Human umbilical cord mesenchymal stem cells-derived exosomal circDLGAP4 promotes angiogenesis after cerebral ischemia-reperfusion injury by regulating miR-320/KLF5 axis.

Accumulating evidence suggests that human umbilical cord mesenchymal stem cell-derived exosomes (hUC-MSCs-Exos) are a promising therapeutic strategy for cerebral ischemia-reperfusion injury (CIRI). However, the underlying mechanism remains unclear. hUC-MSCs-Exos were identified by electron microscopy, NTA, and Western blotting. In the hypoxia/reoxygenation (H/R) cell model, human brain microvascular endothelial cells (HBMECs) were cocultured with hUC-MSCs-Exos. Then, cell viability, migration, apoptosis, and tube formation were measured by MTT, flow cytometry, transwell, and tube formation assays. RT-qPCR and Western blotting were used to detect the changes in RNA and protein. RNA pull-down and dual luciferase reporter assays confirmed the relationship between circDLGAP4, miR-320, and KLF5. Ischemia-reperfusion (I/R) rat model was established for in vivo experiments. hUC-MSCs-Exos increased the expression levels of circDLGAP4 and KLF5 but decreased miR-320 in H/R-treated HBMECs by transferring exosomal circDLGAP4. Knockdown of circDLGAP4 in hUC-MSCs-Exos reversed the promoting effects of hUC-MSCs-Exos on cell viability, migration, and tube formation in H/R-treated HBMECs in vitro and also abolished the protective effects of hUC-MSCs-Exos on cerebrovascular injury in I/R rats. Mechanistically, exosomal circDLGAP4 negatively regulated miR-320 in HBMECs, which directly bound to KLF5. In addition, the downregulation of miR-320 could reverse the regulatory effect of exosomal shcircDLGAL5 in H/R-treated HBMECs by upregulating KLF5. hUC-MSCs-Exos-derived circDLGAP4 reduced cerebrovascular injury by regulating miR-320/KLF5 signaling. These results provide a stem cell-based approach to treat CIRI.

TNFSF4 gene polymorphism rs3861950 but not rs3850641 is associated with the risk of cerebral infarction in a Chinese population.

Tumor necrosis factor superfamily member 4 (TNFSF4) plays a key role in the process of atherosclerosis, a common risk factor for both myocardial and cerebral infarctions. Recent studies indicate that the single nucleotide polymorphism (SNP) rs3850641 in TNFSF4 is associated with higher risk of myocardial infarction, but little is known about the association between TNFSF4 variation and cerebral infarction (CI). A case-control study involving 385 CI patients and 385 age-matched, sex-matched non-CI controls was conducted in a Chinese population, only the most common subtype, atherosclerosis CI, was recruited. Two SNPs of TNFSF4, rs3850641 and rs3861950, were genotyped by the TaqMan SNP genotyping method, and verified partly by genomic DNA sequencing. The results revealed a significant allelic association between rs3861950 and CI (Odds ration = 1.733, 95 % confidence interval = 1.333-2.254, P = 0.000). Genotypic association analysis demonstrated that the CC genotype of rs3861950 confers susceptibility to CI (Odds ration = 2.896, 95 % confidence interval = 1.368-6.132), and it was associated with a significantly higher risk of ischemic stroke (Odds ration = 3.520, 95 % confidence interval = 1.546-8.015, P = 0.003) after adjusting for the other confirmed risk factors such as the history of hypertension, diabetes, CAD, smoking and alcohol drinking. While the odds ratio of the T allele to the C allele was 1.733 (95 % confidence interval: 1.333-2.254). However, there was no significant association between rs3850641 and CI (Odds ration = 1.288, 95 % confidence interval = 0.993-1.670, P = 0.056). TNFSF4 gene polymorphism rs3861950, but not rs3850641, is associated with the risk of atherosclerosis CI in a Chinese population.

Energy restriction induced SIRT6 inhibits microglia activation and promotes angiogenesis in cerebral ischemia via transcriptional inhibition of TXNIP.

Energy restriction (ER) protects against cerebral ischemic injury, but the underlying mechanism remains largely unclear. Here, rats were fed ad libitum (AL) or on an alternate-day food deprivation intermittent fasting (IF) diet for 3 months, followed by middle cerebral artery occlusion (MCAO) surgery. The body weight, infarct volume, and neurological deficit score were accessed at the designated time points. ELISA, qRT-PCR, and Western blotting were used to determine cytokine secretion and the expression of SIRT6, TXNIP, and signaling molecules, respectively. Immunofluorescence evaluated microglial activation and angiogenesis in vivo. For in vitro study, oxygen-glucose deprivation/reoxygenation (OGD/R)-treated cell model was generated. MTT and tube formation assays were employed to determine cell viability and tube formation capability. ChIP assay detected chromatin occupancy of SIRT6 and SIRT6-mediated H3 deacetylation. We found that IF or ER mimetics ameliorated cerebral ischemic brain damage and microglial activation, and potentiated angiogenesis in vivo. ER mimetics or SIRT6 overexpression alleviated cerebral ischemia and reperfusion (I/R)-induced injury in vitro. SIRT6 suppressed TXNIP via deacetylation of H3K9ac and H3K56ac in HAPI cells and BMVECs. Downregulation of SIRT6 reversed ER mimetics-mediated protection during cerebral I/R in vitro. Our study demonstrated that ER-mediated upregulation of SIRT6 inhibited microglia activation and potentiated angiogenesis in cerebral ischemia via suppressing TXNIP.

[Negative association of FGA gene 128C/G polymorphism with cerebral infarction and its effect on plasma fibrinogen in Hunan Hans].

OBJECTIVE: To investigate the relationship of FGA gene 128C/G polymorphism and cerebral infarction (CI) and evaluate the effect of FGA-128C/G polymorphism on plasma fibrinogen in Hunan Hans. METHODS: FGA-128C/G polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing in 194 CI patients and 114 healthy controls. RESULTS: There were CG and CC genotypes in the FGA-128C/G locus. No GG genotype was observed in Hunan Hans. There was no significant difference in genotype and allele frequencies between the controls and CI group (P> 0.05), and statistically significant difference was not found in fibrinogen (Fg) level between the CG and CC genotypes (P>0.05). After analyzing blood plasma Fg using the influencing factor multiple regression analysis, it was shown that the Fg level had no relationship with the FGA-128C/G genotype, but it increased with age. And the Fg level in males was higher than that in females. CONCLUSION: There was FGA gene 128C/G polymorphism in the Hunan Han population. There was no association of this polymorphism with the increased Fg level of CI patient in the population. FGA-128C/G might not be the predisposing gene of CI in Hunan Han population. The age and sex were the major factors affecting the plasma Fg level in this population.

Could Sporadic Creutzfeldt-Jakob Disease Be Underdiagnosed in China? Experience From Four Cases.

Background: Creutzfeldt-Jakob Disease (CJD) is a rapidly progressive neurodegenerative disease caused by the misfolded version of the cellular prion protein. Here we report four cases of sporadic CJD (sCJD) and describe the diagnostic methods available in order avoid missed or delayed recognition of CJD in China. Case presentation: We report four patients diagnosed with sCJD between March 2018 and December 2019 at Xiangya Hospital and the Second Xiangya Hospital of Central South University. All patients were admitted to the hospital because of a progressive cognitive decline. Although their routine tests and biochemical indicators in the cerebrospinal fluid (CSF), as well as computed tomography (CT) imaging, did not reveal any apparent abnormalities, the presence of "cortical ribboning" was incidentally found on diffusion-weighted imaging (DWI). The patients were subsequently diagnosed with CJD based on positive testing for 14-3-3 protein in their CSF, and the presence of periodic sharp and slow wave complexes (PSWCs) on their electroencephalograms (EEG). Additionally, two of patients was confirmed pathological examination of cerebral biopsies demonstrating neuronal loss, gliosis, and spongiform changes. Conclusions: CJD is a rare disease and is easily misdiagnosed by clinician in China due to a lack of recognition and awareness of CJD. Based on our experience described in this report, enhanced vigilance for CJD is required for patients with rapidly progressive dementia in China and other developing countries. DWI, EEG and detection of 14-3-3 protein in CSF should be performed in order to achieve a timely diagnosis of CJD.

SCN1B and SCN2B gene variants analysis in dravet syndrome patients: Analysis of 22 cases.

Previous research identified SCN1B variants in some cases of Dravet syndrome (DS). We investigated whether SCN1B and SCN2B variants are commonly happened in DS patients without SCN1A variants. A total of 22 DS patients without SCN1A variants and 100 healthy controls were enrolled in this genetic study. DNA from DS patients was sequenced by Sanger method in whole exons of SCN1B and SCN2B genes. We identified two exon variants (c.351C>T, p.G117G and c.467C>T, p.T156M), which were present both in 1000 egenomes database and in healthy controls with a frequency of 0.54% and 4%, 0.06% and 0%, respectively. Additionally, eight intron or 3 prime UTR variants showing benign clinical significance have also been identified. Our results suggest that variants of SCN1B and SCN2B may not be common causes of DS according to our data. Further large sample-size cohort studies are needed to confirm our conclusion.

Effect of ephrin-B2 on the expressions of angiopoietin-1 and -2 after focal cerebral ischemia/reperfusion.

Ephrin-B2 has been shown to participate in angiogenesis, but the underlying mechanisms involved remain unclear. In this study, a rat model of focal cerebral ischemia was prepared by focal middle cerebral artery occlusion, followed by 24-hour reperfusion. Then, ephrin-B2 protein was administered intracerebroventricularly for 3 consecutive days via a micro-osmotic pump. Western blot assay and quantitative real-time reverse transcription PCR demonstrated the expression levels of angiopoietin-1 (Ang-1) mRNA and protein in the penumbra cortex of the ephrin-B2 treated group were decreased at day 4 after reperfusion, and increased at day 28, while the expression levels of angiopoietin-2 (Ang-2) were highly up-regulated at all time points tested. Double immunofluorescent staining indicated that Ang-1 and Ang-2 were both expressed in vascular endothelial cells positive for CD31. These findings indicate that ephrin-B2 influences the expressions of Ang-1 and Ang-2 during angiogenesis following transient focal cerebral ischemia.

[Surveillance of incidence of cerebral hemorrhage in community populations in Changsha].

OBJECTIVE: To observe the incidence of cerebral apoplexy among the community populations in Changsha, Hunan Province. METHODS: Surveillance of the incidence of cerebral apoplexy and analysis of its subtypes were conducted among 10,000 urban residents in Changsha selected by random cluster sampling from 1986 to 1990. The number of people under surveillance was amplified to 50,000 in 1991 and the studies lasted to December 2000. RESULTS: Among the Chamgsha populations, the annual average incidence of cerebral hemorrhage was 171.7 per 100,000 during the period 1986 - 1990. The annual average incidence of cerebral apoplexy was 238.9/100,000 with an adjusted rate of 200.2/100,000, and the annual average incidence of cerebral hemorrhage, accounting for 53.8% of cerebral apoplexy, was 128.5/100,000 with an adjusted rate of 100.3/100,000). CONCLUSION: The annual average incidence of cerebral hemorrhage in community populations is 128.5 per 100,000 during the period of fifteen years, 1986 - 2000, in Changsha, one of the areas with high incidence of cerebral hemorrhage.

[Expression and distribution of aquaporin-2 in nasal polyps].

OBJECTIVE: To study the distribution and expression of aquaporion-2 (water channel protein; AQP-2) in nasal polyps and to evaluate the role of AQP-2 in the formation of nasal polyps. METHODS: Eleven samples of normal inferior turbinates and forty-six samples of nasal polyps were used. In the group of nasal polyps, there were ten cases of type II phase 1, twelve type II phase 2, ten type II phase 3 and fourteen type III. The expression of AQP-2 in both groups was studied with immunohistochemical technique. RESULTS: 1. Both the number of AQP-2 positive cells and the area density in the epithelial cell layer from nasal polyps were higher than those from inferior turbinates (P < 0.01, P < 0.05 respectively); In the group of nasal polyps, the area density from type III, type II phase 2 and type II phase 3 was significantly higher than that from type II phase 1 (P < 0.05); 2. Both the number of AQP-2 positive cells and the area density in the subepithelial tissue from type III, type II phase 2 and type II phase 3 nasal polyps were higher than those from type II phase 1 nasal polyps and inferior turbinates(P < 0.05), whereas the difference between the latter two was not significant(P > 0.05). CONCLUSION: The high expression of AQP-2 protein in nasal polyps may contribute to the genesis and development of nasal polyps.

Incidence of cerebral hemorrhage in the Changsha community. A prospective study from 1986 to 2000.

BACKGROUND AND PURPOSE: Intracranial hemorrhage (ICH) constitutes an important subtype of stroke. In a prior study, we found that the city of Changsha, People's Republic of China, not only had a high incidence of stroke, but also had almost the highest incidence of cerebral hemorrhage. To provide information for preventing and managing ICH, we undertook this study supervising and determining the annual first-ever average incidence of cerebral hemorrhage from 1986 to 2000 in Changsha. METHODS: In January 1986, people from 6 well-defined communes in the Kai-fu district in Changsha were selected as the study population. Since 1991, we added another 18 well-defined communes of the same district. Data on these residents were obtained from the census register of the local administrative office. Persons who had a history of stroke were excluded from our study at the beginning. Every year, we checked up on the population and carried out door-to-door inquiries to verify the new cases of ICH and stroke. If the patients were diagnosed in hospital, as many as possible were examined with CT and/or MRI. The incidence rate adjusted to the World population in 1985 for age and sex, as well as the prevalence of risk factors were calculated. RESULTS: An accumulative total of 551,163 people were supervised in 15 years (from 1986 to 2000); the annual average incidences of stroke and ICH were 236.6/100,000 (adjusted rate: 154.7) and 131.0/100,000 (adjusted rate: 73.1), respectively. From 1986 to 2000, the total annual average rate of stroke confirmed by CT and/or MRI in Changsha was 70.8%, and for ICH it was 64.7%. The incidences of stroke and ICH confirmed by image analysis were 167.5 (adjusted rate: 110.8) and 84.7 (adjusted rate: 55.1), respectively. The mean annual mortality rate of ICH was 78.3/100,000 (adjusted rate: 49.3) and that of stroke was 124.5/100,000 (adjusted rate: 97.0). The percentage of ICH among stroke cases was 55.4%. Among patients with ICH, 79.8% had hypertension, 30.6% had cardiovascular disease, 7.6% had diabetes mellitus, and 12.5% had an abnormal high level of lipid. Among patients with other subtypes of stroke apart from ICH, the percentages were 76.6, 40.6, 15.5, and 22.5%. CONCLUSIONS: The incidence of ICH in Changsha is very high, so is the proportion of ICH among all stroke cases. Changsha is an area with a high incidence of ICH in the world. Hypertension is the prominent risk factor. The study demonstrated the importance of ICH as a significant subtype of stroke.