Mitochondrial dysfunction plays a key role in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH). As is known to play a key role in mitochondria, ECSIT, in relation to oxidized mitochondrial DNA is still unclear. This study examines mitochondrial ECSIT expression in MASH mouse models. Mitochondria-targeted ECSIT transgenic (ECSITMTG) mice and wild-type (WT) controls are fed a high-fat, high-cholesterol (HFHC) diet for 16 weeks or a methionine- and choline-deficient (MCD) diet for 8 weeks. Results demonstrate that mitochondrial ECSIT overexpression alleviates diet-induced MASH phenotypes. Mechanistically, we demonstrate that mitochondrial ECSIT promotes the localization of the deubiquitinase OTUD3 to mitochondria. OTUD3 then stabilizes SIRT3 via deubiquitination, thereby inhibiting mtDNA oxidation and alleviating steatosis-induced metabolic disorders. Overall, these findings indicate that mitochondrial ECSIT protects against MASH progression by stabilizing SIRT3, suggesting its potential as a therapeutic target.

Mitochondrial dysfunction is a key factor in exacerbating pressure overload-induced cardiac hypertrophy and is linked to increased morbidity and mortality. ECSIT, a crucial adaptor for inflammation and mitochondrial function, has been reported to express multiple transcripts in various species and tissues, leading to distinct protein isoforms with diverse subcellular localizations and functions. However, whether an unknown ECSIT isoform exists in cardiac cells and its potential role in regulating mitochondrial function and pathological cardiac hypertrophy has remained unclear. This study identified a 42-kDa ECSIT isoform encoded by the transcript variant Ecsit-X4, which is highly expressed in the mitochondria of adult cardiomyocytes but down-regulated in hypertrophic human heart samples and TAC-treated mouse hearts. AAV9-mediated Ecsit-X4 gene therapy, administered either before or after TAC surgery, significantly attenuated cardiac hypertrophy. Cardiomyocyte-specific Ecsit deficiency worsened TAC-induced cardiac hypertrophy, while Ecsit-X4 compensation independently rescued hypertrophic phenotypes in EcsitcKO mice. Mechanistically, ECSIT-X4 localized to the mitochondria and interacted with STAT3, leading to increased STAT3 levels and enhanced serine 727 phosphorylation in cardiomyocyte mitochondria, thereby promoting strong mitochondrial bioenergetics. This study identified a novel transcript variant of ECSIT localized in the mitochondria of adult cardiomyocytes and highlights its potential as a therapeutic target for heart failure.

BACKGROUND: The infiltration of macrophages into the lungs is a common characteristic of perivascular inflammation, contributing to vascular remodeling in pulmonary hypertension (PH). Peli1 (pellino E3 ubiquitin-protein ligase 1) plays a critical role in regulating the production of proinflammatory cytokines and the polarization of macrophages in various diseases. However, the role of Peli1 in PH remains to be investigated. METHODS: The expression and biological function of Peli1 were investigated in both human and experimental models of PH. Peli1-deficient mice and bone marrow transplant mice were utilized to explore the roles of Peli1 in macrophages in vivo. Proteomic analysis and molecular biology techniques were used to uncover the underlying mechanisms. RESULTS: The upregulation of Peli1 in the lungs and alveolar macrophages was observed in hypoxia-treated mice. Peli1 knockout mice and myeloid Peli1-deficient mice significantly ameliorated hypoxia-induced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Mechanistically, Peli1 facilitated the ubiquitination and subsequent proteasomal degradation of Foxp1 (forkhead box p1), thereby alleviating its suppression of IL ( interleukin ) -6 transcription and contributing to macrophage activation. Furthermore, myeloid Foxp1 deficiency partially eliminates the protective effect of myeloid Peli1 deficiency in hypoxia-induced PH mice. CONCLUSIONS: Our findings demonstrate that the Peli1-Foxp1-IL-6 pathway plays a crucial role in macrophage activation and recruitment during the development of PH, underscoring the potential of Peli1 as a therapeutic target for PH.

Impaired fatty acid oxidation (FAO) is a prominent feature of metabolic remodeling observed in pathological myocardial hypertrophy. Hepatocyte nuclear factor 4alpha (HNF4α) is closely associated with FAO in both cellular processes and disease conditions. Pellino 1 (Peli1), an E3 ligase containing a RING-like domain, plays a crucial role in catalyzing polyubiquitination of various substrates. In this study, we aimed to investigate the involvement of HNF4α and its ubiquitination, facilitated by Peli1, in FAO during pressure overload-induced cardiac hypertrophy. Peli1 systemic knockout mice (Peli1KO) display improved myocardial hypertrophy and cardiac function following transverse aortic constriction (TAC). RNA-seq analysis revealed that changes in gene expression related to lipid metabolism caused by TAC were reversed in Peli1KO mice. Importantly, both HNF4α and its downstream genes involved in FAO showed a significant increase in Peli1KO mice. We further used the antagonist BI6015 to inhibit HNF4α and delivered rAAV9-HNF4α to elevate myocardial HNF4α level, and confirmed that HNF4α inhibits the development of cardiac hypertrophy after TAC and is essential for the enhancement of FAO mediated by Peli1 knockout. In vitro experiments using BODIPY incorporation and FAO stress assay demonstrated that HNF4α enhances FAO in cardiomyocytes stimulated with angiotension II (Ang II), while Peli1 suppresses the effect of HNF4α. Mechanistically, immunoprecipitation and mass spectrometry analyses confirmed that Peli1 binds to HNF4α via its RING-like domain and promotes HNF4α ubiquitination at residues K307 and K309. These findings shed light on the underlying mechanisms contributing to impaired FAO and offer valuable insights into a promising therapeutic strategy for addressing pathological cardiac hypertrophy.

Myocardial fibrosis is a major pathogenic factor contributing to cardiac remodeling and heart failure. Recent research has indicated that micro RNAs play a crucial role in the progression of cardiac fibrosis. Bone morphogenetic protein and activin membrane-bound inhibitor(BAMBI) have been shown to alleviate myocardial fibrosis by inhibiting the transforming growth factor β1(TGF-β1) signaling pathway. Therefore, the current study aimed to elucidate the post-transcriptional regulation of BAMBI by miR-19a-3p and its role in TGF-β1-induced cardiac fibroblast activation. We found that transverse aortic constriction induced both myocardial interstitial and perivascular collagen deposition. Quantitative reverse transcription-PCR(q RT-PCR) analysis showed that the expression level of miR-19a-3p was increased in the myocardial tissues of cardiac fibrosis, and TGF-β1 induced an upregulation in miR-19a-3p expression in cardiac fibroblasts. The dual-luciferase reporter assay and q RT-PCR verified that miR-19a-3p directly bound to the 3 ′ untranslated regions of BAMBI m RNA, thereby reducing BAMBI expression and diminishing its ability to inhibit the TGF-β1 signaling pathway. Furthermore,overexpression of miR-19a-3p mimic increased the activation of TGF-β1/SMAD2/3 pathway signaling,promoting cardiac fibroblast activation. However, this activation was blocked by BAMBI overexpression. These findings imply that miR-19a-3p enhances the activation of TGF-β1/SMAD2/3 by inhibiting BAMBI, further boosting the activation of cardiac fibroblasts and contributing to myocardial fibrosis.

Recent studies have attempted to characterize the layer-specific mechanical and microstructural properties of the aortic tissues in either normal or pathological state to understand its structural-mechanical property relationships. However, layer-specific tissue mechanics and compositions of normal and dissected ascending aortas have not been thoroughly compared with a statistical conclusion obtained. Eighteen ascending aortic specimens were harvested from 13 patients with type A aortic dissection and 5 donors without aortic diseases, with each specimen further excised to obtain three tissue samples including an intact wall, an intima-media layer and an adventitia layer. For each tissue sample, biaxial tensile testing was performed to obtain the experimental stress-stretch ratio data, which were further fed into the Fung-type model to quantify the tissue stiffness, and Elastin Van Gieson stain and Masson's trichrome stain were employed to quantify the elastic and collagen fiber densities. Statistical analyses were performed to determine whether any significant differences exist in mechanical properties and compositions between diseased and normal aortic tissues. The tissue stiffness of intima-media samples was significant higher in diseased group than that of normal group in longitudinal direction at the stretch ratio 1.30 (p = 0.0068), while no significant differences were found in the other direction or other tissue types. Even though there was no significant difference in elastic or collagen fiber densities between two groups, the diseased group generally had lower elastic fiber density, but higher collagen fiber density for all three tissue layers. Compared to normal aortic tissues, the elastic fiber density of the intima-media layer in the dissected aortic tissue was lower, while its tissue stiffness was significantly higher, indicating the tissue stiffness of the intima-media layer could be a potential indicator for aortic dissection.

The polarization of macrophages to the M1 or M2 phenotype has a pivotal role in inflammatory response following myocardial ischemia/reperfusion injury. Peli1, an E3 ubiquitin ligase, is closely associated with inflammation and autoimmunity as an important regulatory protein in the Toll-like receptor signaling pathway. We aimed to explore the function of Peli1 in macrophage polarization under myocardial ischemia/reperfusion injury and elucidate the possible mechanisms. We show here that Peli1 is upregulated in peripheral blood mononuclear cells from patients with myocardial ischemia/reperfusion, which is correlated with myocardial injury and cardiac dysfunction. We also found that the proportion of M1 macrophages was reduced and myocardial infarct size was decreased, paralleling improvement of cardiac function in mice with Peli1 deletion in hematopoietic cells or macrophages. Macrophage Peli1 deletion lessened M1 polarization and reduced the migratory ability in vitro. Mechanistically, Peli1 contributed to M1 polarization by promoting K63-linked ubiquitination and nuclear translocation of IRF5. Moreover, Peli1 deficiency in macrophages reduced the apoptosis of cardiomyocytes in vivo and in vitro. Together, our study demonstrates that Peli1 deficiency in macrophages suppresses macrophage M1 polarization and alleviates myocardial ischemia/reperfusion injury by inhibiting the nuclear translocation of IRF5, which may serve as a potential intervention target for myocardial ischemia/reperfusion injury.

Background: Aortic dissection and atherosclerosis are two common pathological conditions affecting the aorta. Aortic biomechanics are believed to be closely associated with the pathological development of these diseases. However, the biomechanical environment that predisposes the aortic wall to these pathological conditions remains unclear. Methods: Sixteen ascending aortic specimens were harvested from 16 human subjects and further categorized into three groups according to their disease states: aortic dissection group, aortic dissection with accompanied atherosclerosis group and healthy group. Experimental stress-strain data from biaxial tensile testing were used to fit the anisotropic Mooney-Rivlin model to determine material parameters. Computed tomography images or transesophageal echocardiography images were collected to construct computational models to simulate the stress/strain distributions in aortas at the pre-dissection state. Statistical analyses were performed to identify the biomechanical factors to distinguish three groups of aortic tissues. Results: Material parameters of anisotropic Mooney-Rivlin model were fitted with average R2 value 0.9749. The aortic diameter showed no significant difference among three groups. Changes of maximum and average stress values from minimum pressure to maximum pressure (oMaxStress and oAveStress) had significantly difference between dissection group and dissection with accompanied atherosclerosis group (p = 0.0201 and 0.0102). Changes of maximum and average strain values from minimum pressure to maximum pressure (oMaxStrain and oAveStrain) from dissection group were significant different from healthy group (p = 0.0171 and 0.0281). Conclusion: Changes of stress and strain values during the cardiac cycle are good biomechanical factors for predicting potential aortic dissection and aortic dissection accompanied with atherosclerosis.

Cardiac fibrosis is an essential pathological process in pressure overload (PO)-induced heart failure. Recently, myocyte-fibroblast communication is proven to be critical in heart failure, in which, pathological growth of cardiomyocytes (CMs) may promote fibrosis via miRNAs-containing exosomes (Exos). Peli1 regulates the activation of NF-κB and AP-1, which has been demonstrated to engage in miRNA transcription in cardiomyocytes. Therefore, we hypothesized that Peli1 in CMs regulates the activation of cardiac fibroblasts (CFs) through an exosomal miRNA-mediated paracrine mechanism, thereby promoting cardiac fibrosis. We found that CM-conditional deletion of Peli1 improved PO-induced cardiac fibrosis. Moreover, Exos from mechanical stretch (MS)-induced WT CMs (WT MS-Exos) promote activation of CFs, Peli1-/- MS-Exos reversed it. Furthermore, miRNA microarray and qPCR analysis showed that miR-494-3p was increased in WT MS-Exos while being down regulated in Peli1-/- MS-Exos. Mechanistically, Peli1 promoted miR-494-3p expression via NF-κB/AP-1 in CMs, and then miR-494-3p induced CFs activation by inhibiting PTEN and amplifying the phosphorylation of AKT, SMAD2/3, and ERK. Collectively, our study suggests that CMs Peli1 contributes to myocardial fibrosis via CMs-derived miR-494-3p-enriched exosomes under PO, and provides a potential exosomal miRNA-based therapy for cardiac fibrosis.

目的:研究TIR/BB环拟似物AS-1对小鼠肾脏缺血再灌注损伤的保护作用及机制.方法:雄性C57BL/6小鼠随机分成4组:假手术组(sham)、肾缺血再灌注组(RIR)、AS-1+肾缺血再灌注组(AS-1+RIR)、溶剂+肾缺血再灌注组(溶剂+RIR).采用夹闭双侧肾动、静脉45 min,再灌注24 h的方法构建肾缺血再灌注损伤模型.AS-1+RIR组与溶剂+RIR组在术前30 min按剂量50 mg/kg分别腹腔注射AS-1和溶剂,再灌注24 h后检测肾功能参数、血清炎症因子、凋亡指标及磷酸化NF-κB p65(p-p65)表达水平.结果:与假手术组比较,缺血再灌注组血清肌酐(Scr)、尿素氮(BUN)、TNF-α、IL-6、p-p65、Cleaved caspase3及Bax的表达水平均明显提高(P＜0.01),肾间质CD68+和MPO炎症细胞浸润增多,Bcl-2的表达量明显降低(P＜0.01),Bcl-2/Bax的比值也降低.给予AS-1处理后可以使Scr、BUN、TNF-α、IL-6、p-p65、Cleaved caspase3、及Bax的水平明显降低(P＜0.01),CD68+和MPO炎症细胞浸润减少;Bcl-2的表达量升高(P＜0.01),Bcl-2/Bax的比值也升高.结论:TIR/BB环拟似物AS-1对小鼠肾缺血再灌注损伤有保护作用,其机制可能与其抑制NF-κB信号通路的活化,产生抗炎作用和抗凋亡作用有关.

Autophagy flux is impaired during myocardial ischemia/reperfusion (M-I/R) via the accumulation of autopha-gosome and insufficient clearance, which exacerbates cardiomyocyte death. Peli1 (Pellion1) is a RING finger domain-containing ubiquitin E3 ligase that could catalyze the polyubiquitination of substrate proteins. Peli1 has been demonstrated to play an important role in ischemic cardiac diseases. However, little is known about whether Peli1 is involved in the regulation of autophagy flux during M-I/R. The present study investigated whether M-I/R induced impaired autophagy flux could be mediated through Peli1 dependent mechanisms. We induced M-I/R injury in wild type (WT) and Peli1 knockout mice and observed that M-I/R significantly decreased cardiac function that was associated with increased cardiac Peli1 expression and upregulated autophagy-associated protein LC3II and P62. In contrast, Peli1 knockout mice exhibited significant improvement of M-I/ R induced cardiac dysfunction and decreased LC3II and P62 expression. Besides, inhibitors of autophagy also increased the infarct size in Peli1 knockout mice after 24 h of reperfusion. Mechanistic studies demonstrated that in vivo I/R or in vitro hypoxia/reoxygenation (H/R) markedly increased the Peli1 E3 ligase activity which directly promoted the ubiquitination of P62 at lysine(K)7 via K63-linkage to inhibit its dimerization and auto-phagic degradation. Co-immunoprecipitation and GST-pull down assay indicated that Peli1 interacted with P62 via the Ring domain. In addition, Peli1 deficiency also decreased cardiomyocyte apoptosis. Together, our work demonstrated a critical link between increased expression and activity of Peli1 and autophagy flux blockage in M-I/R injury, providing insight into a promising strategy for treating myocardium M-I/R injury.

目的:鉴定成年小鼠Toll途径进化保守信号介导因子(evolutionarily conserved signaling intermediate in Toll path-ways,ECSIT)的3'非翻译区(3'-untranslatedregion,3'-UTR)序列,并在细胞中验证非编码RNA对ECSIT表达的影响.方法:采用RACE技术克隆得到小鼠ECSIT3'-UTR序列,并与基因组数据库进行比对;预测ECSIT3'-UTR可能结合的微小RNA(mi-croRNA,miRNA),并针对ECSIT的全长序列设计小干扰RNA(siRNA);通过Western blot分别检测使用不同miRNA和siRNA干扰后,细胞中ECSIT的表达.结果:成功鉴定了346bp的小鼠ECSIT3'-UTR,与NCBI上序列一致率达到99％;在细胞中miR-7-5p和siRNA1、2可以干扰ECSIT的表达.结论:成功鉴定得到成年小鼠心脏ECSIT mRNA3'-UTR序列,该非编码区可作为非编码RNA的调控区域,为进一步从分子水平探明ECSIT在小鼠生长发育及疾病中的作用提供科学依据.

Activation of TLRs mediated the NF-κB signaling pathway plays an important pathophysiological role in cardiac hypertrophy. Triad3A, a ubiquitin E3 ligase, has been reported to negatively regulate NF-κB activation pathway via promoting ubiquitination and degradation of TLR4 and TLR9 in innate immune cells. The role of Triad3A in cardiac hypertrophic development remains unknown. The present study investigated whether there is a link between Triad3A and TLR4 and TLR9 in pressure overload induced cardiac hypertrophy. We observed that Triad3A levels were markedly reduced following transverse aortic constriction (TAC) induced cardiac hypertrophy. Similarly, stimulation of neonatal rat cardiac myocytes (NRCMs) with angiotensin-II (Ang II) significantly decreased Triad3A expression. To determine the role of Triad3A in TAC-induced cardiac hypertrophy, we transduced the myocardium with adenovirus expressing Triad3A followed by induction of TAC. We observed that increased expression of Triad3A significantly attenuated cardiac hypertrophy and improved cardiac function. To investigate the mechanisms by which Triad3A attenuated cardiac hypertrophy, we examined the Triad3A E3 ubiquitination on TLR4 and TLR9. We found that Triad3A promoted TLR4 and TLR9 degradation through ubiquitination. Triad3A mediated TLR4 and TLR9 degradation resulted in suppression of NF-κB activation. Our data suggest that Triad3A plays a protective role in the development of cardiac hypertrophy, at least through catalyzing ubiquitination-mediated degradation of TLR4 and TLR9, thus negatively regulating NF-κB activation.

Morphological and functional abnormalities of vascular endothelial cells (VECs) are risk factors of ischemia-reperfusion in skin flaps. Signaling pathway mediated by interleukin-1 receptor (IL-1R) is essential to hypoxia/reoxygenation (H/R) injury of VECs. While the TIR/BB-loop mimetic (AS-1) disrupts the interaction between IL-1R and myeloid differentiation primary-response protein 88 (MyD88), its role in the VECs dysfunction under H/R is unclear. In this study, we first showed that there was an infiltration of inflammatory cells and the apoptosis of VECs by using a skin flap section from patients who received flap transplantation. We then showed that the H/R treatment induced apoptosis and loss of cell migration of endothelial cell line H926 were attenuated by AS-1. Furthermore, our data suggested that AS-1 inhibits the interaction between IL-1R and MyD88, and subsequent phosphorylation of IκB and p38 pathway, as well as the nuclear localization of NF-KB subunit p65/p50. Thus, this study indicated that the protective role of AS-1 in H/R induced cellular injury may be due to the AS-1 mediated down-regulation of IL-1R signaling pathway.

Ameliorating cardiac microvascular injury is the most effective means to mitigate diabetes-induced cardiovascular complications. Inositol-requiring 1α (IRE1α), a sensor of endoplasmic reticulum stress, is activated by Toll like receptors (TLRs), and then promotes cardiac microvascular injury. Peli1 is a master regulator of TLRs and activates IRE1α. This study aims to investigate whether Peli1 in endothelial cells promotes diabetes-induced cardiac microvascular injury through activating IRE1α. Here we found that Peli1 was markedly up-regulated in cardiac endothelial cells of both diabetic mice and in AGEs-treated cardiac microvascular endothelial cells (CMECs). Peli1 deficiency in endothelial cells significantly alleviated diabetes-induced cardiac microvascular permeability, promoted microvascular regeneration, and suppressed apoptosis, accompanied by the attenuation of adverse cardiac remodeling. Furthermore, Peli1 deletion in CMECs ameliorated AGEs-induced damages in vitro. We identified heat shock protein 90 (Hsp90) as a potential binding partner for Peli1, and the Ring domain of Peli1 directly bound with Hsp90 to enhance IRE1α phosphorylation. Our study suggests that blocking Peli1 in endothelial cells may protect against diabetes-induced cardiac microvascular injury by restraining ER stress.

目的:构建线粒体靶向过表达ECSIT转基因小鼠,并对其进行鉴定和心功能分析,建立ECSIT基因相关功能研究模型动物.方法:构建过表达打靶载体pCAG-OTCL-ECSIT-3Xflag-BPA,采用电转导方法将线性化打靶载体转入胚胎干细胞(ES细胞);将含有过表达载体的ES细胞进行囊胚腔注射,并将嵌合囊胚移植至代孕小鼠体内,繁殖嵌合体小鼠.嵌合体小鼠和C57BL/6J鼠交配繁殖出杂合子,PCR筛选阳性过表达小鼠.采用小动物超声分析线粒体靶向过表达ECSIT小鼠的心功能.结果:成功构建了线粒体靶向过表达ECSIT载体pCAG-OTCL-ECSIT-3Xflag-BPA,经PCR鉴定为阳性;完成线粒体靶向过表达ECSIT基因打靶及囊胚注射,经PCR鉴定为阳性;分别提取转基因小鼠心肌组织线粒体和胞浆蛋白,检测发现线粒体特异性过表达ECSIT.8周龄转基因小鼠心功能与同龄野生型小鼠无明显差异.结论:成功构建出线粒体靶向过表达ECSIT小鼠;线粒体过表达ECSIT对8周龄小鼠心功能无明显影响.