Loss of Gucy1a3 causes poor post-stroke recovery by reducing angiogenesis via the HIF-1α/VEGFA signaling pathway in mice.

OBJECTIVES: Ischemic stroke is a common and debilitating disease that can cause permanent neurological damage. Gucy1a3, which encodes the α1 subunit of soluble guanylyl cyclase, has been reported to be associated with functional recovery after ischemic stroke. However, the mechanism is still not well understood. In the present study, we investigated the effects of Gucy1a3 on (i) post-stroke recovery; (ii) vascular endothelial growth factor A (VEGFA) and hypoxia inducible factor 1 alpha (HIF-1α) expression; and (iii) angiogenesis after ischemic stroke. MATERIALS AND METHODS: Wild-type and Gucy1a3 knockout C57BL/6J male mice were respectively used to establish the models of permanent middle cerebral artery occlusion (pMCAO). Neurological deficit scores were evaluated at 24 h and 96 h after pMCAO. Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. For determining microvessel density, immunohistochemical analysis was performed with CD31. The expression of VEGFA and HIF-1α was detected by western blotting. RESULTS: Our results suggest that loss of Gucy1a3 increased the infarct volume and aggravated neurological deficits after pMCAO. In addition, the Gucy1a3 knockout brains exhibited significantly lower microvessel densities and VEGFA and HIF-1α expression levels than the wild-type brains at 96 h post-pMCAO. CONCLUSIONS: Our study indicates that GUCY1A3 might be involved in angiogenesis after ischemic stroke. Further investigation of GUCY1A3 will provide a new therapeutic target for stroke.

RNF213 loss-of-function promotes pathological angiogenesis in moyamoya disease via the Hippo pathway.

Moyamoya disease is an uncommon cerebrovascular disorder characterized by steno-occlusive changes in the circle of Willis and abnormal vascular network development. Ring finger protein 213 (RNF213) has been identified as an important susceptibility gene for Asian patients, but researchers have not completely elucidated whether RNF213 mutations affect the pathogenesis of moyamoya disease. Using donor superficial temporal artery samples, whole-genome sequencing was performed to identify RNF213 mutation types in patients with moyamoya disease, and histopathology was performed to compare morphological differences between patients with moyamoya disease and intracranial aneurysm. The vascular phenotype of RNF213-deficient mice and zebrafish was explored in vivo, and RNF213 knockdown in human brain microvascular endothelial cells was employed to analyse cell proliferation, migration and tube formation abilities in vitro. After bioinformatics analysis of both cell and bulk RNA-seq data, potential signalling pathways were measured in RNF213-knockdown or RNF213-knockout endothelial cells. We found that patients with moyamoya disease carried pathogenic mutations of RNF213 that were positively associated with moyamoya disease histopathology. RNF213 deletion exacerbated pathological angiogenesis in the cortex and retina. Reduced RNF213 expression led to increased endothelial cell proliferation, migration and tube formation. Endothelial knockdown of RNF213 activated the Hippo pathway effector Yes-associated protein (YAP)/tafazzin (TAZ) and promoted the overexpression of the downstream effector VEGFR2. Additionally, inhibition of YAP/TAZ resulted in altered cellular VEGFR2 distribution due to defects in trafficking from the Golgi apparatus to the plasma membrane and reversed RNF213 knockdown-induced angiogenesis. All these key molecules were validated in ECs isolated from RNF213-deficient animals. Our findings may suggest that loss-of-function of RNF213 mediates the pathogenesis of moyamoya disease via the Hippo pathway.

Rnf-213 Knockout Induces Pericyte Reduction and Blood-Brain Barrier Impairment in Mouse.

Moyamoya disease (MMD) is a rare cerebrovascular disorder characterized by progressive occlusion of the internal carotid artery and the formation of an abnormal compensatory capillary network at the base of the brain. Genomics studies identified Ring finger protein 213 (RNF213) as a common genetic factor that increases the susceptibility to MMD in East Asian people. However, the function of RNF213 and its roles in pathogenesis of MMD is unclear. Here, we showed that genetic knockout of Rnf213 in mice causes significant pericyte reduction and blood-brain barrier impairment in the cortex. These phenotypes are accompanied with microglia activation and elevated level of proinflammatory cytokines. Additionally, Rnf213-deficient mice showed reduced expression of tight junction proteins, including Occludin, Claudin-5, and ZO-1. Together, these data suggested that RNF213 might contribute to the pathogenesis of MMD through disruption of pericyte homeostasis and blood-brain barrier integrity by dysregulation of inflammatory responses and tight junction formation.

Identification of immune-associated gene signature and immune cell infiltration related to overall survival in progressive multiple sclerosis.

BACKGROUND: Delayed diagnosis and noneffective treatment contribute to the shorter life expectancy in patients with progressive multiple sclerosis (PMS). Studies demonstrate the key role of autoimmunity in PMS, but the prognostic value of immune-associated factors remains unknown. Thus, this study aimed to develop an immune-associated gene (IAG) signature related to overall survival (OS) and conduct an immune cell infiltration analysis using PMS data. METHODS: The differentially expressed IAGs were identified based on gene expression profiles (from the Gene Expression Omnibus database) and IAGs (from the ImmPort database). Univariate and least absolute shrinkage and selection operator (LASSO)-penalized Cox regression analyses were used to develop the IAG signature related to OS. Kaplan-Meier analyses were conducted, and receiver operating characteristic (ROC) curves were generated to assess the performance. Additionally, the differential distribution of immune cells was identified by Wilcoxon rank-sum tests and correlations with IAGs were analyzed using Spearman correlation analyses. Moreover, univariate and multivariate Cox regression analyses were used to identify the independent prognostic factors to develop a prognostic nomogram. RESULTS: The training group, consisting of 57 PMS lesions and 52 control tissues, was obtained through batch normalization to remove the inter-batch difference. A total of 206 differentially expressed IAGs were identified, and 38 of them were associated with OS. Thereafter, a 4-IAG signature was constructed to calculate the risk score and thus classify PMS patients into high- and low-risk groups according to mean risk score. Patients in the high-risk group had a lower survival time than those in the low-risk group. The Kaplan-Meier plots and ROC curves demonstrated a good performance in both the training and internal validation groups. Additionally, five differentially abundant immune cell types were identified and their relationships with IAGs were analyzed. Finally, risk score, cortical region, and naive B cells were identified as independent prognostic factors, and a nomogram incorporating these factors was developed to predict the OS in PMS. CONCLUSION: The novel IAG signature may be a reliable tool for assisting neurologists in predicting the OS for PMS patients in clinical settings. These findings may facilitate personalized treatment and provide insights into the complex mechanism of PMS.

N6-Methyladenosine RNA modification in cerebrospinal fluid as a novel potential diagnostic biomarker for progressive multiple sclerosis.

BACKGROUND: Progressive multiple sclerosis (PMS) is an uncommon and severe subtype of MS that worsens gradually and leads to irreversible disabilities in young adults. Currently, there are no applicable or reliable biomarkers to distinguish PMS from relapsing-remitting multiple sclerosis (RRMS). Previous studies have demonstrated that dysfunction of N6-methyladenosine (m6A) RNA modification is relevant to many neurological disorders. Thus, the aim of this study was to explore the diagnostic biomarkers for PMS based on m6A regulatory genes in the cerebrospinal fluid (CSF). METHODS: Gene expression matrices were downloaded from the ArrayExpress database. Then, we identified differentially expressed m6A regulatory genes between MS and non-MS patients. MS clusters were identified by consensus clustering analysis. Next, we analyzed the correlation between clusters and clinical characteristics. The random forest (RF) algorithm was applied to select key m6A-related genes. The support vector machine (SVM) was then used to construct a diagnostic gene signature. Receiver operating characteristic (ROC) curves were plotted to evaluate the accuracy of the diagnostic model. In addition, CSF samples from MS and non-MS patients were collected and used for external validation, as evaluated by an m6A RNA Methylation Quantification Kit and by real-time quantitative polymerase chain reaction. RESULTS: The 13 central m6A RNA methylation regulators were all upregulated in MS patients when compared with non-MS patients. Consensus clustering analysis identified two clusters, both of which were significantly associated with MS subtypes. Next, we divided 61 MS patients into a training set (n = 41) and a test set (n = 20). The RF algorithm identified eight feature genes, and the SVM method was successfully applied to construct a diagnostic model. ROC curves revealed good performance. Finally, the analysis of 11 CSF samples demonstrated that RRMS samples exhibited significantly higher levels of m6A RNA methylation and higher gene expression levels of m6A-related genes than PMS samples. CONCLUSIONS: The dynamic modification of m6A RNA methylation is involved in the progression of MS and could potentially represent a novel CSF biomarker for diagnosing MS and distinguishing PMS from RRMS in the early stages of the disease.

Associations between SNP83 of phosphodiesterase 4D gene and carotid atherosclerosis in a southern Chinese Han population: a case-control study.

Atherosclerosis was an important pathophysiological basis of atherothrombotic stroke, and phosphodiesterase 4D (PDE4D) polymorphism (SNP83/rs966221) was reported to be associated with the susceptibility to atherothrombotic stroke. Aim of the present study was to explore the potential association between SNP83 and carotid atherosclerosis (CAS). 204 southern Chinese Han participants were divided into two groups according to the carotid intima-media thickness (IMT) of the carotid artery: CAS group (carotid IMT ≥ 1.0 mm) and non-CAS group (carotid IMT < 1.0 mm). Carotid IMT was measured by color Doppler ultrasound. The PDE4D SNP83 polymorphism was determined by SNaPshot technique. Our study found that SNP83 was associated significantly with CAS susceptibility under the dominant, overdominant and codominant models. After adjusting for age, gender, low-density lipoprotein cholesterol, Hemoglobin A1c, cigarette smoking, hypertension history, and diabetes mellitus history, the association still remained significant (dominant model: crude OR = 2.373, 95% CI: 1.268-4.442, P = 0.007; adjusted OR = 3.129, 95% CI: 1.104-8.866, P = 0.032; overdominant model: crude OR = 1.968, 95% CI: 1.043-3.714, P = 0.037; adjusted OR = 2.854, 95% CI: 1.005-8.108, P = 0.049; codominant: crude OR = 2.102, 95% CI: 1.110-3.979, P = 0.023; adjusted OR = 2.984, 95% CI: 1.047-8.502, P = 0.041). Carotid IMT of carriers with CT + CC genotypes was higher than carriers with TT genotype (P = 0.016). Our results indicated that the SNP83/rs966221 located on PDE4D gene was significantly associated between CAS susceptibility and carotid IMT independently of conventional risk factors in a southern Chinese Han population.

Mutations of RNF213 are responsible for sporadic cerebral cavernous malformation and lead to a mulberry-like cluster in zebrafish.

Although familial forms of cerebral cavernous malformation are mainly attributed to three CCM genes (KRIT1, CCM2 and PDCD10), no mutation is identified in sporadic cerebral cavernous malformation cases with a unique lesion, indicating additional genes for sporadic cerebral cavernous malformation. To screen the candidate genes, we conducted whole exome sequencing in 31 sporadic cerebral cavernous malformation patients and 32 healthy controls, and identified 5 affected individuals carrying 6 heterozygous deleterious mutations in RNF213 but no RNF213 mutation in healthy individuals. To further confirm RNF213 was associated with cerebral cavernous malformation, we generated rnf213a homozygous knockout zebrafish and found mutation of rnf213a in zebrafish led to a mulberry-like cluster of disordered-flow vascular channels which was reminiscent of human cerebral cavernous malformation. In addition, we revealed kbtbd7 and anxa6 were significantly downregulated due to rnf213a mutation through transcriptomic sequencing and RT-qPCR analysis. Based on the mulberry-like phenotype partly rescued by mRNA of kbtbd7 as well as anxa6, we suggested that rnf213a promoted mulberry-like cluster via downregulation of kbtbd7 and anxa6. Altogether, we firstly demonstrate RNF213is a novel candidate gene for sporadic cerebral cavernous malformation and the mutation of rnf213a is responsible for the mulberry-like cluster in zebrafish.

Prevalence of RNF213 variants in symptomatic intracranial arterial stenosis/occlusion in China.

The ring finger protein 213 gene (RNF213) rs112735431 was significantly associated with intracranial artery stenosis/occlusion disease (ICASO) in Japan and Korea and to a lesser degree in China. We conducted a case-control study to examine the prevalence and correlates of the RNF213 rare variants in Chinese patients with symptomatic ICASO. A total of 503 cases including 390 ischemic stroke patients (ICASO-IS), 113 intracranial hemorrhage patients (ICASO-ICH) and 227 control subjects were recruited. The snapshot technique was used for RNF213 rare variants analysis, including rs112735431, rs148731719, rs37144111 and rs138130613. Moreover, a meta-analysis was performed to explore the relationship between RNF213 variants and ICASO in Asian. In our case-control study, we found that the rs138130613 variant was significantly associated with ICASO-IS (OR = 9.92, 95% CI 1.24-79.19, p = 0.03). The mean age of first ischemic stroke onset of variant carriers was earlier than the noncarriers (51.3 ± 18.0 versus 66.0 ± 12.9 years old, p = 0.02), but the conventional atherosclerotic risk factors and the characteristics of artery stenosis did not differ between them. In addition, the meta-analysis showed significant association between the rs112735431 polymorphism and the ICASO or ICASO-IS, and this variant was found more often in women and young-onset patients in Asia. This study suggests that the RNF213 rs112735431 and rs138130613 are genetic risk variants for ischemic stroke with intracranial artery stenosis/occlusion in China and rs112735431 is also associated with the high risk of ICASO in Asia. Further large-scale investigation of the RNF213 gene will provide new insights into pathogenetic mechanisms of symptomatic ICASO.

Decreased Pituitary Height and Stunted Linear Growth After Radiotherapy in Survivors of Childhood Nasopharyngeal Carcinoma Cases.

To examine the morphological changes of the pituitary glands and linear growth of childhood nasopharyngeal carcinoma (NPC) cases who accepted radiotherapy. A total of 90 children (i.e., age less than 18 years) who were diagnosed as NPC at Sun Yat-sen University Cancer Center from January 2009 to January 2016 were identified by reviewing medical records. Two radiologists reviewed and measured the pre-radiation, post-radiation, and the latest available pituitary gland heights independently. Patients' current height information was collected by telephone interviews. We compared the pituitary height differences using paired t-tests and estimated the pituitary height trajectories within each sex by mixed regression models. Height-for-age Z-score was calculated for each patient using the WHO growth reference data for 5-19 years as reference. Most of the included participants were of male sex (75.6%) and over half were diagnosed at stage IV (58.4%). Among the 90 included participants, 89 had one repeated measurement of the pituitary height and 79 had two repeated measurements of the pituitary height. Seventy six of the 89 childhood NPC participants had reduced pituitary heights after radiation and accounted for 85.4% of the whole population. The means of the pituitary heights before and after radiotherapy were 6.4 ± 1.3 mm and 5.6 ± 1.2 mm (P < 0.001), respectively. The mean of height-for-age Z-score for childhood NPC cases was significantly below zero (-0.54, 95% CI = -0.74, -0.34). We concluded that childhood NPC cases had decreased pituitary heights and stunted linear growth after radiotherapy.

Thymectomy is a beneficial therapy for patients with non-thymomatous ocular myasthenia gravis: a systematic review and meta-analysis.

Ocular myasthenia gravis, an autoimmune disease, is characterized by extraocular muscle weakness. Myasthenia gravis is closely associated with the functional status of the thymus gland. The efficacy of thymectomy for non-thymomatous ocular myasthenia gravis remains controversial. Here, we present the first systematic review and meta-analysis of studies assessing the outcome of thymectomy in patients with non-thymomatous ocular myasthenia gravis and found that the pooled rate of complete stable remission was 0.5074 with considerable heterogeneity. Furthermore, subgroup analysis showed that the efficacy of thymectomy differed according to geographical location. Furthermore, thymectomy outcomes are better in children than they are in adults. Thymectomy clearly represents an effective treatment for patients with non-thymomatous ocular myasthenia gravis. However, more multicenter, randomized, controlled clinical trials are now required to confirm these conclusions.

The association between the ring finger protein 213 (RNF213) polymorphisms and moyamoya disease susceptibility: a meta-analysis based on case-control studies.

A number of studies assessed the association of ring finger protein 213 (RNF213) gene polymorphisms with moyamoya disease (MMD), but the results were not entirely consistent. This meta-analysis was performed to explore the relationship between RNF213 polymorphisms and moyamoya disease in Asian population. A systematic search from the PubMed, MEDLINE, EMBASE, ISI web of science, CNKI, China CBM and WANFANG DATA databases was conducted to retrieve published studies until March 2015. Statistical analyses were performed using the STATA12.0 software. Fixed or random effects model, subgroup analysis, sensitivity analysis, and publication bias were used to improve the comprehensive analysis. Eight papers including 904 MMD patients and 2258 controls were recruited in the meta-analysis. rs112735431 was closely associated with the risk of MMD among Asian population in all genetic models (dominant model: OR 103.39, 95 % CI 52.25-204.55, P = 1.69e-40; recessive model: OR 16.45, 95 % CI 6.00-45.10, P = 5.33e-08; additive model: OR 61.49, 95 % CI 22.07-171.33, P = 3.32e-15), especially in the Japanese population. Subgroup analysis revealed highly statistically significant higher risk in the patients with family histories. Although another polymorphism rs148731719 showed no significant association with the MMD, rs138130613 was found to be related to the higher risk in Chinese population (dominant model: OR 8.34, 95 % CI 1.72-40.47, P = 0.008). Our meta-analysis strengthens RNF213 rs112735431 is closely associated with the increased risk of MMD in Japanese, and the screening combined with rs112735431 and rs138130613may improve the detection rate for MMD in China.