Neuregulin 1 as a potential biomarker for disease progression in moyamoya disease: A case-control study in Chinese population.

OBJECTIVE: Moyamoya disease (MMD) is a rare and progressive stenosis of cerebral arteries characterized by abnormally proliferative vasculopathy. Current studies have demonstrated that Neuregulin 1 (NRG1) plays a key role in angiogenesis-related disorders. Thus, the aim of our study is to investigate the serum NRG1 levels and their clinical correlations in MMD patients. METHODS: In this study, thirty adult patients with MMD and age-gender matched healthy controls were enrolled from our hospital between July 2020 and April 2022. Peripheral blood samples were collected at baseline, and clinical data were obtained from the electronic medical record system. Serum NRG1 concentrations were measured by enzyme-linked immunosorbent assay. Sanger sequencing was applied to detect the RNF213 p.R4810K mutation. RESULTS: The serum NRG1 levels were significantly higher in MMD patients compared to controls (14.48 ± 10.81 vs.7.54 ± 6.35mmol/L, p < 0.001). No statistical difference in baseline clinical characteristics was found between both groups. Correlation analyses showed that NRG1 levels were positively associated with Suzuki staging (r = 0.4137, p = 0.023) while not related to other clinical features (reduced cerebral blood flow, posterior cerebral artery involvement, bilateral or unilateral steno-occlusive changes). Furthermore, subgroup analysis revealed that MMD patients with the RNF213 p.R4810K mutation presented with significantly higher NRG1 levels than those without the mutation (9.60 ± 0.929 vs. 25.89 ± 4.338 mmol/L, p = 0.001). CONCLUSIONS: Our study suggests that increased serum NRG1 levels may constitute a characteristic feature of MMD, indicating a potential positive correlation with disease progression and the presence of the RNF213 mutation. This positions NRG1 as a potentially crucial target for further studies aimed at comprehending the pathogenesis of MMD.

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.

RTN4/Nogo-A-S1PR2 negatively regulates angiogenesis and secondary neural repair through enhancing vascular autophagy in the thalamus after cerebral cortical infarction.

Cerebral infarction induces angiogenesis in the thalamus and influences functional recovery. The mechanisms underlying angiogenesis remain unclear. This study aimed to investigate the role of RTN4/Nogo-A in mediating macroautophagy/autophagy and angiogenesis in the thalamus following middle cerebral artery occlusion (MCAO). We assessed secondary neuronal damage, angiogenesis, vascular autophagy, RTN4 and S1PR2 signaling in the thalamus. The effects of RTN4-S1PR2 on vascular autophagy and angiogenesis were evaluated using lentiviral and pharmacological approaches. The results showed that RTN4 and S1PR2 signaling molecules were upregulated in parallel with angiogenesis in the ipsilateral thalamus after MCAO. Knockdown of Rtn4 by siRNA markedly reduced MAP1LC3B-II conversion and levels of BECN1 and SQSTM1 in vessels, coinciding with enhanced angiogenesis in the ipsilateral thalamus. This effect coincided with rescued neuronal loss of the thalamus and improved cognitive function. Conversely, activating S1PR2 augmented vascular autophagy, along with suppressed angiogenesis and aggravated neuronal damage of the thalamus. Further inhibition of autophagic initiation with 3-methyladenine or spautin-1 enhanced angiogenesis while blockade of lysosomal degradation by bafilomycin A1 suppressed angiogenesis in the ipsilateral thalamus. The control of autophagic flux by RTN4-S1PR2 was verified in vitro. Additionally, ROCK1-BECN1 interaction along with phosphorylation of BECN1 (Thr119) was identified in the thalamic vessels after MCAO. Knockdown of Rtn4 markedly reduced BECN1 phosphorylation whereas activating S1PR2 increased its phosphorylation in vessels. These results suggest that blockade of RTN4-S1PR2 interaction promotes angiogenesis and secondary neural repair in the thalamus by suppressing autophagic activation and alleviating dysfunction of lysosomal degradation in vessels after cerebral infarction.Abbreviations: 3-MA: 3-methyladenine; ACTA2/ɑ-SMA: actin alpha 2, smooth muscle, aorta; AIF1/Iba1: allograft inflammatory factor 1; BafA1: bafilomycin A1; BMVECs: brain microvascular endothelial cells; BrdU: 5-bromo-2'-deoxyuridine; CLDN11/OSP: claudin 11; GFAP: glial fibrillary acidic protein; HUVECs: human umbilical vein endothelial cells; LAMA1: laminin, alpha 1; MAP2: microtubule-associated protein 2; MBP2: myelin basic protein 2; MCAO: middle cerebral artery occlusion; PDGFRB/PDGFRß: platelet derived growth factor receptor, beta polypeptide; RECA-1: rat endothelial cell antigen-1; RHOA: ras homolog family member A; RHRSP: stroke-prone renovascular hypertensive rats; ROCK1: Rho-associated coiled-coil containing protein kinase 1; RTN4/Nogo-A: reticulon 4; RTN4R/NgR1: reticulon 4 receptor; S1PR2: sphingosine-1-phosphate receptor 2; SQSTM1: sequestosome 1.

Anti-DR5 mAb inhibits proliferation of human fibroblast-like synovial cells and reduces their cytokine secretion in vitro.

BACKGROUND: We have previously reported that anti-death receptor 5 (DR5) monoclonal antibody (mAb) is therapeutically effective in the treatment of rheumatoid arthritis (RA) in a collagen-induced arthritis rat model. However, the molecular mechanism and the effect of anti-DR5 mAb on proapoptotic genes and cytokine secretion in the human fibroblast-like synovial cells (FLS) requires further clarification. This study may provide new evidence for the application of anti-DR5 mAb as a treatment for RA. METHODS: Human FLS were isolated from patients with RA and were treated with anti-DR5 mAb. An MTT assay and flow cytometry were used to detect the induction of apoptosis in vitro. Cytokine secretion by the FLS was detected using the enzyme-linked immunosorbent assay. The mRNA expression was assessed by reverse transcription polymerase chain reaction, and the protein expression was analyzed by Western blot. The apoptotic pathway was investigated further using a caspase inhibition assay. RESULTS: Anti-DR5 mAb-induced apoptosis in human RA FLS in vitro. The protein expressions of caspase-8, -3, and -9 were decreased in human anti-DR5 mAb-treated FLS in a dose-dependent manner through exposure to a caspase inhibitor, indicating that anti-DR5 mAb induction of apoptosis is through the caspase pathway. Decreased levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were detected after treatment with anti-DR5 mAb in vitro. CONCLUSION: Anti-DR5 mAb may induce apoptosis in human FLS through the caspase pathway and through decreased secretions of TNF-α and IFN-γ.