CARMA3 Deficiency Aggravates Angiotensin II-Induced Abdominal Aortic Aneurysm Development Interacting Between Endoplasmic Reticulum and Mitochondria.

BACKGROUND: Abdominal aortic aneurysm (AAA) is life threatening and associated with vascular walls' chronic inflammation. However, a detailed understanding of the underlying mechanisms is yet to be elucidated. CARMA3 assembles the CARMA3-BCL10-MALT1 (CBM) complex in inflammatory diseases and is proven to mediate angiotensin II (Ang II) response to inflammatory signals by modulating DNA damage-induced cell pyroptosis. In addition, interaction between endoplasmic reticulum (ER) stress and mitochondrial damage is one of the main causes of cell pyroptosis. METHODS: Male wild type (WT) or CARMA3-/- mice aged 8 to 10 weeks were subcutaneously implanted with osmotic minipumps, delivering saline or Ang II at the rate of 1 µg/kg/min for 1, 2, and 4 weeks. RESULTS: We discovered that CARMA3 knockout promoted formation of AAA and prominently increased diameter and severity of the mice abdominal aorta infused with Ang II. Moreover, a significant increase in the excretion of inflammatory cytokines, expression levels of matrix metalloproteinases (MMPs) and cell death was found in the aneurysmal aortic wall of CARMA3-/- mice infused with Ang II compared with WT mice. Further studies found that the degree of ER stress and mitochondrial damage in the abdominal aorta of CARMA3-/- mice was more severe than that in WT mice. Mechanistically, CARMA3 deficiency exacerbates the interaction between ER stress and mitochondrial damage by activating the p38MAPK pathway, ultimately contributing to the pyroptosis of vascular smooth muscle cells (VSMCs). CONCLUSIONS: CARMA3 appears to play a key role in AAA formation and might be a potential target for therapeutic interventions of AAA.

I-κB kinase-ε deficiency improves doxorubicin-induced dilated cardiomyopathy by inhibiting the NF-κB pathway.

Dilated cardiomyopathy (DCM) can lead to heart expansion and severe heart failure, but its specific pathogenesis is still elusive. In many cardiovascular diseases, I-κB kinase-ε (IKKε) has been recognized as a pro-inflammatory molecule. In this study, wild-type mice (WT, n = 14) and IKKε knockout mice (IKKε-KO, n = 14) were intraperitoneally injected with a cumulative dose of 25 mg/kg with Dox or Saline five times in 30 days. Finally, the experimental mice were divided into WT + Saline group、WT + DOX group、IKKε-KO + Saline group and IKKε-KO + Dox group. Echocardiography was performed to assess cardiac structure and function. Moreover, the mechanism was validated by immunohistochemistry and western blotting. Our results demonstrated that compared to WT + Dox mice, IKKε-KO + Dox mice exhibited attenuation of dilated cardiomyopathy-related morphological changes and alleviation of heart failure. Additionally, compared to the WT mice after Dox-injected, the expression of fibrosis and proinflammatory were decreased in IKKε-KO mice, and the expression of cardiac gap junction proteins was much higher in IKKε-KO mice. Further testing found that pyroptosis and apoptosis in the myocardium were also ameliorated in IKKε-KO mice compared to WT mice after Dox was injected. Mechanistically, our results showed that deficiency of IKKε might inhibit the phosphorylation of IκBα, p65, RelB, and p100 in mouse heart tissues after Dox stimulation. In summary, our research suggests that IKKε might play an essential role in the development of Dox-induced dilated cardiomyopathy and may be a potential target for the treatment of dilated cardiomyopathy in the future.

IKKε knockout alleviates angiotensin II-induced apoptosis and excessive autophagy in vascular smooth muscle cells by regulating the ERK1/2 pathway.

Inhibitor of nuclear factor-κB kinase subunit ε (IKKε) is an important signal regulator in the formation of abdominal aortic aneurysm (AAA). However, the underlying mechanism remains to be elucidated. Therefore, the present study aimed to investigate the mechanism underlying IKKε function in AAA formation by studying apoptosis and autophagy in angiotensin II (Ang II)-induced vascular smooth muscle cells (VSMCs). AngII was used to stimulate VSMCs for 24 h to simulate the process of AAA formation. VSMCs were transfected with IKKε small interfering RNA to investigate the effect of IKKε on AAA formation, cell apoptosis and autophagy. IKKε deficiency led to reduced mitochondrial damage and apoptosis in VSMCs in the early stage of apoptosis in vitro, as demonstrated using a JC-1 probe. IKKε deficiency also reduced autophagy and decreased the formation of autophagic vacuoles in VSMCs, demonstrated using transmission electron microscopy. The decrease in apoptosis caused by IKKε knockdown was reversed when the autophagic flow was blocked using bafilomycin A1. Western blot analysis further revealed that IKKε deficiency negatively regulated the ERK1/2 signaling pathway to reduce autophagy. Collectively, the results of the present study revealed that IKKε played a key role in apoptosis by inducing excessive autophagy, thereby potentially contributing to AAA formation. These findings further revealed the mechanism underlying IKKε function in the formation of AAA.

I-κB Kinase-ε Deficiency Attenuates the Development of Angiotensin II-Induced Myocardial Hypertrophy in Mice.

I-κB kinase-ε (IKKε) is a member of the IKK complex and a proinflammatory regulator that is active in many diseases. Angiotensin II (Ang II) is a vasoconstricting peptide hormone, and Ang II-induced myocardial hypertrophy is a common cardiovascular disease that can result in heart failure. In this study, we sought to determine the role of IKKε in the development of Ang II-induced myocardial hypertrophy in mice. Wild-type (WT) and IKKε-knockout (IKKε-KO) mice were generated and infused with saline or Ang II for 8 weeks. We found that WT mouse hearts have increased IKKε expression after 8 weeks of Ang II infusion. Our results further indicated that IKKε-KO mice have attenuated myocardial hypertrophy and alleviated heart failure compared with WT mice. Additionally, Ang II-induced expression of proinflammatory and collagen factors was much lower in the IKKε-KO mice than in the WT mice. Apoptosis and pyroptosis were also ameliorated in IKKε-KO mice. Mechanistically, IKKε bound to extracellular signal-regulated kinase (ERK) and the mitogen-activated protein kinase p38, resulting in MAPK/ERK kinase (MEK) phosphorylation, and IKKε deficiency inhibited the phosphorylation of MEK-ERK1/2 and p38 in mouse heart tissues after 8 weeks of Ang II infusion. The findings of our study reveal that IKKε plays an important role in the development of Ang II-induced myocardial hypertrophy and may represent a potential therapeutic target for the management of myocardial hypertrophy.

miR-133a-3p attenuates cardiomyocyte hypertrophy through inhibiting pyroptosis activation by targeting IKKε.

OBJECTIVE: Cardiac hypertrophy is an adaptive response to physiological and pathological stimuli, the latter of which frequently progresses to valvulopathy, heart failure and sudden death. Recent reports revealed that pyroptosis is involved in regulating multiple cardiovascular diseases progression, including cardiac hypertrophy. However, the underlying mechanisms remain poorly understood. This study aims to extensively investigate the regulation of miR-133a-3p on pyroptosis in angiotensin II (Ang II)-induced cardiac hypertrophyin vitro. METHODS: The in vitro model of cardiac hypertrophy was induced by Ang II, which was validated by qPCR combined with measurement of cell surface area by immunofluorescence assay. CCK-8 assay and Hochest33342/PI staining was performed to assess pyroptosis. Dual luciferase reporter system was used to verify the direct interaction between miR-133a-3p and IKKε. The effects of miR-133a-3p/IKKε on pyroptosis activation and cardiac hypertrophy markers (Caspase-1, NLRP3, IL-1ß, IL-18, GSDMD, ASC, ANP, BNP and ß-MHC) were evaluated by western blot, ELISA and qPCR. RESULTS: Ang II treatment could induce cardiomyocyte hypertrophy and pyroptosis. The expression of miR-133a-3p was repressed in Ang II-treated HCM cells, and its overexpression could attenuate both pyroptosis and cardiac hypertrophyin vitro. Additionally, IKKε expression was significantly up-regulated in Ang II-induced HCM cells. Dual luciferase reporter system and qPCR validated that miR-133a-3p directly targeted the 3'-UTR of IKKε and suppressed its expression. Moreover, IKKε overexpression impaired the protective function of miR-133a-3p in cardiomyocyte hypertrophy. CONCLUSION: Collectively, miR-133a-3p attenuates Ang II induced cardiomyocyte hypertrophy via inhibition of pyroptosis by targeting IKKε. Therefore, miR-133a-3p up-regulation may be a promising strategy for cardiac hypertrophy treatment.

A Novel Molecular Mechanism of IKKε-Mediated Akt/mTOR Inhibition in the Cardiomyocyte Autophagy after Myocardial Infarction.

Autophagy of cardiomyocytes after myocardial infarction (MI) is an important factor affecting the prognosis of MI. Excessive autophagy can lead to massive death of cardiomyocytes, which will seriously affect cardiac function. IKKε plays a crucial role in the occurrence of autophagy, but the functional role in MI remains largely unknown. To evaluate the impact of IKKε on the autophagy of cardiomyocytes after MI, MI was induced by surgical left anterior descending coronary artery ligation in IKKε knockout (KO) mice and wild-type (WT) mice. Starvation of H9c2 cells with IKKε siRNA and rescued with IKKε overexpressed afterwards to test the mechanism of IKKε in autophagy in vitro. Our results demonstrated that the expression of IKKε was upregulated in mice myocardial tissues which were consistent with cardiomyocyte autophagy after MI. Significantly, the IKKε KO mice showed increased infarct size, decreased viable cardiomyocytes, and exacerbated cardiac dysfunction when compared with the wild-type mice. Western blot and electron micrography analysis also revealed that loss of IKKε induces excessive cardiomyocyte autophagy and reduced the expression of p-Akt and p-mTOR. Similar results were observed in IKKε siRNA H9c2 cells in vitro which were under starvation injury. Notably, the levels of p-Akt and p-mTOR can restore in IKKε rescued cells. In conclusion, our results indicated that IKKε protects cardiomyocyte survival by reduced autophagy following MI via regulation of the Akt/mTOR signaling pathway. Thus, our study suggests that IKKε might represent a potential therapeutic target for the treatment of MI.

High expression levels and localization of Sox5 in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease that can lead to heart expansion and severe heart failure, but the specific pathogenesis remains unclear. Sox5 is a member of the Sox family with a key role in cardiac function. However, the role of Sox5 in DCM remains unclear. In the present study, wild­type mice were intraperitoneally injected with doxorubicin (Dox) to induce DCM, and heart specimens from human patients with DCM were used to investigate the preliminary role of Sox5 in DCM. The present study demonstrated that, compared with control human hearts, the hearts of patients with DCM exhibited high expression levels of Sox5 and activation of the wnt/ß­catenin pathway. This result was consistent with Dox­induced DCM in mice. Furthermore, in Dox­treated mice, apoptosis was activated during the development of DCM. Inflammation and collagen deposition also increased in DCM mice. The results of the present study indicate that Sox5 may be associated with the development of DCM. Sox5 may be a novel potential factor that regulates DCM.

De Ritis ratio as a significant prognostic factor of international normalized ratio ≥4 in the initial 10 days of warfarin therapy.

Aim: To assess the relationship between the De Ritis ratio on admission and warfarin sensitivity in the initial 10 days of anticoagulation therapy. Methods: We retrospectively reviewed data from 906 patients who underwent heart valve replacement surgery. Results: A De Ritis ratio of 1.19 was identified as the optimal cutoff according to the ROC curve. Patients with a high De Ritis ratio achieved an international normalized ratio (INR) ≥4 more easily and earlier than those with a low De Ritis ratio in the initial 10 days of warfarin therapy. Multivariate analysis showed that a high De Ritis ratio was an independent predictor of an INR ≥4 (HR: 2.567; p < 0.001). Conclusion: A De Ritis ratio ≥1.19 on admission was significantly associated with an INR ≥4 in the initial 10 days of warfarin therapy among patients underwent heart valve replacement surgery.

Macrophage-Specific IκB Kinase α Contributes to Ventricular Remodelling and Dysfunction After Myocardial Infarction.

BACKGROUND: The IκB kinase (IKK) complex has been found to have critical functions in cancer and the immune system. In particular, IKKα, which is a member of the IKK complex, has been shown to influence the inflammatory response and malignant diseases. However, the role of IKKα in macrophages after myocardial infarction (MI) remains largely unknown. METHODS: Sham or MI operations were performed on macrophage-specific IKKɑ knockout (mIKKɑ-/-) mice and IKKɑflox/flox littermates. We ligated the left anterior descending coronary artery of the MI group and observed the results at 3, 7, and 30 days after MI. RESULTS: We discovered more severe cardiac dysfunction with reduced angiogenesis, fibrosis, and collagen deposition in mIKKɑ-/- than in IKKɑflox/flox. In addition, we also observed that macrophages in mIKKɑ-/- were easier to polarize to the M1 phenotype and expressed more proinflammatory factors than IKKɑflox/flox. Mechanistically, IKKα deficiency in macrophages inhibited the alternative nuclear factor-κB/RelB pathway and enhanced the MEK1/2/ERK1/2 pathway. CONCLUSIONS: Overall, our data identified IKKɑ in the heart as a novel mediator that protected the heart from a severe inflammatory response and attenuated ventricular remodelling after MI by negatively regulating macrophage polarization to the M1 phenotype. Therefore, IKKα may serve as a potential therapeutic target for treatment after MI.

Regulatory role of IKKɑ in myocardial ischemia/reperfusion injury by the determination of M1 versus M2 polarization of macrophages.

The IκB kinase (IKK) complex plays a well-documented role in cancer and immune system. This function has been widely attributed to its role as the master regulator of the NF-κB family. Particularly, IKKɑ, a member of IKK complex, is reported to have various regulating effects in inflammatory and malignant diseases. However, its role as well as its mechanism of function in macrophages following myocardial ischemia and reperfusion (I/R) injury remains unexplored. In vivo, sham or I/R operations were performed on macrophage-specific IKKɑ knockout (mIKKɑ-/-) mice and their IKKɑflox/flox littermates. We ligated the left anterior descending (LAD) coronary artery of I/R groups simulating ischemia for 30 min, followed by a reperfusion period of 3 days and 7 days, respectively. The hearts of mIKKɑ-/- mice exhibited significantly increased inflammation and macrophage aggregation as compared to their IKKɑflox/flox littermates. Moreover, in the mIKKɑ-/- group subjected to I/R macrophages had a tendency to polarize to M1 phenotype. In vitro, we stimulated RAW264.7 cells with Lipopolysaccharides (LPS) after infection by the lentivirus, either knocking-down or overexpressing IKKɑ. We discovered that a deficiency of IKKɑ in RAW264.7 caused increased expression of pro-inflammatory markers compared to normal RAW264.7 after LPS stimulation. Inversely, pro-inflammatory factors were inhibited with IKKɑ overexpression. Mechanistically, IKKɑ directly combined with RelB to regulate macrophage polarization. Furthermore, IKKɑ regulated MEK1/2-ERK1/2 and downstream p65 signaling cascades after LPS stimulation. Overall, our data reveals that IKKɑ is a novel mediator protecting against the development of myocardial I/R injury via negative regulation of macrophage polarization to M1 phenotype. Thus, IKKɑ may serve as a valuable therapeutic target for the treatment of myocardial I/R injury.

Risk factors for early death in primary malignant cardiac tumors: An analysis of over 40 years and 500 patients.

OBJECTIVES: To investigate risk factors contributing to early death in patients diagnosed with primary malignant cardiac tumors (PMCTs) and derive better understanding of these poorly characterized individuals. METHOD: Data from the Surveillance, Epidemiology and End-Results (SEER) registries on 564 patients diagnosed with PMCTs between 1973 and 2014 were analyzed. Early death was defined as survival of ≤3 months from the time of diagnosis. Two-tailed χ2 or fisher's exact test were used for association between categorical variables and occurrence of early death. Logistic regression analysis was used to assess independent risk factors of early death. Time trends in early death rates of PMCTs were described using scatter plot. RESULTS: Of the 564 patients with PMCTs, early death was identified in 214 individuals (37.9%). Patients with unspecified soft tissue sarcomas and blood vessel tumors had the highest risk of early death. Age > 80 years and non-consent for surgery were strong predictors of early death in all PMCT subtypes. In sarcomas, disadvantaged income was associated with an increase in early mortality, while black race was associated with a reduction in early mortality. In mesotheliomas and others, male sex was a risk factor for early mortality, while Hispanic ethnicity was associated with a reduction in early mortality. Percentages of early death slightly decreased over the past 40 years. CONCLUSIONS: Predictors of early death are primarily related to age older than 80 years, no surgery and specific histopathology types but also include disadvantaged socioeconomic status and male sex. Initiatives to identify those at risk and develop preventive interventions should be prioritized.

Association between apolipoprotein E genotype and warfarin response during initial anticoagulation.

Apolipoprotein E (APOE) genotypes are associated with warfarin dose requirements in various populations. Whether APOE genotypes mediate the warfarin response is unknown. The aim of this study was to evaluate the genetic contributions of different APOE genotypes to the early phase of anticoagulation in Han Chinese patients. We conducted a retrospective cohort study and assessed APOE genotypes, clinical characteristics, international normalized ratio (INR) responses, warfarin dose requirements and bleeding events in 429 Han Chinese patients. The study outcomes were the time to the first INR within the therapeutic range, the time to the first INR of more than 4, the INR response over time, and the warfarin dose requirement. Compared with patients with the ε3/ε3 genotype, patients with at least one ε4 allele had significantly longer times to the first INR of more than 4 during both the initial 20 days (P = 0.001, HR 2.9; 95%CI, 1.54-5.45) and the entire follow-up period (P < 0.001, HR 3.26; 95%CI,1.94-5.47), but this allele was not a significant predictor of the time to the first INR within the therapeutic range. No association was observed between the ε2 allele and INR response, and both the ε4 allele and the ε2 allele did not significantly affect the required warfarin dose during the follow-up. These observations suggest that genetic variants of APOE are associated with an increased risk of overanticoagulation among the Han Chinese population. However, these variants may not be useful in predicting warfarin maintenance dose requirements.

Upregulation of ADAMTS­7 and downregulation of COMP are associated with aortic aneurysm.

Aortic aneurysm (AA) remains a fatal condition with high rates of morbidity and mortality, and the associated underlying mechanism influencing its pathology remains to be elucidated. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­7 has previously been demonstrated to be involved in the pathogenesis of vascular atherosclerosis via degradation of cartilage oligomeric matrix protein (COMP). The ADAMTS­7/COMP pathway may therefore act as a potential therapeutic target for vascular disorders. To the best of the author's knowledge, the present study aimed to investigate for the first time, the expression of ADAMTS­7 and COMP in human AA. Human aortic aneurysm samples were collected from patients with AA (n=24), and ascending aorta control samples were harvested from dilated cardiomyopathy patients who underwent heart transplantation (n=18). Expression levels of ADAMTS­7 and matrix metalloproteinase­9 were significantly increased in the AA group, as detected by immunohistochemistry (P<0.05). The COMP protein level was markedly decreased in the AA group when compared with the control group, as demonstrated via immunohistochemistry and western blot analysis (P<0.05). The findings suggest that upregulation of ADAMTS­7 and downregulation of COMP are associated with induction of human AA. ADAMTS­7/COMP pathway may provide therefore act as a potential therapeutic target in human AA for efficient, optimal treatment interventions in the future.

Prevalence of primary cardiac tumor malignancies in retrospective studies over six decades: a systematic review and meta-analysis.

The incidence of patients diagnosed with primary malignant cardiac tumors (PMCTs) has increased greatly in the past few decades. Whether this rising prevalence is due to overdiagnosis or an increased malignancy rate of primary cardiac tumors (PCTs) remains unclear. Therefore, we performed a systematic review and meta-analysis of published retrospective studies to determine whether the malignancy rate has been increasing over time. Published studies containing relevant data between 1956 and 2014 were evaluated. Two authors searched for all retrospective studies that included patients diagnosed with PCT and PMCT. Two other investigators independently extracted the data, and discrepancies were resolved by consensus. A random-effects meta-analysis model and cumulative meta-analysis model were used to evaluate the pooled prevalence and trend of dynamic change in PCT malignancies. The effects of time, study period and sample size were studied using a logit-linear regression model with robust error variance and a time variable. Thirty-eight studies involving 5,586 patients were analyzed. The pooled prevalence of PMCT among the patients diagnosed with PCT was 9.9% (95% CI, 8.4% to 11.4%) (I2=70%; P< 0.001), and this prevalence has been stable since around 2003. In the regression model, the malignancy odds ratio remained stable from 1975 onward, and no time effect was observed. Our study confirms that PMCT is uncommon, and the prevalence of PCT malignancies remained stable in the past few decades. The clinically observed increase in incidence is unlikely to reflect a true population-level increase in tumorigenesis. This result strongly suggests that the observed increase in incidence of PMCT most likely reflects increased diagnostic detection over time.