Background Autoantibodies against the second extracellular loop of the β 1 ‐adrenoceptor (β 1 ‐ AA ) act similarly to agonist of β 1 ‐adrenergic receptor, which plays an important role in the pathophysiological characteristics of ventricular remodeling. Recently, considerable lines of evidence have suggested that CTRP9 (C1q tumor necrosis factor–related protein 9) is a potent cardioprotective cardiokine and protects the heart from ventricular remodeling. The aim of this study was to determine the role of CTRP 9 in ventricular remodeling induced by β 1 ‐ AA . Methods and Results Blood samples were collected from 131 patients with coronary heart disease and 131 healthy subjects. The serum levels of β 1 ‐ AA and CTRP 9 were detected using ELISA . The results revealed that CTRP 9 levels in β 1 ‐ AA –positive patients were lower than those in β 1 ‐ AA –negative patients, and serum CTRP 9 concentrations were inversely correlated with β 1 ‐ AA . β 1 ‐ AA monoclonal antibodies (β 1 ‐ AA mAbs) were administered in mice with and without rAAV 9‐ cTnT ‐Full Ctrp9‐ FLAG virus for 8 weeks. Reverse transcription–polymerase chain reaction/Western analysis showed that cardiomyocyte CTRP 9 expression was significantly reduced in β 1 ‐ AA mAb–treated mice. Moreover, compared with the β 1 ‐ AA mAb alone group, cardiac‐specific CTRP 9 overexpression improved cardiac function, attenuated adverse remodeling, and ameliorated cardiomyocyte apoptosis and fibrosis. Mechanistic studies demonstrated that CTRP 9 overexpression decreased the levels of G‐protein–coupled receptor kinase 2 and promoted the activation of AMP‐dependent kinase pathway. However, cardiac‐specific overexpression of CTRP 9 had no effect on the levels of cAMP and protein kinase A activity elevated by β 1 ‐AAmAb. Conclusions This study provides the first evidence that the long‐term existence of β 1 ‐ AA mAb suppresses cardiac CTRP 9 expression and exaggerates cardiac remodeling, suggesting that CTRP 9 may be a novel therapeutic target against pathologic remodeling in β 1 ‐ AA –positive patients with coronary heart disease.
Abstract Background Although agonistic autoantibodies against type-1 angiotensin-II receptor (AT 1 -AA) are frequently detected in women with preeclampsia, the clinical significance of AT 1 -AA in association with epithelial ovarian cancer (EOC) has not been identified. Methods In an attempt to clarify this issue, we measured serum AT 1 -AA titer from EOC patients (n = 89) and healthy normal subjects (n = 55), correlated AT 1 -AA titer with EOC stage and grade, and demonstrated the effects of purified AT 1 -AA on migration of ovarian cancer cells and angiogenesis of chick embryo chorioallantoic membrane. Results We found that the AT 1 -AA titer was significantly higher in EOC patients compared with healthy control subjects (1.77 ± 0.28 vs. 0.35 ± 0.05, P < 0.01). The positive rate was averaged by 72.1±2.5% in EOC patients and 15.5 ±1.5% in control (P < 0.01). Increased AT 1 -AA titer in EOC patients was associated with advanced stages and grades of EOC, and positively correlated with level of vascular endothelial growth factor (r = 0.855, P < 0.01). Furthermore, AT 1 -AA directly stimulated migration of ovarian cancer cells and enhanced microvascular density of chick embryo chorioallantoic membrane. These AT 1 -AA-mediated effects were significantly blocked either by an autoantibody-neutralizing peptide or an angiotensin II type I receptor antagonist, losartan. Conclusion Taken together, we found that a higher serum AT 1 -AA titer may be associated with advanced progression of EOC in patients and play an important role in development of EOC by promoting cancer cell migration and angiogenesis. These findings implicate that AT 1 -AA might be selected as a detectable biomarker and potential therapeutic target in diagnosis and treatment of EOC patients.
Numerous studies have found that the age-associated structural and functional alterations in arteries were characterized by increased endothelial dysfunction. In this study, young (3 months), adult (9 months), and aging (20 months) male Sprague-Dawley rats were randomly divided into 6 groups, including control groups and FeTMPyP (peroxynitrite scavenger) groups receiving saline and FeTMPyP, respectively, for 5 administrations once every 3 days through intraperitoneal injection. The aged-related proteins beta-galactosidase, p53, and p16 as well as the nitrotyrosine and endothelial marker endothelial nitric oxide synthase and von Willebrand factor (vWF) in vascular tissues were measured by immunohistochemistry. Endothelium-dependent vasorelaxation and endothelium-independent vasorelaxation of rat thoracic aortas and mesenteric arteries were measured by acetylcholine and sodium nitroprusside, respectively. The amount of circulating endothelial progenitor cells (EPCs) was determined by flow cytometry. The endothelium-dependent/independent relaxation in mesenteric arteries and the amount of circulating EPCs (CD31/CD34) in peripheral blood of aging rats were reduced significantly compared with young and adult rats. Immunohistochemistry results showed that the nitrotyrosine levels and morphological damage in mesenteric arteries were increased significantly in aging rats. Adoption of peroxynitrite scavenger FeTMPyP intervention may not only improve the endothelium-dependent relaxation and the amount of circulating EPCs in aging rats but also reverse endothelial injury. In conclusion, this study demonstrates that enhanced nitrative stress may aggravate the endothelial injury and vascular dysfunction of resistance arteries in aging rats. Antiperoxynitrite treatment can ameliorate the vasorelaxation and may be involved with the protection of circulating EPCs.
Poor oocyte quality is detrimental to fertilization and embryo development, which causes infertility. Cystathionine β-synthase (CBS) is one of the key enzymes modulating the metabolism of homocysteine (Hcy). Studies have shown that CBS plays an important role in female reproduction. However, the role of CBS in regulating oocyte quality during meiotic maturation still needs further investigation.Immunohistochemistry, immunofluorescence, drug treatment, western blot, cRNA construct and in vitro transcription, microinjection of morpholino oligo and cRNA were performed for this study.We found that CBS was expressed both in human and mouse oocytes of follicles. In mouse oocytes, CBS was distributed in the nucleus at germinal vesicle (GV) stage and localized to spindle from germinal vesicle breakdown (GVBD) to metaphase II (MII). The expression of CBS was reduced in ovaries and oocytes of aged mice. CBS depletion resulted in meiotic arrest, spindle abnormality and chromosome misalignment, disrupted kinetochore-microtubule attachments and provoked spindle assembly checkpoint (SAC). CBS was disassembled when microtubules were disrupted with nocodazole, and co-localized with the stabilized microtubules after taxol treatment. Furthermore, CBS depletion decreased the acetylation of α-tubulin.These results reveal that CBS is required for the acetylation of α-tubulin to ensure proper spindle assembly in regulating oocyte quality during meiotic maturation.
Abstract BackgroundCardiomyocyte autophagy is essential for maintaining cardiac function. Our previous studies have found that β 1 -adrenergic receptor autoantibody (β 1 -AA) induced the decreased myocardial autophagic flux, which resulted in cardiomyocytes death and cardiac dysfunction. And other studies demonstrated that β 1 -AA induced the decrease of AMPK phosphorylation, the key hub of autophagy pathway, while adiponectin up-regulated autophagic flux mediated by AMPK. However, it is not clear whether adiponectin improves the inhibition of myocardial autophagic flux induced by β 1 -AA by up-regulating the level of AMPK phosphorylation.MethodAPN-KO and WT mice were subcutaneously injected withthe peptide of the second extracellular loop of β 1 -adrenergic receptor (β 1 -AR-ECII) as antigen to establish β 1 -AA active immunization model. The OD value of β 1 -AA in serum of mice was detected by SA-ELISA to ensure the successful construction of active immunization model. The changes of cardiac function and myocardial fibrosis were detected by small animal ultrasound and Masson trichrome staining. The cardiomyocytes viability was assessed using a Cell Counting Kit-8. The mRNA levels of LC3B and Beclin1 in cardiomyocytes were measured by Real-time PCR. The protein levels of LC3 II, p62, AMP dependent protein kinase (AMP) - activated protein kinase (AMPK) and phosphorylated AMP dependent protein kinase (p-AMPK) in cardiomyocytes were detected by Western blotting.ResultsIn this study, it has been confirmed that β 1 -AA induced the decrease of AMPK phosphorylation level in both vivo and vitro. Moreover, pretreatment of cardiomyocytes with AMPK inhibitor Compound C could further reduce the autophagic flux induced by β 1 -AA. Adiponectin deficiency could aggravate the decrease of myocardial AMPK phosphorylation level, autophagic flux and cardiac function induced by β 1 -AA. Further, exogenous adiponectin could reverse the decline of AMPK phosphorylation level and autophagic flux induced by β 1 -AA, and even reduce cardiomyocytes death. While pre-treated with the Compound C, the adiponectin treatment did not improve the decreased autophagosome formation, but still improved the decreased autophagosome clearance induced by β 1 -AA in cardiomyocytes, suggesting that adiponectin up-regulated β 1 -AA-induced decreased autophagic flux of cardiomyocytes partly depended on the AMPK.ConclusionThis study is the first time to confirm that β 1 -AA could inhibit myocardial autophagic flux by down regulating AMPK phosphorylation level. Adiponectin could improve the inhibition of myocardial autophagic flux induced by β 1 -AA partly dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β 1 -AA-positive cardiac dysfunction.
Since the autoantibodies against the second extracellular loop of β(1)-adrenoceptor (β(1)-AABs) have been found in the sera of patients with idiopathic dilated cardiomyopathy (IDCM), the involvement of autoimmune mechanisms in the pathogenesis of many cardiovascular diseases has extensively been investigated. Our previous study found that urinary occult blood and protein excretion were frequently found in the rats with positive β(1)-AABs, but the mechanisms are unclear. Therefore, we infused the β(1)-AABs into the vein periodically in an attempt to investigate whether β(1)-AABs could induce morphological and functional changes in the kidneys of adult and aged rats and explore the possible mechanisms. The synthetic peptide according to the sequences of the second extracellular loop of β(1)-adrenoceptor (β(1)-AR-ECII) was used to immunize the adult rats to acquire enough β(1)-AABs for use. Neonatal rat ventricular myocytes (NRVMs) culture was used to observe the biological effects of β(1)-AABs on the beating rate. The purified β(1)-AABs were transfused into the vein of rats. The sera level of blood urea nitrogen (BUN), creatinine (CR), uric acid (UA), urinary specific gravity, protein excretion, occult blood and urinary glucose were detected at the different time points by biochemistry and urine analyzers. HE and Masson's trichrome staining were used to detect the changes in kidney structure of passively immunized rats. Enhanced green fluorescent protein (EGFP) and β(1)-AR-EGFP plasmids were transfected into the human embryonic kidney 293 (HEK293) cells in order to observe the changes in cell injury with the treatment of β(1)-AABs. It was found that the sera level of BUN, CR and UA increased gradually and the ratio of BUN to CR decreased progressively with the administration of β(1)-AABs. The increasing of proteinuria, urinary occult blood and urinary glucose was detected by urine analyzer in β(1)-AABs group. By HE and Masson's coloration, lots of mononuclear cell infiltration and collagen fibers deposition could be observed at the 24th week of immunization. After the treatment of β(1)-AABs, the caspase-3 activity increased significantly in the HEK293 cells transfected with β(1)-AR-EGFP plasmids, while no significant changes were observed for lactate dehydrogenase (LDH) activity. The results indicate that long-term presence of β(1)-AABs can induce the morphological and functional damage of the kidneys in adult and aged rats.
HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.
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