Abnormal expression of PRKAG2-AS1 in endothelial cells induced inflammation and apoptosis by reducing PRKAG2 expression.

PRKAG2 is required for the maintenance of cellular energy balance. PRKAG2-AS1, a long non-coding RNA (lncRNA), was found within the promoter region of PRKAG2. Despite the extensive expression of PRKAG2-AS1 in endothelial cells, the precise function and mechanism of this gene in endothelial cells have yet to be elucidated. The localization of PRKAG2-AS1 was predominantly observed in the nucleus, as revealed using nuclear and cytoplasmic fractionation and fluorescence in situ hybridization. The manipulation of PRKAG2-AS1 by knockdown and overexpression within the nucleus significantly altered PRKAG2 expression in a cis-regulatory manner. The expression of PRKAG2-AS1 and its target genes, PRKAG2b and PRKAG2d, was down-regulated in endothelial cells subjected to oxLDL and Hcy-induced injury. This finding suggests that PRKAG2-AS1 may be involved in the mechanism behind endothelial injury. The suppression of PRKAG2-AS1 specifically in the nucleus led to an upregulation of inflammatory molecules such as cytokines, adhesion molecules, and chemokines in endothelial cells. Additionally, this nuclear suppression of PRKAG2-AS1 facilitated the adherence of THP1 cells to endothelial cells. We confirmed the role of nuclear knockdown PRKAG2-AS1 in the induction of apoptosis and inhibition of cell proliferation, migration, and lumen formation through flow cytometry, TUNEL test, CCK8 assay, and cell scratching. Finally, it was determined that PRKAG2-AS1 exerts direct control over the transcription of PRKAG2 by its binding to their promoters. In conclusion, downregulation of PRKAG2-AS1 suppressed the proliferation and migration, promoted inflammation and apoptosis of endothelial cells, and thus contributed to the development of atherosclerosis resulting from endothelial cell injury.

Abnormal expression of PRKAG2-AS results in dysfunction of cardiomyocytes through regulating PRKAG2 transcription by interacting with PPARG.

The role of PRKAG2 in the maintenance of heart function is well established, but little is known about how PRKAG2 is regulated in cardiomyocytes. In this study, we investigated the role of the lncRNA PRKAG2-AS, which is present at the PRKAG2 promoter, in the regulation of PRKAG2 expression. PRKAG2-AS expression was predominantly nuclear, as determined by RNA nucleoplasmic separation and fluorescence in situ hybridization. Knockdown of PRKAG2-AS in the nucleus, but not the cytoplasm, significantly decreased the expression of PRKAG2b and PRKAG2d. Interestingly, we found that PRKAG2-AS and its target genes, PRKAG2b and PRKAG2d, were reduced in the hearts of patients with ischemic cardiomyopathy, suggesting a potential role for PRKAG2-AS in myocardial ischemia. Indeed, knockdown of PRKAG2-AS in the nucleus resulted in apoptosis of cardiomyocytes. We further elucidated the mechanism by which PRKAG2-AS regulates PRKAG2 transcription by identifying 58 PRKAG2-AS interacting proteins. Among them, PPARG was selected for further investigation based on its correlation and potential interaction with PRKAG2-AS in regulating transcription. Overexpression of PPARG, or its activation with rosiglitazone, led to a significant increase in the expression of PRKAG2b and PRKAG2d in cardiomyocytes, which could be attenuated by PRKAG2-AS knockdown. This finding suggests that PRKAG2-AS mediates, at least partially, the protective effects of rosiglitazone on hypoxia-induced apoptosis. However, given the risk of rosiglitazone in heart failure, we also examined the involvement of PRKAG2-AS in this condition and found that PRKAG2-AS, as well as PRKAG2b and PRKAG2d, was elevated in hearts with dilated cardiomyopathy (DCM) and that overexpression of PRKAG2-AS led to a significant increase in PRKAG2b and PRKAG2d expression, indicating that up-regulation of PRKAG2-AS may contribute to the mechanism of heart failure by promoting transcription of PRKAG2. Consequently, proper expression of PRKAG2-AS is essential for maintaining cardiomyocyte function, and aberrant PRKAG2-AS expression induced by hypoxia or other stimuli may cause cardiac dysfunction.

Downregulation of NUP93 aggravates hypoxia-induced death of cardiomyocytes in vitro through abnormal regulation of gene transcription.

Nuclear pore complex in the nuclear envelope plays an important role in controlling the transportation of RNAs, proteins and other macromolecules between the nucleus and cytoplasm. The relationship between abnormal expression of nucleoporins and cardiovascular diseases is unclear. In this study we investigated how myocardial infarction affected the expression and function of nucleoporins in cardiomyocytes. We separately knocked down 27 nucleoporins in rat primary myocardial cells. Among 27 nucleoporins, knockdown of Nup93, Nup210 and Nup214 markedly increased the expression of ANP and BNP, two molecular markers of cardiomyocyte function. We showed that Nup93 was significantly downregulated in hypoxic cardiomyocytes. Knockdown of Nup93 aggravated hypoxia-induced injury and cell death of cardiomyocytes, whereas overexpression of Nup93 led to the opposite effects. RNA-seq and bioinformatics analysis revealed that knockdown of Nup93 did not affect the overall transportation of mRNAs from the nucleus to the cytoplasm, but regulated the transcription of a large number of mRNAs in cardiomyocytes, which are mainly involved in oxidative phosphorylation and ribosome subunits. Most of the down-regulated genes by Nup93 knockdown overlapped with the genes whose promoters could be directly bound by Nup93. Among these genes, we demonstrated that Nup93 knockdown significantly down-regulated the expression of YAP1. Overexpression of YAP1 partially rescued the function of Nup93 knockdown and attenuated the effects of hypoxia on cell injury and cardiomyocyte death. We conclude that down-regulation of Nup93, at least partially, contributes to hypoxia-induced injury and cardiomyocyte death through abnormal interaction with the genome to dynamically regulate the transcription of YAP1 and other genes. These results reveal a new mechanism of Nup93 and might provide new therapeutic targets for the treatment of ischemia-induced heart failure.

Caspase-6-cleaved tau is relevant in Alzheimer's disease and marginal in four-repeat tauopathies: Diagnostic and therapeutic implications.

AIM: Tau truncation (tr-tau) by active caspase-6 (aCasp-6) generates tau fragments that may be toxic. Yet the relationship between aCasp-6, different forms of tr-tau and hyperphosphorylated tau (p-tau) accumulation in human brains with Alzheimer's disease (AD) and other tauopathies remains unclear. METHODS: We generated two neoepitope monoclonal antibodies against tr-tau sites (D402 and D13) targeted by aCasp-6. Then, we used five-plex immunofluorescence to quantify the neuronal and astroglial burden of aCasp-6, tr-tau, p-tau and their co-occurrence in healthy controls, AD and primary tauopathies. RESULTS: Casp-6 activation was strongest in AD and Pick's disease (PiD) but almost absent in 4-repeat (4R) tauopathies. In neurons, the tr-tau burden was much more abundant in AD and PiD than in 4R tauopathies and disproportionally higher when normalising by p-tau pathology. Tr-tau astrogliopathy was detected in low numbers in 4R tauopathies. Unexpectedly, about half of tr-tau positive neurons in AD and PiD lacked p-tau aggregates, a finding we confirmed using several p-tau antibodies. CONCLUSIONS: Early modulation of aCasp-6 to reduce tr-tau pathology is a promising therapeutic strategy for AD and PiD but is unlikely to benefit 4R tauopathies. The large percentage of tr-tau-positive neurons lacking p-tau suggests that many vulnerable neurons to tau pathology go undetected when using conventional p-tau antibodies. Therapeutic strategies against tr-tau pathology could be necessary to modulate the extent of tau abnormalities in AD. The disproportionally higher burden of tr-tau in AD and PiD supports the development of biofluid biomarkers against tr-tau to detect AD and PiD and differentiate them from 4R tauopathies at a patient level.

Cardiovascular-specific PSEN1 deletion leads to abnormalities in calcium homeostasis.

Mutations of PSEN1 have been reported in dilated cardiomyopathy pedigrees. Understanding the effects and mechanisms of PSEN1 in cardiomyocytes might have important implications for treatment of heart diseases. Here, we showed that PSEN1 was downregulated in ischemia-induced failing hearts. Functionally, cardiovascular specific PSEN1 deletion led to spontaneous death of the mice due to cardiomyopathy. At the age of 11 months, the ratio of the heart weight/body weight was slightly lower in the Sm22a-PSEN1-KO mice compared with that of the WT mice. Echocardiography showed that the percentage of ejection fraction and fractional shortening was significantly reduced in the Sm22a-PSEN1-KO group compared with the percent of these measures in the WT group, indicating that PSEN1-KO resulted in heart failure. The abnormally regulated genes resulted from PSEN1-KO were detected to be enriched in muscle development and dilated cardiomyopathy. Among them, several genes encode Ca2+ ion channels, promoting us to investigate the effects of PSEN1 KO on regulation of Ca2+ in isolated adult cardiomyocytes. Consistently, in isolated adult cardiomyocytes, PSEN1-KO increased the concentration of cytosolic Ca2+ and reduced Ca2+ concentration inside the sarcoplasmic reticulum (SR) lumen at the resting stage. Additionally, SR Ca2+ was decreased in the failing hearts of WT mice, but with the lowest levels observed in the failing hearts of PSEN1 knockout mice. These results indicate that the process of Ca2+ release from SR into cytoplasm was affected by PSEN1 KO. Therefore, the abnormalities in Ca2+ homeostasis resulted from downregulation of PSEN1 in failing hearts might contribute to aging-related cardiomyopathy, which might had important implications for the treatment of aging-related heart diseases.

Molecular characterization of selectively vulnerable neurons in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus-brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively-from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience.

A manual multiplex immunofluorescence method for investigating neurodegenerative diseases.

BACKGROUND: Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps. NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing. RESULTS: A protocol using ß-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue. COMPARISON WITH EXISTING METHODS: Our proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping. CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.

Plasma miR-451 with echocardiography serves as a diagnostic reference for pulmonary hypertension.

Due to the lack of typical clinical symptoms, the average delay time for diagnosis of pulmonary hypertension (PH) is longer than 2 years. It is urgent to find biomarkers for PH diagnosis. In this study we investigated whether plasma microRNAs (miRNAs) can be used as biomarkers for PH diagnosis. We used microarray to identify dynamic miRNAs between PH and non-PH patients. The candidate miRNAs were verified using qRT-PCR in a mouse model of PH, which was induced by monocrotaline (MCT) injection. We observed that miR-21, miR-126, miR-145, miR-191 and miR-150 had no differences between control mice and MCT-treated mice; but plasma miR-451 was significantly decreased in the 2wk-MCT group, with no further decrease in the 4wk-MCT group. Plasma miR-451 was also markedly decreased in PH patients, whereas miR-21, miR-126, miR-150 and miR-320 did not show differences between 53 PH patients and 54 non-PH patients. Receiver operating characteristic curves (ROCs) were constructed from the patient data to assess the clinical diagnostic values of circulating miR-451 and Doppler echocardiography (D-ECHO). The areas under the curve (AUCs) of ROCs for miR-451 and D-ECHO were 0.710 and 0.766, respectively. Combination of miR-451 and D-ECHO with AUC of 0.825 was superior to the use of either miR-451 or D-ECHO alone for PH diagnosis. In conclusion, plasma miR-451 has a moderate diagnostic value in PH comparable to that of D-ECHO, and the combination of miR-451 with D-ECHO has better diagnostic value than either method alone, which may have implications for PH diagnosis.

Dynamic autophagic activity affected the development of thoracic aortic dissection by regulating functional properties of smooth muscle cells.

The aortic medial degeneration is the key histopathologic feature of Thoracic aortic dissection (TAD). The aim of this study was to identify the change of autophagic activity in the aortic wall during TAD development, and to explore the roles of autophagy on regulating functional properties of smooth muscle cells (SMCs). Firstly, compared with control group (n = 11), the increased expression of autophagic markers Beclin1 and LC3 was detected in the aortic wall from TAD group (n = 23) by immunochemistry and western blot. We found that more autophagic vacuoles were present in the aortic wall of TAD patients using Transmission electron microscopy. Next, autophagic activity was examined in AD mice model established by ß-aminopropionitrile fumarate (BAPN) and angiotensin II. Immunochemistry proved that autophagic activity was dynamically changed during AD development. Beclin1 and LC3 were detected up-regulated in the aortic wall in the second week after BAPN feeding, earlier than the fragmentation or loss of elastic fibers. When AD occurred in the 4th week, the expression of Beclin1 and LC3 began to decrease, but still higher than the control. Furthermore, autophagy was found to inhibit starvation-induced apoptosis of SMCs. Meanwhile, blockage of autophagy could suppress PDGF-induced phenotypic switch of SMCs. Taken together, autophagic activity was dynamically changed in the aortic wall during TAD development. The abnormal autophagy could regulate the functional properties of aortic SMCs, which might be the potential pathogenesis of TAD.

Inhibition of heat shock protein 90 improves pulmonary arteriole remodeling in pulmonary arterial hypertension.

While the molecular chaperone heat shock protein 90 (HSP90) is involved in a multitude of physiological and pathological processes, its role relating to pulmonary arterial hypertension (PAH) remains unclear. In the present study, we investigated the effect in which HSP90 improves pulmonary arteriole remodeling, and explored the therapeutic utility of targeting HSP90 as therapeutic drug for PAH. By Elisa and immunohistochemistry, HSP90 was found to be increased in both plasma and membrane walls of pulmonary arterioles from PAH patients. Moreover, plasma HSP90 levels positively correlated with mean pulmonary arterial pressure and C-reactive protein. In a monocrotaline-induced rat model of PH, we found that 17-AAG, a HSP90-inhibitor, alleviated the progress of PH, demonstrated by lower pulmonary arterial pressure and absence of right ventricular hypertrophy. Immunohistochemical staining demonstrated that 17-AAG improved pulmonary arteriole remodeling on the basis of reduced wall thickness and wall area. The inflammatory response attributed to PH could be attenuated by 17-AAG through reduction of NF-κB signaling. Moreover, 17-AAG was found to suppress PDGF-stimulated proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through induction of cell cycle arrest in the G1 phase. In conclusion, HSP90 inhibitor 17-AAG could improve pulmonary arteriole remodeling via inhibiting the excessive proliferation of PASMCs, and inhibition of HSP90 may represent a therapeutic avenue for the treatment of PAH.

Intra-muscular arteries are also affected in Takayasu's arteritis.

Takayasu's arteritis (TA) is a chronic vasculitis, primarily affecting large vessels, such as the aorta and its main branches. Several reports suggest that the vascular inflammatory process is not always confined to large vessels. Here, a new case of vasculitis in intra-muscular arteries associated with TA is reported. This case provides further support for the idea that TA also involves small arterial vessels.

[Genetic polymorphisms of six Y chromosome short tandem repeat loci in Chinese Korean ethnic group].

OBJECTIVE: To determine the allelic sequences and genetic polymorphism of six short tandem repeats (STRs) loci on Y chromosome, including DYS441, DYS442, DYS443, DYS444, DYS445 and DYS446 in Chinese Korean ethnic males from Yanbian region of Jilin province, China, and to construct a preliminary database. METHODS: Allele frequencies of the six STR loci in 205 Chinese Korean ethnic individuals were analyzed by multiplex polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE). RESULTS: Respectively, 8, 7, 7, 5, 6 and 9 alleles were detected for each of the locus. Together they have formed 151 haplotypes, with a diversity of 0.9937. CONCLUSION: The six STR loci included in this study were found to be highly polymorphic, and may provide useful markers for genetic analysis.