Interleukin-5 alleviates cardiac remodelling via the STAT3 pathway in angiotensin II-infused mice.

Interleukin-5 (IL-5) has been reported to be involved in cardiovascular diseases, such as atherosclerosis and cardiac injury. This study aimed to investigate the effects of IL-5 on cardiac remodelling. Mice were infused with angiotensin II (Ang II), and the expression and source of cardiac IL-5 were analysed. The results showed that cardiac IL-5 expression was time- and dose-dependently decreased after Ang II infusion, and was mainly derived from cardiac macrophages. Additionally, IL-5-knockout (IL-5-/-) mice were used to observe the effects of IL-5 knockout on Ang II-induced cardiac remodelling. We found knockout of IL-5 significantly increased the expression of cardiac hypertrophy markers, elevated myocardial cell cross-sectional areas and worsened cardiac dysfunction in Ang II-infused mice. IL-5 deletion also promoted M2 macrophage differentiation and exacerbated cardiac fibrosis. Furthermore, the effects of IL-5 deletion on cardiac remodelling was detected after the STAT3 pathway was inhibited by S31-201. The effects of IL-5 on cardiac remodelling and M2 macrophage differentiation were reversed by S31-201. Finally, the effects of IL-5 on macrophage differentiation and macrophage-related cardiac hypertrophy and fibrosis were analysed in vitro. IL-5 knockout significantly increased the Ang II-induced mRNA expression of cardiac hypertrophy markers in myocardial cells that were co-cultured with macrophages, and this effect was reversed by S31-201. Similar trends in the mRNA levels of fibrosis markers were observed when cardiac fibroblasts and macrophages were co-cultured. In conclusions, IL-5 deficiency promote the differentiation of M2 macrophages by activating the STAT3 pathway, thereby exacerbating cardiac remodelling in Ang II-infused mice. IL-5 may be a potential target for the clinical prevention of cardiac remodelling.

Single-cell RNA sequencing reveals that NRF2 regulates vascular smooth muscle cell phenotypic switching in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a life-threatening disease characterized by extensive membrane destruction in the vascular wall that is closely associated with vascular smooth muscle cell (VSMC) phenotypic switching. A thorough understanding of the changes in regulatory factors during VSMC phenotypic switching is essential for managing AAA therapy. In this study, we revealed the impact of NRF2 on the modulation of VSMC phenotype and the development of AAA based on single-cell RNA sequencing analysis. By utilizing a murine model of VSMC-specific knockout of nuclear factor E2-related factor 2 (NRF2), we observed that the absence of NRF2 in VSMCs exacerbated AAA formation in an angiotensin II-induced AAA model. The downregulation of NRF2 promoted VSMC phenotypic switching, leading to an enhanced inflammatory response. Through genome-wide transcriptome analysis and loss- or gain-of-function experiments, we discovered that NRF2 upregulated the expression of VSMC contractile phenotype-specific genes by facilitating microRNA-145 (miR-145) expression. Our data identified NRF2 as a novel regulator involved in maintaining the VSMC contractile phenotype while also influencing AAA formation through an miR-145-dependent regulatory mechanism.

Angiotensin II-independent abnormal renal vascular reactivity during puromycin nephropathy.

Experimental glomerulonephritis results in hypertension that is sensitive to salt. Nevertheless, salt retention alone cannot explain the increase in blood pressure. Angiotensin antagonistic therapy reduces hypertension caused by puromycin amino nucleosides (PAN). We investigated the hypothesis that PAN modifies renal vascular reactivity through processes dependent on angiotensin. Long-Evans rats were given an intraperitoneal injection of either puromycin (150 mg/kg) or saline (controls). Group 1 was fed a normal sodium diet (NSD, n = 9). Group 2 was given 30 mg/L of quinapril (Q) in addition to NSD (NSD + Q; n = 6). Group 3 received a high sodium diet (HSD, n = 7), and Group 4 received HSD + Q (n = 7). Systolic blood pressure (SBP), plasma creatinine, proteinuria, and sodium balance were monitored for 12 days. On day 15, renal vascular reactivity was assessed by administering increasing doses of angiotensin II, acetylcholine (ACh), and sodium nitroprusside (SNP) directly into the renal artery. SBP progressively increased in all PAN groups. This increase in SBP was greater in the HSD groups and was not significantly altered by Q treatment. SBP increased by 22 ± 4% (NSD), 51 ± 5% (NSD + Q), 81 ± 10% (HSD), and 65 ± 8% (HSD + Q). The renal blood flow of PAN rats did not return to baseline despite their normal renal vasoconstrictor responses to angiotensin II. Additionally, they showed reduced renal vasodilator responses to SNP and Ach. The vasodilator responses to both vasodilators were surprisingly unaffected by the inhibition of the angiotensin-converting enzyme (ACE). Renal vasodilator responses to both endothelium-dependent and independent variables were reduced in early PAN-induced hypertension. We found that the angiotensin-mediated mechanism is not responsible for this altered renal vasoreactivity.

SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

[Mechanisms by which Mettl3 regulates pericyte-myofibroblast transdifferentiation through PI3K/AKT signaling pathway].

Objective: To investigate the role and underlying mechanisms of methyltransferase (Mettl) 3 in the process of angiotensin Ⅱ (Ang Ⅱ)-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis. Methods: C57BL/6J mice were used, in cell experiments, mouse renal pericytes were isolated and cultured using magnetic bead sorting. These pericytes were then induced to transdifferentiate into myofibroblasts with 1×106 mmol/L Ang Ⅱ, which was the Ang Ⅱ group, while pericytes cultured in normal conditions served as the control group. Successful transdifferentiation was verified by immunofluorescence staining, Western blotting, and real-time reverse transcription PCR (RT-qPCR) for α-smooth muscle actin (α-SMA). The levels of m6A modifications and related enzymes (Mettl3, Mettl14), Wilms tumor 1-associated protein (WTAP), fat mass and obesity protein (FTO), ALKBH5, YTHDF1, YTHDF2, YTHDC1, YTHDC2, YTHDC3 were assessed by Dot blot, RT-qPCR and Western blot. Mettl3 expression was inhibited in cells using lentivirus-mediated Mettl3-shRNA transfection, creating sh-Mettl3 and Ang Ⅱ+sh-Mettl3 groups, while lentivirus empty vector transfection served as the negative control (Ang Ⅱ+sh-NC group). The impact of Ang Ⅱ on pericyte transdifferentiation was observed, and the expression of downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway proteins, including PI3K, AKT, phosphorylated AKT at serine 473 (p-AKT (S473)), and phosphorylated AKT at threonine 308 (p-AKT (T308)), were examined. PI3K gene transcription was inhibited by co-culturing cells with actinomycin D, and the half-life of PI3K mRNA was calculated by measuring residual PI3K mRNA expression over different co-culture time. The reversibility of Mettl3 inhibition on Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation was assessed by adding the AKT activator SC79 to the Ang Ⅱ+sh-Mettl3 group. In animal experiments, mice were divided into these groups: sham group (administered 0.9% sterile saline), Ang Ⅱ group (infused with Ang Ⅱ solution), sh-Mettl3 group (injected with Mettl3 shRNA lentivirus solution), Ang Ⅱ+sh-Mettl3 group (infused with Ang Ⅱ solution and injected with Mettl3 shRNA lentivirus solution), and Ang Ⅱ+sh-Mettl3+SC79 group (administered Ang Ⅱ solution and Mettl3 shRNA lentivirus, with an additional injection of SC79). Each group consisted of six subject mice. Blood pressure was measured using the tail-cuff method before and after surgery, and serum creatinine, urea, and urinary albumin levels were determined 4 weeks post-surgery. Kidney tissues were collected at 28 days and stained using hematoxylin-eosin (HE) and Masson's trichrome to assess the extent of renal fibrosis. Results: Primary renal pericytes were successfully obtained by magnetic bead sorting, and intervened with 1×106 mmol/L Ang Ⅱ for 48 hours to induce pericyte-to-myofibroblast transdifferentiation. Dot blot results indicated higher m6A modification levels in the Ang Ⅱ group compared to the control group (P<0.05). RT-qPCR and Western blot results showed upregulation of Mettl3 mRNA and protein levels in the Ang Ⅱ group compared to the control group (both P<0.05). In the Ang Ⅱ+sh-Mettl3 group, Mettl3 protein expression was lower than that in the Ang Ⅱ group, with reduced expression levels of α-SMA, vimentin, desmin, fibroblast agonist protein (FAPa) and type Ⅰ collagen (all P<0.05). Compared to the control group, PI3K mRNA expression level was elevated in the Ang Ⅱ group, along with increased p-AKT (S473) and p-AKT (T308) expressions. In the Ang Ⅱ+sh-Mettl3 group, PI3K mRNA expression and p-AKT (S473) and p-AKT (T308) levels were decreased (all P<0.05). The half-life of PI3K mRNA was shorter in the Ang Ⅱ+sh-Mettl3 group than that in the Ang Ⅱ+sh-NC group (2.34 h vs. 3.42 h). The ameliorative effect of Mettl3 inhibition on Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation was reversible by SC79. Animal experiments showed higher blood pressure, serum creatinine, urea, and 24-hour urinary protein levels, and a larger fibrosis area in the Ang Ⅱ group compared to the sham group (all P<0.05). The fibrosis area was smaller in the Ang Ⅱ+sh-Mettl3 group than that in the Ang Ⅱ group (P<0.05), but increased again upon addition of SC79. Conclusion: Mettl3-mediated RNA m6A epigenetic regulation is involved in Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis, potentially by affecting PI3K stability and regulating the PI3K/AKT signaling pathway.

Molecular changes in endometrium origin stromal cells during initiation of cardiomyogenic differentiation induced with Decitabine, Angiotensin II and TGF- ß1.

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells - menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-ß1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems.

Anemoside B4 attenuates abdominal aortic aneurysm by limiting smooth muscle cell transdifferentiation and its mediated inflammation.

Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by local abnormal dilation of the aorta accompanied by vascular smooth muscle cell (VSMC) dysfunction and chronic inflammation. VSMC dedifferentiation, transdifferentiation, and increased expression of matrix metalloproteinases (MMPs) are essential causes of AAA formation. Previous studies from us and others have shown that Anemoside B4 (AB4), a saponin from Pulsatilla chinensis, has anti-inflammatory, anti-tumor, and regulatory effects on VSMC dedifferentiation. The current study aimed to investigate whether AB4 inhibits AAA development and its underlying mechanisms. By using an Ang II induced AAA model in vivo and cholesterol loading mediated VSMC to macrophage transdifferentiation model in vitro, our study demonstrated that AB4 could attenuate AAA pathogenesis, prevent VSMC dedifferentiation and transdifferentiation to macrophage-like cells, decrease vascular inflammation, and suppress MMP expression and activity. Furthermore, KLF4 overexpression attenuated the effects of AB4 on VSMC to macrophage-like cell transition and VSMC inflammation in vitro. In conclusion, AB4 protects against AAA formation in mice by inhibiting KLF4 mediated VSMC transdifferentiation and inflammation. Our study provides the first proof of concept of using AB4 for AAA management.

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.

Oxidative modification of miR-30c promotes cardiac fibroblast proliferation via CDKN2C mismatch.

The response of cardiac fibroblast proliferation to detrimental stimuli is one of the main pathological factors causing heart remodeling. Reactive oxygen species (ROS) mediate the proliferation of cardiac fibroblasts. However, the exact molecular mechanism remains unclear. In vivo, we examined the oxidative modification of miRNAs with miRNA immunoprecipitation with O8G in animal models of cardiac fibrosis induced by Ang II injection or ischemia‒reperfusion injury. Furthermore, in vitro, we constructed oxidation-modified miR-30c and investigated its effects on the proliferation of cardiac fibroblasts. Additionally, luciferase reporter assays were used to identify the target of oxidized miR-30c. We found that miR-30c oxidation was modified by Ang II and PDGF treatment and mediated by excess ROS. We demonstrated that oxidative modification of G to O8G occurred at positions 4 and 5 of the 5' end of miR-30c (4,5-oxo-miR-30c), and this modification promoted cardiac fibroblast proliferation. Furthermore, CDKN2C is a negative regulator of cardiac fibroblast proliferation. 4,5-oxo-miR-30c misrecognizes CDKN2C mRNA, resulting in a reduction in protein expression. Oxidized miR-30c promotes cardiac fibroblast proliferation by mismatch mRNA of CDKN2C.

A new procedure to induce aortic aneurysms in mice.

Aortic aneurysms (AAs) are a major public health challenge, featured by a progressive impairs in aortic wall integrity that drives to aortic dilation and, in end stage, to its rupture. Despite important advances in the surgical treatment of aortic aneurysms, there is currently no pharmacological intervention that prevents their development, reduces their expansion, or avoids their rupture. In addition to classic risk factors such age or gender, several heritable connective tissue disorders have been associated with AA developing, highlighting the role of extracellular matrix (ECM) genes alterations in the developing of AA. In this sense, we have recently demonstrated that global deletion of the cellular communicating network factor 2 (CCN2), previously known as connective tissue growth factor (CTGF) due to its role in the extracellular matrix formation, predisposes to early and lethal AAs development after Angiotensin II (Ang II) infusion in mice. Here, we detail the protocol to induce and detect AAs generation in inducible global CCN2 knockout mice after Ang II infusion which allow the characterization of CCN role in AA development and may help to the development of pharmacological target for AA treatment.

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

Modulation of Angiotensin II-Induced Cellular Hypertrophy by Cannflavin-C: Unveiling the Impact on Cytochrome P450 1B1 and Arachidonic Acid Metabolites.

This research aimed to clarify the impacts of cannflavin-C on angiotensin II (Ang II)-induced cardiac hypertrophy and their potential role in modulating cytochrome P450 1B1 (CYP1B1) and arachidonic acid (AA) metabolites. Currently there is no evidence to suggest that cannflavin-C, a prenylated flavonoid, has any significant effects on the heart or cardiac hypertrophy. The metabolism of arachidonic acid (AA) into midchain hydroxyeicosatetraenoic acids (HETEs), facilitated by CYP1B1 enzyme, plays a role in the development of cardiac hypertrophy, which is marked by enlarged cardiac cells. Adult human ventricular cardiomyocyte (AC16) cell line was cultured and exposed to cannflavin-C in the presence and absence of Ang II. The assessment of mRNA expression pertaining to cardiac hypertrophic markers and cytochromes P450 (P450s) was conducted via real-time polymerase chain reaction (PCR), whereas the quantification of P450 protein levels was carried out through western blot analysis. Ang II induced hypertrophic markers myosin heavy chain (ß/α-MHC), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) and increased cell surface area, whereas cannflavin-C mitigated these effects. Gene and protein expression analysis revealed that cannflavin-C downregulated CYP1B1 gene expression, protein level, and enzyme activity assessed by 7-methoxyresorufin O-deethylase (MROD). Arachidonic acid metabolites analysis, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), demonstrated that Ang II increased midchain (R/S)-HETE concentrations, which were attenuated by cannflavin-C. This study provides novel insights into the potential of cannflavin-C in modulating arachidonic acid metabolites and attenuating Ang II-induced cardiac hypertrophy, highlighting the importance of this compound as potential therapeutic agents for cardiac hypertrophy. SIGNIFICANCE STATEMENT: This study demonstrates that cannflavin-C offers protection against cellular hypertrophy induced by angiotensin II. The significance of this research lies in its novel discovery, which elucidates a mechanistic pathway involving the inhibition of CYP1B1 by cannflavin-C. This discovery opens up new avenues for leveraging this compound in the treatment of heart failure.

Osteoprotegerin Is Essential for the Development of Endothelial Dysfunction Induced by Angiotensin II in Mice.

Opinions on the effects of osteoprotegerin (OPG) have evolved over the years from a protein protecting the vasculature from calcification to a cardiovascular risk factor contributing to inflammation within the vascular wall. Nowadays, the link between OPG and angiotensin II (Ang II) appears to be particularly important. In this study, the endothelial function was investigated in OPG-knockout mice (B6.129.S4-OPG, OPG-) and wild-type (C57BL/6J, OPG+) mice under basic conditions and after Ang II exposure by assessing the endothelium-dependent diastolic response of aortic rings to acetylcholine in vitro. A further aim of the study was to compare the effect of Ang II on the expression of cytokines in the aortic wall of both groups of mice. Our study shows that rings from OPG- mice had their normal endothelial function preserved after incubation with Ang II, whereas those from OPG+ mice showed significant endothelial dysfunction. We conclude that the absence of OPG, although associated with a pro-inflammatory cytokine profile in the vascular wall, simultaneously protects against Ang II-induced increases in pro-inflammatory cytokines in the murine vascular wall. Our study also demonstrates that the absence of OPG can result in a decrease in the concentration of pro-inflammatory cytokines in the vascular wall after Ang II exposure. The presence of OPG is therefore crucial for the development of Ang II-induced inflammation in the vascular wall and for the development of Ang II-induced endothelial dysfunction.

BRD4 as a therapeutic target for atrial fibrosis and atrial fibrillation.

OBJECTIVE: This study aimed to elucidate the molecular mechanisms by which BRD4 play a role in atrial fibrillation (AF). METHODS AND RESULTS: We used a discovery-driven approach to detect BRD4 expression in the atria of patients with AF and in various murine models of atrial fibrosis. We used a BRD4 inhibitor (JQ1) and atrial fibroblast (aFB)-specific BRD4-knockout mice to elucidate the role of BRD4 in AF. We further examined the underlying mechanisms using RNA-seq and ChIP-seq analyses in vitro, to identify key downstream targets of BRD4. We found that BRD4 expression is significantly increased in patients with AF, with accompanying atrial fibrosis and aFB differentiation. We showed that JQ1 treatment and shRNA-based molecular silencing of BRD4 blocked ANG-II-induced extracellular matrix production and cell-cycle progression in aFBs. BRD4-related RNA-seq and ChIP-seq analyses in aFBs demonstrated enrichment of a subset of promoters related to the expression of profibrotic and proliferation-related genes. The pharmacological inhibition of BRD4 in vivo or in aFB-specific BRD4-knockout in mice limited ANG-II-induced atrial fibrosis, atrial enlargement, and AF susceptibility. CONCLUSION: Our findings suggest that BRD4 plays a key role in pathological AF, at least partially by activating aFB proliferation and ECM synthesis. This study provides mechanistic insights into the development of BRD4 inhibitors as targeted antiarrhythmic therapies.

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.

The zinc-finger transcription factor KLF6 regulates cardiac fibrosis.

AIMS: Heart failure (HF) is one of the most devastating consequences of cardiovascular diseases. Regardless of etiology, cardiac fibrosis is present and promotes the loss of heart function in HF patients. Cardiac resident fibroblasts, in response to a host of pro-fibrogenic stimuli, trans-differentiate into myofibroblasts to mediate cardiac fibrosis, the underlying mechanism of which remains incompletely understood. METHODS: Fibroblast-myofibroblast transition was induced in vitro by exposure to transforming growth factor (TGF-ß). Cardiac fibrosis was induced in mice by either transverse aortic constriction (TAC) or by chronic infusion with angiotensin II (Ang II). RESULTS: Through bioinformatic screening, we identified Kruppel-like factor 6 (KLF6) as a transcription factor preferentially up-regulated in cardiac fibroblasts from individuals with non-ischemic cardiomyopathy (NICM) compared to the healthy donors. Further analysis showed that nuclear factor kappa B (NF-κB) bound to the KLF6 promoter and mediated KLF6 trans-activation by pro-fibrogenic stimuli. KLF6 knockdown attenuated whereas KLF6 over-expression enhanced TGF-ß induced fibroblast-myofibroblast transition in vitro. More importantly, myofibroblast-specific KLF6 depletion ameliorated cardiac fibrosis and rescued heart function in mice subjected to the TAC procedure or chronic Ang II infusion. SIGNIFICANCE: In conclusion, our data support a role for KLF6 in cardiac fibrosis.

Glutamine Protects against Mouse Abdominal Aortic Aneurysm through Modulating VSMC Apoptosis and M1 Macrophage Activation.

Glutamine (Gln), known as the most abundant free amino acid, is widely spread in human body. In this study, we demonstrated the protective effects of glutamine against mouse abdominal aortic aneurysm (AAA) induced by both angiotensin II (AngII) and calcium phosphate (Ca3(PO4)2) in vivo, which was characterized with lower incidence of mouse AAA. Moreover, histomorphological staining visually presented more intact elastic fiber and less collagen deposition in abdominal aortas of mice treated by glutamine. Further, we found glutamine inhibited the excessive production of reactive oxide species (ROS), activity of matrix metalloproteinase (MMP), M1 macrophage activation, and apoptosis of vascular smooth muscle cells (VSMCs) in suprarenal abdominal aortas of mice, what's more, the high expressions of MMP-2 protein, MMP-9 protein, pro-apoptotic proteins, and IL-6 as well as TNF-α in protein and mRNA levels in cells treated by AngII were down-regulated by glutamine. Collectively, these results revealed that glutamine protected against mouse AAA through inhibiting apoptosis of VSMCs, M1 macrophage activation, oxidative stress, and extracellular matrix degradation.

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes.

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with ß-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.

Neferine mitigates angiotensin II-induced atrial fibrillation and fibrosis via upregulation of Nrf2/HO-1 and inhibition of TGF-ß/p-Smad2/3 pathways.

BACKGROUND: Atrial fibrillation (AF) is often associated with atrial fibrosis and oxidative stress. Neferine, a bisbenzylisoquinoline alkaloid, has been reported to exert an antiarrhythmic effect. However, its impact on Angiotensin II (Ang II) infusion-induced AF and the underlying mechanism remains unclear. This study aimed to investigate whether neferine alleviates Ang II-induced AF and explore the underlying mechanisms. METHODS: Mice subjected to Ang II infusion to induce AF were concurrently treated with neferine or saline. AF incidence, myocardial cell size, fibrosis, and oxidative stress were then examined. RESULTS: Neferine treatment inhibited Ang II-induced AF, atrial size augmentation, and atrial fibrosis. Additionally, we observed that Ang II increased reactive oxygen species (ROS) generation, induced mitochondrial membrane potential depolarization, and reduced glutathione (GSH) and superoxide dismutase (SOD) levels, which were reversed to some extent by neferine. Mechanistically, neferine activated the Nrf2/HO-1 signaling pathway and inhibited TGF-ß/p-Smad2/3 in Ang II-infused atria. Zinc Protoporphyrin (ZnPP), an HO-1 inhibitor, reduced the anti-oxidative effect of neferine to some extent and subsequently abolished the beneficial effect of neferine on Ang II-induced AF. CONCLUSIONS: These findings provide hitherto undocumented evidence that the protective role of neferine in Ang II-induced AF is dependent on HO-1.