growth Factor 1 Receptor Deficiency Alleviates Angiotensin II-Induced Cardiac Fibrosis Through the Protein Kinase B/Extracellular Signal-Regulated Kinase/Nuclear Factor-κB Pathway.

Background The renin-angiotensin system plays a crucial role in the development of heart failure, and Ang II (angiotensin II) acts as the critical effector of the renin-angiotensin system in regulating cardiac fibrosis. However, the mechanisms of cardiac fibrosis are complex and still not fully understood. IGF1R (insulin-like growth factor 1 receptor) has multiple functions in maintaining cardiovascular homeostasis, and low-dose IGF1 treatment is effective in relieving Ang II-induced cardiac fibrosis. Here, we aimed to investigate the molecular mechanism of IGF1R in Ang II-induced cardiac fibrosis. Methods and Results Using primary mouse cardiac microvascular endothelial cells and fibroblasts, in vitro experiments were performed. Using C57BL/6J mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated IGF1R heterozygous knockout (Igf1r+/-) mice, cardiac fibrosis mouse models were induced by Ang II for 2 weeks. The expression of IGF1R was examined by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot. Mice heart histologic changes were evaluated using Masson and picro sirius red staining. Fibrotic markers and signal molecules indicating the function of the Akt (protein kinase B)/ERK (extracellular signal-regulated kinase)/nuclear factor-κB pathway were detected using quantitative reverse transcription polymerase chain reaction and Western blot. RNA sequencing was used to explore IGF1R-mediated target genes in the hearts of mice, and the association of IGF1R and G-protein-coupled receptor kinase 5 was identified by coimmunoprecipitation. More important, blocking IGF1R signaling significantly suppressed endothelial-mesenchymal transition in primary mouse cardiac microvascular endothelial cells and mice in response to transforming growth factor-β1 or Ang II, respectively. Deficiency or inhibition of IGF1R signaling remarkably attenuated Ang II-induced cardiac fibrosis in primary mouse cardiac fibroblasts and mice. We further observed that the patients with heart failure exhibited higher blood levels of IGF1 and IGF1R than healthy individuals. Moreover, Ang II treatment significantly increased cardiac IGF1R in wild type mice but led to a slight downregulation in Igf1r+/- mice. Interestingly, IGF1R deficiency significantly alleviated cardiac fibrosis in Ang II-treated mice. Mechanistically, the phosphorylation level of Akt and ERK was upregulated in Ang II-treated mice, whereas blocking IGF1R signaling in mice inhibited these changes of Akt and ERK phosphorylation. Concurrently, phosphorylated p65 of nuclear factor-κB exhibited similar alterations in the corresponding group of mice. Intriguingly, IGF1R directly interacted with G-protein-coupled receptor kinase 5, and this association decreased ≈50% in Igf1r+/- mice. In addition, Grk5 deletion downregulated expression of the Akt/ERK/nuclear factor-κB signaling pathway in primary mouse cardiac fibroblasts. Conclusions IGF1R signaling deficiency alleviates Ang II-induced cardiac fibrosis, at least partially through inhibiting endothelial-mesenchymal transition via the Akt/ERK/nuclear factor-κB pathway. Interestingly, G-protein-coupled receptor kinase 5 associates with IGF1R signaling directly, and it concurrently acts as an IGF1R downstream effector. This study suggests the promising potential of IGF1R as a therapeutic target for cardiac fibrosis.

PS-B06-8: EXPLORING THE ROLE OF NECROPTOSIS IN HYPERTENSION

Objective: Altered immune cell activation plays a key role in promoting hypertension. A major question remains as to the immune system becomes activated during hypertension. A possibility is necroptosis, which a newly described form of cell death alerts the immune system to dying cells, causing inflammation. Using mice genetically unable to undergo necroptosis, we aimed to determine whether necroptosis contributes to inflammation and fibrosis or alters blood pressure during hypertension. To determine whether factors present during hypertension impact necroptosis, we examined whether Angiotensin II sensitizes human and murine cells to necroptosis. Design and Methods: Monocytes were sorted from peripheral blood mononuclear cells from 3 normotensive and 3 untreated hypertensive participants diagnosed with 24-h blood pressure (BP) monitoring. Necroptosis was induced in human monocytes using hTNFα (10ug/ml), SMAC mimetic (500 nM), zVAD (20uM), and in primary neonatal murine cardiomyocytes and fibroblasts from 3 litters of C57BL/6J mice using mTNFα (10ug/ml), SMAC mimetic (100 nM), zVAD (10uM), in the presence or absence of angiotensin II (Ang-II, 5 ug/ml). Viability of human cells was measured using propidium iodide by flow cytometry, and in murine cells using CellTitre-Glo Luminescent Cell Viability Assay. Hypertension was induced in male Mlkl-/- mice, which cannot undergo necroptosis, and wild-type (WT) littermate controls by implanting minipumps containing Ang-II (0.5 mg/kg/day for 28 days; n = 6/genotype). Blood pressure (BP) was measured weekly by tail-cuff. Results: Monocytes from hypertensive participants were not more susceptible to necroptosis than normotensive participants (% viability ± SD; 48% ± 0.11 vs 51% ± 0.04, P = 0.79). This was not altered by Ang-II treatment in normotensive (51% ± 0.04 vs 56% ± 0.11 with Ang-II, P = 0.98) or hypertensive participants (48% ± 0.11 vs 56% ± 0.18 with Ang-II, P = 0.92). Moreover, Ang-II did not alter the viability of primary murine cardiomyocytes in vitro (42% ± 23 vs 40.1 ± 17.8 with Ang-II, P = 0.79). Primary mouse cardiac fibroblasts were unable to undergo necroptosis. In vivo, there was no difference in BP of Ang-II-treated Mlkl-/- and WT control mice at 4 weeks (P = 0.87). Conclusions: Our findings showed that, compared to normotensive participants, monocytes from hypertensive participants were not more susceptible to necroptosis. Furthermore, Ang-II did not directly act on the necroptosis pathway in both mouse cardiac fibroblasts or human monocytes. Changes in BP were not observed between WT and MLKL knockout mice upon Ang-II treatment, suggesting necroptosis does not impact BP regulation. Further analyses are being performed to determine whether inflammation or tissue fibrosis are altered in the absence of necroptosis.

Inhibition of Wdr5 Attenuates Ang-II-Induced Fibroblast-to-Myofibroblast Transition in Cardiac Fibrosis by Regulating Mdm2/P53/P21 Pathway.

Cardiac fibrosis is an important pathological process in many diseases. Wdr5 catalyzes the trimethylation of lysine K4 on histone H3. The effects of Wdr5 on the cardiac fibrosis phenotype and the activation or transformation of cardiac fibroblasts were investigated by Ang-II-infused mice by osmotic mini-pump and isolated primary neonatal rat cardiac fibroblasts. We found that the Wdr5 expression and histone H3K4me3 modification were significantly increased in Ang-II-infused mice. By stimulating primary neonatal rat cardiac fibroblasts with Ang II, we detected that the expression of Wdr5 and H3K4me3 modification were also significantly increased. Two Wdr5-specific inhibitors, and the lentivirus that transfected Sh-Wdr5, were used to treat primary mouse cardiac fibroblasts, which not only inhibited the histone methylation by Wdr5 but also significantly reduced the activation and migration ability of Ang-II-treated fibroblasts. To explore its mechanism, we found that the inhibition of Wdr5 increased the expression of P53, P21. Cut&Tag-qPCR showed that the inhibition of Wdr5 significantly reduced the enrichment of H3K4me3 in the Mdm2 promoter region. For in vivo experiments, we finally proved that the Wdr5 inhibitor OICR9429 significantly reduced Ang-II-induced cardiac fibrosis and increased the expression of P21 in cardiac fibroblasts. Inhibition of Wdr5 may mediate cardiac fibroblast cycle arrest through the Mdm2/P53/P21 pathway and alleviate cardiac fibrosis.

Tangshen Formula Improves Diabetes-Associated Myocardial Fibrosis by Inhibiting TGF-β/Smads and Wnt/β-Catenin Pathways.

Cardiovascular disease has become the main cause of death among complications of diabetes. Myocardial fibrosis is a crucial pathological change of cardiovascular disease. Tangshen Formula (TSF) shows a good clinical effect in the treatment of diabetic kidney disease (DKD). However, whether TSF alleviates diabetes-associated myocardial fibrosis is still unknown. In the present research, we studied the effect and mechanism of TSF in the treatment of myocardial fibrosis in vivo and in vitro. We found that TSF treatment significantly downregulates myocardial fibrosis-related markers, including collagens I and III, and α-SMA. TSF also protects primary mouse cardiac fibroblast (CF) from transforming growth factor-β1- (TGF-β1-) induced damage. Moreover, TSF decreased the expression levels of TGF-β/Smad-related genes (α-SMA, collagens I and III, TGF-β1, and pSmad2/3), and increased Smad7 gene expression. Finally, TSF decreased the expressions of wnt1, active-β-catenin, FN, and MMP7 to regulate the Wnt/β-catenin pathway. Taken together, TSF seems to attenuate myocardial fibrosis in KKAy mice by inhibiting TGF-β/Smad2/3 and Wnt/β-catenin signaling pathways.

FABP5 Deficiency Impairs Mitochondrial Function and Aggravates Pathological Cardiac Remodeling and Dysfunction.

Fatty acid-binding protein 5 (FABP5) is an important member of the FABP family and plays a vital role in the metabolism of fatty acids. However, few studies have examined the role of FABP5 in pathological cardiac remodeling and heart failure. The aim of this study was to explore the role of FABP5 in transverse aortic constriction (TAC)-induced pathological cardiac remodeling and dysfunction in mice. Quantitative RT-PCR (qRT-PCR) and western blotting (WB) analysis showed that the levels of FABP5 mRNA and protein, respectively, were upregulated in hearts of the TAC model. Ten weeks after TAC in FABP5 knockout and wild type control mice, echocardiography, histopathology, qRT-PCR, and WB demonstrated that FABP5 deficiency aggravated cardiac injury (both cardiac hypertrophy and fibrosis) and dysfunction. In addition, transmission electron microscopy, ATP detection, and WB revealed that TAC caused severe impairment to mitochondria in the hearts of FABP5-deficient mice compared with that in control mice. When FABP5 was downregulated by siRNA in primary mouse cardiac fibroblasts, FABP5 silencing increased oxidative stress, reduced mitochondrial respiration, and increased the expression of myofibroblast activation marker genes in response to treatment with transforming growth factor-β. Our findings demonstrate that FABP5 deficiency aggravates cardiac pathological remodeling and dysfunction by damaging cardiac mitochondrial function.

MiR-34a attenuates myocardial fibrosis in diabetic cardiomyopathy mice via targeting Pin-1.

Diabetic cardiomyopathy (DCM) is characterized by myocardial hypertrophy and fibrosis. This study aimed to investigate the effects of microRNA (miR)-34a on myocardial fibrosis in DCM and its potential mechanism of targeting Pin-1 signaling. Vimentin and Pin-1 proteins in mouse cardiac tissues were detected by immunohistochemical staining. Locked nucleic acid in situ hybridization was used to measure miR-34a expression in cardiac tissues. Primary mouse cardiac fibroblasts (CFs) were transfected with a mimics control/miR-34a mimics or Pin-1 plasmid and cultured in high-glucose (HG) Dulbecco's modified Eagle's medium. The miR-34a levels were measured by quantitative polymerase chain reaction. The apoptosis and viability of transfected cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling and Cell Counting Kit-8 assays respectively. A cell migration experiment and dual-luciferase reporter assay were also performed. The body weight and fasting blood glucose of DCM mice were significantly higher than those in the control (CTL) group. In addition, DCM mice had decreased serum insulin levels and impaired cardiac function. The number of CFs in the DCM group was higher than in the CTL group and Pin-1 expression was upregulated. The expression level of miR-34a in the cardiac tissue of mice in the DCM group was obviously downregulated compared with the CTL group. The HG stimulation of CFs for 48 h significantly downregulated the expression level of miR-34a and was associated with increased Type I collagen expression, cell viability, and migration and decreased apoptosis. However, these effects could be reversed by overexpressing miR-34a in HG-induced CFs. Furthermore, we found that Pin-1 was a direct target of miR-34a. Our results suggest that miR-34a can attenuate myocardial fibrosis in DCM by reducing Type I collagen production, cell viability, and migration and increasing the apoptosis of CFs by targeting Pin-1 signaling.

EphrinA1-Fc Attenuates Ventricular Remodeling and Dysfunction in Chronically Nonreperfused WT but not EphA2-R-M mice.

Background: EphrinA1-Fc abolishes acute I/R injury and attenuates nonreperfused cardiac injury 4 days after permanent occlusion in mice. The goal of this study was to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of coronary artery ligation, to determine the degree to which early salvage effects translate to reduced adverse remodeling after 4 weeks of nonreperfused myocardial infarction (MI) in wild-type B6 and EphA2-R-M (EphA2 receptor null) mice. Methods: At 4 weeks post-MI, echocardiography, histologic and immunohistochemical analyses of B6 mouse hearts were performed. Primary mouse cardiac fibroblasts (FBs) isolated from B6 mice cultured in the presence of low and high dose ephrinA1-Fc, both with and without pro-fibrotic TGF-β stimulation and Western blots, were probed for relative expression of remodeling proteins MMP-2, MMP-9 and TIMP-1, in addition to DDR2 and (p)SMAD2/3/totalSMAD2/3. Results: EphrinA1-Fc preserved a significant degree of contractile function, decreased adverse left ventricular remodeling, attenuated excessive compensatory hypertrophy, and decreased interstitial fibrosis in wild-type (WT) B6 mouse hearts. In contrast, most of these parameters were poorer in ephrinA1-Fc-treated EphA2-R-M mice. Of note, fibrosis was proportionately decreased, implying that other EphA receptor(s) are more important in regulating the pro-fibrotic response. Primary FBs showed disparate alteration of MMP-2, MMP-9 and TIMP-1, as well as DDR2 and p-SMAD2/3/totalSMAD2/3, which indicates that matrix remodeling and cardiac fibrosis in the injured heart are influenced by ephrinA1-Fc. Conclusion: This study demonstrates the capacity of a single administration of ephrinA1-Fc at the onset of injury to attenuate long-term nonreperfused post-MI ventricular remodeling that results in progressive heart failure, and the important role of EphA2 in mitigating the deleterious effects.

P6296The role of extracellular matrix protein 1 (ECM1) - a novel link between inflammation and cardiac fibrosis

Abstract Introduction Cardiac fibrosis is a severe consequence of cardiovascular disease and aging, in which we currently have no effective treatments. The mechanisms underpinning the development of cardiac fibrosis remains poorly understood. Our preliminary data suggested extracellular matrix protein 1 (ECM1) is involved in cardiac fibrosis. We therefore aimed to investigate the role of ECM1 in several fibrotic cardiac diseases. Methods Young and ageing (3m/18m) male C57BL/6 mice, and primary mouse cardiac fibroblast (cFB) cultures, commercial human cardiac fibroblasts (Hu-cFB), human coronary artery endothelial cell (HCAEC)/smooth muscle cell (HCASMC), and human cardiac myocyte (HCM) cell lines were used. Young mice were subject to myocardial infarction (MI, 3-day/28-day, n=6/6), or pressure overload (TAC, 3-day/13-week, n=4/4). Left ventricle (LV) was collected at all time-points, and at 18m (ageing; n=3). Spleen and bone marrow was extracted from young control mice. Hu-cFB cells were treated with recombinant ECM1 (20ng/ml) for either 10, 30 or 50 min, or 48h. Immunoblotting was conducted on all samples, qPCR on LV tissue only, density gradient centrifugation and multicolour flow cytometry coupled with fluorescent ECM1 mRNA in-situ hybridisation (FISH-Flow) on bone marrow cells. Results ECM1 expression was upregulated in ageing LV (mRNA 2.2±0.1-fold, p=0.0002; protein 2.0-fold, p=0.0006), day-3 post-MI (mRNA, 4.9±2.0-fold, p=0.004; protein, 3.0-fold, p=0.004), a trend of ECM1 upregulation was observed at day-28 post-MI (mRNA, 13.2±12.0-fold, p=0.003; protein, 1.8-fold, p=0.2), but no change post-TAC. Both ERK1/2 and AKT phosphorylation was upregulated 10 min post-ECM1 treatment of Hu-cFBs (ERK1/2, 2.0-fold, p<0.0001; AKT, 1.9-fold, p<0.0001), and Collagen-I protein expression was upregulated 48h post-ECM1 treatment (1.9-fold, p=0.004). ECM1 protein was not expressed in cFB, Hu-cFB, HCAEC, HCASMC or HCM, however ECM1 protein was highly expressed in spleen and bone marrow; to a greater extent in granulocytes compared to monocytes (p=0.004). tSNE analysis of ECM1 mRNA FISH-Flow revealed ECM1+ are highly granular, moderate to large in size, and express (to varying levels) CD45, CD11b, CD11c, F4/80, Ly6-C, Ly-6G, and FcεrI-α. However ECM1+ cells did not express markers indicative of smaller cells (CD3 or MHC II). Conclusions These data demonstrate that ECM1 plays a role in ageing and post-MI fibrosis. Although ECM1 was not produced by resident cardiac cells, it was highly expressed in spleen and bone marrow; specifically, large, granular bone marrow cell sub-types such as granulocytes and/or macrophages. Our data suggest ECM1 is expressed by cardiac infiltrating leukocytes to provoke fibroblast collagen expression in a disease specific manner; potentially via the ERK1/2 and/or AKT pathway activation. Therefore, ECM1 warrants further investigation, and may be a promising target for the treatment of fibrotic cardiac diseases. Acknowledgement/Funding John hunter hospital charitable trust, Hunter medical research institute (HMRI) grants

Abstract 590: EphrinA1-Fc Attenuates Dysfunction and Fibrosis in Nonreperfused Myocardium at 4 weeks Post-MI in WT B6 but Not EphA2-R Mutant Mice

Each year, an estimated 805,000 US citizens have a new or recurrent myocardial infarction (MI), causing over 360,000 deaths. We have previously reported that a single intramyocardial administration of recombinant ephrinA1-Fc, a member of the ephrinA/EphA family of membrane-anchored receptor tyrosine kinases, significantly reduced ischemic injury and remodeling 4 days in nonreperfused WT B6 mice via reduced apoptosis and inflammation and increased autophagic flux. After acute ischemia/reperfusion (30 min I/24hr or 4 days R) in WT B6 mice, ephrinA1-Fc demonstrated anti-inflammatory effects coupled with complete functional preservation due to modulation of cardiomyocyte ultrastructure and metabolism. The goal of this study is to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of permanent coronary artery ligation to maintain the structural and functional salvage effects through the wound healing process to stable scar formation in both B6 and EphA2-R-M (EphA2 receptor null mutant) mice. At 4 weeks post-MI in WT B6 mice, ephrinA1-Fc improved ejection fraction by 11%, attenuated cardiomyocyte hypertrophy by 38%, and decreased interstitial fibrosis by 35%. In contrast, these parameters were not different or worsened in EphA2-R-M mice treated with ephrinA1-Fc, suggesting that this receptor plays an important role in ephrinA1-Fc-mediated mitigation of the deleterious ventricular remodeling caused by permanent coronary artery occlusion. To further investigate the effects of ephrinA1-Fc directly on fibroblast function, isolated primary mouse cardiac fibroblasts were cultured with ephrinA1-Fc both with and without pro-fibrotic TGF-β stimulation to determine the direct effects of ephrinA1-Fc on col I and III production, remodeling mediators MMP-2, MMP-9, and TIMP-1, as well as signaling molecules DDR2 and SMADs2/3 as potential mechanistic pathways involved. The results of these assays are forthcoming. This study demonstrates the potential of a single administration of ephrinA1-Fc to not only attenuate acute ischemic injury but also mitigate pathological accumulation of interstitial fibrosis and remodeling, thereby reducing ventricular stiffening and the resultant progression to heart failure.

Mammalian Target of Rapamycin (mTOR) and the Proteasome Attenuates IL-1β Expression in Primary Mouse Cardiac Fibroblasts.

Background: IL-1β is a highly potent pro-inflammatory cytokine and its secretion is tightly regulated. Inactive pro-IL-1β is transcribed in response to innate immune receptors activating NFκB. If tissue damage occurs, danger signals released from necrotic cells, such as ATP, can activate NLRP3-inflammasomes (multiprotein complexes consisting of NLRP3, ASC, and active caspase-1) which cleaves and activates pro-IL-1β. NLRP3 activation also depends on NEK7 and mitochondrial ROS-production. Thus, IL-1β secretion may be regulated at the level of each involved component. We have previously shown that NLRP3-dependent IL-1β release can be induced in cardiac fibroblasts by pro-inflammatory stimuli. However, anti-inflammatory mechanisms targeting IL-1β release in cardiac cells have not been investigated. mTOR is a key regulator of protein metabolism, including autophagy and proteasome activity. In this study we explored whether autophagy or proteasomal degradation are regulators of NLRP3 inflammasome activation and IL-1β release from cardiac fibroblasts.Methods and Results: Serum starvation selectively reduced LPS/ATP-induced IL-1β secretion from cardiac fibroblasts. However, no other inflammasome components, nor mitochondrial mass, were affected. The mTOR inhibitor rapamycin restored pro-IL-1β protein levels as well as LPS/ATP-induced IL-1β release from serum starved cells. However, neither serum starvation nor rapamycin induced autophagy in cardiac fibroblasts. Conversely, chloroquine and bafilomycin A (inhibitors of autophagy) and betulinic acid (a proteasome activator) effectively reduced LPS-induced pro-IL-1β protein levels. Key findings were reinvestigated in human monocyte-derived macrophages.Conclusion: In cardiac fibroblasts, mTOR inhibition selectively favors pro-IL-1β synthesis while proteasomal degradation and not autophagy is the major catabolic anti-inflammatory mechanism for degradation of this cytokine.

Abstract 12745: Interleukin-4 Promotes Procollagen Type-I Alpha 1 Gene Expression via Activator Protein 1 Activation in Cardiac Fibroblasts

Interleukin-4 (IL-4) activates the signal transducer and activator of transcription 6 signaling pathway, resulting in the upregulation of procollagen genes and promotion of collagen production in cardiac fibroblasts (CFs). In addition, transcription factors may participate in the transcriptional activation of collagen genes. We hypothesize that IL-4 activates activator protein 1 (AP-1) via c-Jun phosphorylation, promoting transcriptional activation of the procollagen type-I alpha 1 (Col1α1) gene and collagen type-I expression in CFs. Primary mouse CFs were used. Western blot analyses revealed that IL-4 (10 ng/ml) increased phosphorylation of c-Jun, a subunit of AP-1. Additionally, our experiments utilizing electrophoretic mobility shift assay (EMSA) revealed a significant increase in DNA binding activity for AP-1 in IL-4-treated CFs compared to control cells (1.00±0.07 to 2.14±0.38 fold change versus control, p <0.05). Here we aimed to determine whether IL-4-activated AP-1 participates in transcriptional activation of the Col1α1 gene leading to increased collagen type-I expression in CFs. To that end, we blocked AP-1 with shRNA lentiviral particles specific for c-Jun in CFs, detected Col1α1 mRNA by qRT-PCR, and measured collagen type-I by Western blot. We found that IL-4 significantly induced Col1α1 mRNA expression after incubation with control shRNA-transduced cells for 6 hours (1.01±0.01 to 1.52±0.07 fold change versus control, p <0.005), and it increased collagen type-I expression after 24 hours of incubation (1.00±0.01 to 1.95±0.12 fold change versus control, p <0.005). In contrast, IL-4 did not increase mRNA levels of Col1α1 (1.09±0.02 to 0.98±0.03, p >0.05) and the collagen type-I (0.95±0.05 to 1.01±0.10, p >0.05) in c-Jun shRNA-transfected cells. Our data demonstrate that IL-4-activated AP-1, an ubiquitous transcription factor containing phosphorylated c-Jun, enhanced Col1α1 gene expression and collagen type-I expression in CFs. Data generated from this study provides a novel molecular basis for potential treatment of cardiac fibrosis by targeting IL-4-induced signaling in CFs.

Resveratrol ameliorates myocardial fibrosis by inhibiting ROS/ERK/TGF-β/periostin pathway in STZ-induced diabetic mice.

BackgroundMyocardial fibrosis is an essential hallmark of diabetic cardiomyopathy (DCM) contributing to cardiac dysfunctions. Resveratrol, an antioxidant, exerts its anti-fibrotic effect via inhibition of oxidative stress, while the underlying molecular mechanism remains largely elusive. Periostin, a fibrogenesis matricellular protein, has been shown to be associated with oxidative stress. In the present study, we investigated the role of periostin in anti-fibrotic effect of resveratrol in streptozocin (STZ)-induced diabetic heart and the underlying mechanisms.MethodsDiabetic mice were induced by STZ injection. After treatment with resveratrol (5 or 25 mg/kg/day i.g) or Saline containing 0.5 % carboxymethyl cellulose (CMC) for 2 months, the hearts were detected for oxidative stress and cardiac fibrosis using western blot, Masson’s trichrome staining and Dihydroethidium (DHE) staining. In in vitro experiments, proliferation and differentiation of fibroblasts under different conditions were investigated through western blot, 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and immunofluorescence staining.ResultsAdministration of resveratrol significantly mitigated oxidative level, interstitial fibrosis and expressions of related proteins in STZ-induced diabetic hearts. In in vitro experiments, resveratrol exhibited anti-proliferative effect on primary mouse cardiac fibroblasts via inhibiting reactive oxygen species (ROS)/extracellular regulated kinase (ERK) pathway and ameliorated myofibroblast differentiation via suppressing ROS/ERK/ transforming growth factor β (TGF-β)/periostin pathway.ConclusionIncreased ROS production, activation of ERK/TGF-β/periostin pathway and myocardial fibrosis are important events in DCM. Alleviated ROS genesis by resveratrol prevents myocardial fibrosis by regulating periostin related signaling pathway. Thus, inhibition of ROS/periostin may represent a novel approach for resveratrol to reverse fibrosis in DCM.

Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration

The omega-3 polyunsaturated fatty acids (ω−3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω−3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.

In This Issue

HomeCirculation ResearchVol. 113, No. 10In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published25 Oct 2013https://doi.org/10.1161/01.res.0000437011.92592.d9Circulation Research. 2013;113:1097Regulation of VSMC Function by miR-663 (p 1117)Download figureDownload PowerPointLi et al identify a new microRNA that regulates phenotype switching in human vascular smooth muscle cells.Fully differentiated vascular smooth muscle cells (VSMCs) in situ are generally non-proliferative, but after a vessel injury these cells dedifferentiate, proliferate and migrate. It is believed that such phenotype switching is an essential part of the healing process, but it can also lead to vessel narrowing, restenosis and other associated problems. Among the factors controlling this phenotype switch are microRNAs (miRs)—that suppress the expression of target mRNAs. While a number of such miRs have been identified in animal VSMCs, little is known about miRs in human VSMCs. Li and colleagues thus analyzed changes in miR expression in human VSMCs following induction of the phenotype switch. They found that miR-663 in particular, was dramatically downregulated. They also found that overexpression of miR-663 in VSMCs induced the expression of differentiation markers, whereas blocking miR-663 expression increased both proliferation and migration. MiR-663 targeted the mRNA of transcription factor JunB, suppressing its expression in human VSMCs. Although miR-663 is not conserved between humans and mice, its target sequence in the JunB mRNA is, and the team found that transfection of miR-663 into mice could suppress JunB expression and could block vessel narrowing. Boosting miR-663 activity or suppressing JunB might thus be strategies for preventing restenosis.Somatic Mesoderm and the Proepicardium (p 1128)Download figureDownload PowerPointSchlueter and Brand investigate the origins of the heart’s proepicardium and discover that some of the cells derive from somatic mesoderm.During embryogenesis, the proepicardium gives rise to epicardial cells as well as cells of the coronary vasculature and cardiac fibroblasts. But the origin of the proepicardium itself is unclear. In addition, it is also unclear how the proepicardium develops its characteristic left-right asymmetry—though the growth factor FGF8 and transcription factor SNAI1 are both known to be involved. Schlueter and Brand now show that a known target gene of SNAI1, a transcription factor called TWIST1, is asymmetrically expressed in lateral plate mesoderm of the early chick embryo, being highly expressed on the right-hand side. Furthermore, the team showed that these cells give rise to a population of proepicardial cells. They found that loss of TWIST1 expression in the right-hand mesoderm led to malformation of the proepicardium, while overexpression of TWIST in the left-hand mesoderm caused these cells to adopt characteristics of the right-hand proepicardium. The findings could have implications not only for understanding cardiac development, but also for understanding disease processes like myocardial fibrosis and for developing approaches for regenerative therapies.MicroRNAs in Cardiac Fibroblasts (p 1138)Download figureDownload PowerPointAbonnenc et al confirm the role of microRNAs in extracellular matrix secretion by cardiac fibroblasts.Changes to the composition of the extracellular matrix (ECM) contribute to the development of fibrosis, a characteristic feature of myocardial remodeling in a number of cardiac diseases. Previous studies have identified several microRNAs (miRs) that regulate cardiac fibrosis, including miR-30c and miR-29b. But a systematic analysis of the targets of these miRs is lacking. Rather than searching for messenger RNA targets of miR-30c and 29b—via computer prediction software or microarray analyses—Abonnenc and colleagues opted to search for proteins secreted by cardiac fibroblasts in response to the two miRs, thus allowing for the identification of both direct and indirect targets. The team used mass spectrometry to study the secretomes of primary mouse cardiac fibroblasts in which the two miRs were either over-expressed or inhibited. While miR-29b suppressed the expression of ECM and fibrosis proteins, including collagens, matrix metalloproteases, leukemia inhibitory factor (LIF) and insulin-like growth factor-1 (IGF-1), miR-30c had little effect on these proteins. The proteomic approach identified previously predicted and novel targets for the two miRs and, say the authors, offers a valuable resource for future studies into cardiac fibrosis. Previous Back to top Next FiguresReferencesRelatedDetails October 25, 2013Vol 113, Issue 10 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/01.res.0000437011.92592.d9 Originally publishedOctober 25, 2013 PDF download Advertisement

Poster session 3

Purpose: Type 2 diabetes (T2D) is an important risk factor for heart failure. T2D is associated with the metabolic syndrome, encompassing cardiovascular risk factors such as obesity, insulin resistance and dyslipidemia, factors also associated with left ventricular diastolic dysfunction. Patients with T2D have elevated plasma levels of free fatty acids and these are proposed to activate inflammation. Interleukin-1b (IL-1b) is activated by the NLRP3 inflammasome and a central mediator in T2D. Palmitate, the most abundant saturated fatty acid in plasma was recently shown to activate the NLRP3 inflammasome in macrophages. The aim of this study was to investigate if this mechanism operates within myocardial cells. Methods and Results: We first replicated previous findings by incubating bone marrow-derived macrophages with palmitate, and found release of active IL-1b. We went on to study primary mouse cardiac fibroblasts (CF). CF were stimulated with LPS, causing priming of the inflammasome, and then with palmitate. This caused release of active IL-1b. Importantly, this was not found in CF from NLRP3 and ASC deficient mice, demonstrating that the effect of palmitate on IL-1b release is mediated through the NLRP3 inflammasome. Other fatty acids such as eicosapentaenoic acid (EPA), oleic acid (OA), docosahexaenoic acid did not cause inflammasome activation. The omega-3 fatty EPA is known to suppress palmitate-induced metabolic stress, and when combining palmitate and EPA, we found markedly reduced release of IL-1b. By viewing CF stained with Bodipy under fluorescence microscopy, we observed an inhibitory effect of EPA on PA-induced lipid accumulation. AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis and also suppresses reactive oxygen species (ROS) production. We found that palmitate markedly reduced AMPK phosphorylation and Mitosox staining revealed increased mitochondrial ROS. Addition of the pharmacological AMPK activator AICAR completely blocked palmitate-induced IL-1b production. AMPK is also involved in regulation of autophagy, and we found that palmitate reduced levels of beclin-1, indicating that reduced autophagy could represent a mechanism for palmitate-induced inflammasome activation. Conclusion: Palmitate causes activation of the NLRP3 inflammasome in cardiac fibroblasts and this may involve increased mitochondrial ROS and defective autophagy. These findings indicate that NLRP3 inflammasome may act as a sensor of metabolic stress in the heart. EPA inhibits PA-induced NLRP3 inflammasome activation, possibly via reduced lipid accumulation. Cardiovascular Research Supplements (2014) 103, S102–S141

Extracellular superoxide dismutase protects cardiovascular syndecan-1 from oxidative shedding

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.

Resveratrol inhibits high glucose-induced PI3K/Akt/ERK-dependent interleukin-17 expression in primary mouse cardiac fibroblasts

We investigated the expression of the proinflammatory cytokine interleukin (IL)-17 in cardiac fibroblasts and its induction by high glucose (HG). Our results show that primary mouse cardiac fibroblasts (mCFs) secrete low basal levels of IL-17 and that HG (25 mM D-glucose) as opposed to low glucose (5 mM D-glucose + 20 mM mannitol) significantly enhances its secretion. HG induces IL-17 mRNA expression by both transcriptional and posttranscriptional mechanisms. HG induces phosphoinositide 3- kinase [PI3K; inhibited by adenoviral (Ad).dominant negative (dn)PI3Kp85], Akt (inhibited by Ad.dnAkt1), and ERK (inhibited by PD-98059) activation and induces IL-17 expression via PI3K-->Akt-->ERK-dependent signaling. Moreover, mCFs express both IL-17 receptors A and C, and although IL-17RA is upregulated, HG fails to modulate IL-17RC expression. Furthermore, IL-17 stimulates net collagen production by mCFs. Pretreatment with the phytoalexin resveratrol blocks HG-induced PI3K-, Akt-, and ERK-dependent IL-17 expression. These results demonstrate that 1) cardiac fibroblasts express IL-17 and its receptors; 2) HG upregulates IL-17 and IL-17RA, suggesting a positive amplification loop in IL-17 signaling in hyperglycemia; 3) IL-17 enhances net collagen production; and 4) resveratrol can inhibit these HG-induced changes. Thus, in hyperglycemic conditions, IL-17 may potentiate myocardial inflammation, injury, and remodeling through autocrine and paracrine mechanisms, and resveratrol has therapeutic potential in ameliorating this effect.