Knockdown the moyamoya disease susceptibility gene, RNF213, upregulates the expression of basic fibroblast growth factor and matrix metalloproteinase-9 in bone marrow derived mesenchymal stem cells.

Moyamoya disease (MMD) is a chronic, progressive cerebrovascular occlusive disease. Ring finger protein 213 (RNF213) is a susceptibility gene of MMD. Previous studies have shown that the expression levels of angiogenic factors increase in MMD patients, but the relationship between the susceptibility gene RNF213 and these angiogenic mediators is still unclear. The aim of the present study was to investigate the pathogenesis of MMD by examining the effect of RNF213 gene knockdown on the expression of matrix metalloproteinase-9 (MMP-9) and basic fibroblast growth factor (bFGF) in rat bone marrow-derived mesenchymal stem cells (rBMSCs). Firstly, 40 patients with MMD and 40 age-matched normal individuals (as the control group) were enrolled in the present study to detect the levels of MMP-9 and bFGF in serum by ELISA. Secondly, Sprague-Dawley male rat BMSCs were isolated and cultured using the whole bone marrow adhesion method, and subsequent phenotypic analysis was performed by flow cytometry. Alizarin red and oil red O staining methods were used to identify osteogenic and adipogenic differentiation, respectively. Finally, third generation rBMSCs were transfected with lentivirus recombinant plasmid to knockout expression of the RNF213 gene. After successful transfection was confirmed by reverse transcription-quantitative PCR and fluorescence imaging, the expression levels of bFGF and MMP-9 mRNA in rBMSCs and the levels of bFGF and MMP-9 protein in the supernatant of the culture medium were detected on the 7th and 14th days after transfection. There was no significant difference in the relative expression level of bFGF among the three groups on the 7th day. For the relative expression level of MMP-9, there were significant differences on the 7th day and 14th day. In addition, there was no statistically significant difference in the expression of bFGF in the supernatant of the RNF213 shRNA group culture medium, while there was a significant difference in the expression level of MMP-9. The knockdown of the RNF213 gene affects the expression of bFGF and MMP-9. However, further studies are needed to determine how they participate in the pathogenesis of MMD. The findings of the present study provide a theoretical basis for clarifying the pathogenesis and clinical treatment of MMD.

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease.

BACKGROUND: Moyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. METHODS: Our study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. RESULTS: The results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. CONCLUSION: These findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

Differentiation of Fibroblasts Into Myofibroblasts in the Arachnoid Membrane of Moyamoya Disease.

BACKGROUND: Moyamoya disease (MMD) is a very specific disorder in terms of spontaneous development of extracranial-to-intracranial collateral circulation through the dura mater, but the underlying mechanisms are unclear. This study aimed to investigate the role of the arachnoid membrane in this unique angiogenesis in MMD. METHODS: A piece of arachnoid membrane and 1- to 2-mL cerebrospinal fluid were simultaneously harvested during surgery from 26 patients with MMD. The specimens were also collected during surgery as the controls from 6 patients with atherosclerotic carotid artery diseases. The arachnoid membrane was subjected to immunohistochemistry and the cerebrospinal fluid was used to measure the concentration of cytokines using ELISA. RESULTS: The number of cells positive for PDGFR (platelet-derived growth factor receptor) α was significantly higher in MMD than in the controls (5.4±3.1 versus 2.3±2.1 cells/field; P=0.02). The results were same in PDGFRß-positive cells (10.1±4.6 versus 4.8±2.8; P=0.01) and α-SMA (alpha-smooth muscle actin)-positive cells (8.8±3.1 versus 2.0±2.5; P<0.01). On multicolor immunofluorescence, 80.5±15.6% of cells positive for PDGFRα in MMD also expressed α-SMA, being significantly higher than 14.6±7.2% in the controls (P<0.01). The density of collagen in the arachnoid membrane was significantly higher in MMD than in the controls (60.3±15.0% versus 40.1±15.3%; P<0.01). In MMD, advanced disease stage was significantly associated with a larger number of α-SMA-positive cells in the arachnoid membrane (P=0.04). On ELISA, the cerebrospinal fluid concentrations of bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), and TGF (transforming growth factor)-ß1 were significantly higher in MMD than in the controls. CONCLUSIONS: Based on these findings, MMD may elevate the concentrations of angiogenic factors in the cerebrospinal fluid and then promote the proliferation of fibroblasts in the arachnoid membrane and their differentiation into myofibroblasts, which may, in turn, enhance the production of collagen essential for spontaneous collateral formation across the arachnoid membrane.

Dysregulation of Rnf 213 gene contributes to T cell response via antigen uptake, processing, and presentation.

Growing evidence suggest the association between Moyamoya disease (MMD) and immune systems, such as antigen presenting cells in particular. Rnf213 gene, a susceptibility gene for MMD, is highly expressed in immune tissues, however, its function remains unclear. In addition, the physiological role of RNF213 gene polymorphism c.14576G > A (rs112735431), susceptibility variant for MMD, is also poorly understood. By studying Rnf213-knockout (Rnf213-KO) mice with deletion of largest exon32 and Rnf213-knockin (Rnf213-KI) mice with insertion of single-nucleotide polymorphism corresponding to c.14576G > A mutation in MMD patients, we aimed to investigate the role of RNF213 in dendritic cell development, and antigen processing and presentation. First, we found a high level of Rnf213 gene expression in conventional DCs and monocytes. Second, flow cytometric and confocal microscopic analysis revealed ovalbumin protein-pulsed Rnf213-KO and Rnf213-KI DCs showed impaired antigen uptake, proteolysis and reduced numbers of endosomes and lysosomes, and thereby failed to activate and proliferate antigen-specific T cells efficiently. In addition, Rnf213-KI DCs showed a similar phenotype to that of Rnf213-KO BMDCs. In conclusion, our findings suggest the critical role of RNF213 in antigen uptake, processing and presentation.

Linking cortical astrocytic neogenin deficiency to the development of Moyamoya disease-like vasculopathy.

Moyamoya-like vasculopathy, the "puff of smoke"-like small vessels in the brain, is initially identified in patients with Moyamoya disease (MMD), a rare cerebrovascular disease, and later found in patients with various types of neurological conditions, including Down syndrome, Stroke, and vascular dementia. It is thus of interest to understand how this vasculopathy is developed. Here, we provided evidence for cortical astrocytic neogenin (NEO1) deficiency to be a risk factor for its development. NEO1, a member of deleted in colorectal cancer (DCC) family netrin receptors, was reduced in brain samples of patients with MMD. Astrocytic Neo1-loss resulted in an increase of small blood vessels (BVs) selectively in the cortex. These BVs were dysfunctional, with leaky blood-brain barrier (BBB), thin arteries, and accelerated hyperplasia in veins and capillaries, resembled to the features of moyamoya-like vasculopathy. Additionally, we found that both MMD patient and Neo1 mutant mice exhibited altered gene expression in their cortex in proteins critical for not only angiogenesis [e.g., an increase in vascular endothelial growth factor (VEGFa)], but also axon guidance (e.g., netrin family proteins) and inflammation. In aggregates, these results suggest a critical role of astrocytic NEO1-loss in the development of Moyamoya-like vasculopathy, providing a mouse model for investigating mechanisms of Moyamoya-like vasculopathy.

Association of Brain-Gut Peptides with Inflammatory Cytokines in Moyamoya Disease.

Systemic inflammation has been shown to play a pivotal role in the pathogenesis of moyamoya disease (MMD). Brain-gut peptides exhibit regulatory effects in the secretion of proinflammatory cytokines. To investigate the association between brain-gut peptides and inflammation in the occurrence of MMD, 41 patients with MMD, as well as 74 age- and sex-matched healthy individuals were enrolled. The levels of four brain-gut peptides (vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), somatostatin (SST), substance P (SP)) and three proinflammatory cytokines (interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-12) in the serum and cerebrospinal fluid (CSF) were measured using the enzyme-linked immunosorbent assay. The associations between brain-gut peptides and proinflammatory cytokines were estimated according to the multiple linear regression and correlation analyses. MMD patients exhibited significantly lower levels of VIP, CCK, and SST and higher levels of IL-1ß, TNF-α, and IL-12 in the serum compared with healthy controls. Multiple logistic regression analysis showed that decreased VIP, CCK, and SST levels were independent predictors of the occurrence of MMD. Negative correlations were observed between the VIP and proinflammatory cytokines, including IL-1ß, TNF-α, and IL-12 (serum vs. CSF). Significant negative correlations were also found between CCK and IL-1ß, as well as IL-12 (serum vs. CSF). SST was negatively correlated with IL-1ß and TNF-α in the serum and IL-1ß only in the CSF. In addition, the levels of VIP, CCK, SST, and proinflammatory cytokines IL-1ß and TNF-α in the serum were correlated with those measured in the CSF. Collectively, lower levels of VIP, CCK, and SST may be associated with the pathogenesis of MMD and act as clinically useful biomarkers along with the levels of proinflammatory cytokines.

Vascular Smooth Muscle Cell Derived from IPS Cell of Moyamoya Disease - Comparative Characterization with Endothelial Cell Transcriptome.

BACKGROUND: Moyamoya disease (MMD) is an occlusive cerebrovascular disease, causing stroke in children and young adults with unknown etiology. The fundamental pathology is fibrocellular intimal thickening of cerebral arteries, in which vascular smooth muscle cells (VSMCs) are observed as one of the major cell types. Although the characteristics of circulating smooth muscle progenitor cells have been previously reported, the VSMCs are poorly characterized in MMD. We aimed to characterize VSMCs in MMD using induced pluripotent stem cell (iPSC)-technology. METHODS: We differentiated VSMCs from neural crest stem cells (NCSCs) using peripheral blood mononuclear cell-derived iPSCs and compared biological and transcriptome features under naïve culture conditions between three independent healthy control (HC) subjects and three MMD patients. VSMC transcriptome profiles were also compared to those of endothelial cells (ECs) differentiated from the same iPSCs. RESULTS: Homogeneous spindle-shaped cells differentiated from iPSCs exhibited smooth muscle cell marker expressions, including α-smooth muscle actin (αSMA, 82.3 ± 6.7% and 81.0 ± 6.7%); calponin (91.3 ± 2.1% and 90.9 ± 1.3%); myosin heavy chain-11 (MYH11, 96.9 ± 0.7% and 97.1 ± 0.3%) without significance of differences between the two groups. Real-time PCR showed few PECAM1 and CD34 gene expressions in both groups, indicating features of differentiated VSMCs. There were no significant differences in cellular proliferation (p = 0.45), migration (p = 0.60), and contractile abilities (p = 0.96) between the two groups. Transcriptome analysis demonstrated similar gene expression profiles of VSMCs in HC subjects and MMD patients with six differentially expressed genes (DEGs); while ECs showed a distinct transcriptome profile in MMD patients with 120 DEGs. The Wnt-signaling pathway was a significant pathway in VSMCs. CONCLUSIONS: This is the first study that established VSMCs from NCSCs using MMD patient-derived iPSCs and demonstrated similar biological function and transcriptome profile of iPSC-derived VMSCs in MMD patients and HC subjects under naïve single culture condition. Comparative transcriptome features between iPSC-derived VSMCs and ECs, displaying distinct transcriptome in the ECs, suggested that pathological traits can be driven by naïve ECs predominantly and VSMCs may require specific environmental factors in MMD, which provides novel insight into the pathophysiology of MMD. Our iPSC derived VSMC model can contribute to further investigations of diagnostic and therapeutic target of MMD in addition to the current iPSC derived EC model.

Cav-1 (Caveolin-1) and Arterial Remodeling in Adult Moyamoya Disease.

Background and Purpose- Moyamoya disease (MMD) is a unique cerebrovascular occlusive disease characterized by progressive stenosis and negative remodeling of the distal internal carotid artery (ICA). We hypothesized that cav-1 (caveolin-1)-a protein that controls the regulation of endothelial vesicular trafficking and signal transduction-is associated with negative remodeling in MMD. Methods- We prospectively recruited 77 consecutive patients with MMD diagnosed via conventional angiography. Seventeen patients with intracranial atherosclerotic stroke and no RNF213 mutation served as controls. The outer distal ICA diameters were examined using high-resolution magnetic resonance imaging. We evaluated whether the degree of negative remodeling in the patients with MMD was associated with RNF213 polymorphism, cav-1 levels, or various clinical and vascular risk factors. We also investigated whether the derived factor was associated with negative remodeling at the cellular level using the tube formation and apoptosis assays. Results- The serum cav-1 level was lower in the patients with MMD than in the controls (0.47±0.29 versus 0.86±0.68 ng/mL; P=0.034). The mean ICA diameter was 2.48±0.98 mm for the 126 affected distal ICAs in patients with MMD and 3.84±0.42 mm for the asymptomatic ICAs in the controls ( P<0.001). After adjusting for confounders, cav-1 levels (coefficient, 1.018; P<0.001) were independently associated with the distal ICA diameter in patients with MMD. In vitro analysis showed that cav-1 downregulation suppressed angiogenesis in the endothelial cells and induced apoptosis in the smooth muscle cells. Conclusions- Our findings suggest that cav-1 may play a major role in negative arterial remodeling in MMD.

Adult moyamoya disease associated with abundant phosphorylated tau accumulation in the brainstem: Report of a case with autopsy findings.

We report the case of a 72-year-old Japanese woman with moyamoya disease (MMD). She experienced her first intracerebral hemorrhage (ICH) at the age of 32 years, and had nine ICHs and/or intraventricular hemorrhages during the following 40 years. Cerebral angiograms and vascular pathologies at autopsy confirmed that the patient suffered from MMD. Macroscopically, there were brown-colored changes in the subarachnoid space, mainly at the base of the brain and around the cerebellar hemispheres. Microscopically, hemosiderin deposits were observed mainly in the old hemorrhagic lesions and on the surface of the brainstem and cerebellum. Many AT8-immunoreactive neurons and neurites were observed in the pons and midbrain, mainly in the locus ceruleus and reticular formation in the midbrain. Several AT8-immunoreactive neurons and neurites were positive for Gallyas silver staining. A few tiny and short AT8-immunoreactive processes were observed in the molecular, Purkinje cell and granular layers of the cerebellum. There were a few phosphorylated tau accumulations in the cerebrum without senile plaques. Lewy pathologies and transactive response DNA-binding protein 43 kDa proteinopathy were not detected. We suspect that oxidative stress after repeated bleedings with long-term courses in the ventricles and subarachnoid space may accelerate phosphorylated tau accumulation in the brainstem. To our knowledge, this is the first report of MMD with tauopathy in the brainstem.

Expression of Connexin43 in Cerebral Arteries of Patients with Moyamoya Disease.

BACKGROUND AND PURPOSE: Moyamoya disease has a high incidence of cerebral vascular accident in children and adolescents, which can endanger the physical and mental health of children and adults seriously. However, the etiology and the pathogenesis of moyamoya disease remain unclear. Connexin43 (Cx43) is a predominant intercellular gap junction protein that plays an important role in the normal function of arteries and the development of several cardiovascular diseases. We aimed to preliminarily investigate pathological changes and the expression of Cx43 in cerebral arteries of patients with moyamoya disease. MATERIALS AND METHODS: This study collected 10 experimental cerebral artery specimens from patients with moyamoya disease and 10 control cerebral artery specimens from patients without moyamoya disease during surgery, then pathological changes and change in Cx43 expression of cerebral artery specimens were investigated in the 2 groups by hematoxylin and eosin staining and immunofluorescence. RESULTS: The intima of cerebral arteries was thin with monolayer endothelial cells in the control group but had asymmetrical thickening for the cerebral arteries in the experimental group. The mean ± standard deviation of the mean optical density of Cx43 in the experimental group was .065 ± .011 (range, .045-.081), whereas that in the control group was .035 ± .005 (range, .028-.042). The expression of Cx43 in the experimental group was statistically significantly higher in comparison with the control group (P < .01). CONCLUSION: The abnormal expression of Cx43 in the cerebral arteries may play an important role in the formation of vascular intima thickening in patients with moyamoya disease.

Loss of α1ß1 soluble guanylate cyclase, the major nitric oxide receptor, leads to moyamoya and achalasia.

Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal "moyamoya" vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1ß1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya.

Transient Global Cerebral Ischemia Induces RNF213, a Moyamoya Disease Susceptibility Gene, in Vulnerable Neurons of the Rat Hippocampus CA1 Subregion and Ischemic Cortex.

The RING finger protein 213 (RNF213) is an important susceptibility gene for moyamoya disease (MMD) and is also implicated in other types of intracranial major artery stenosis/occlusion (ICAS); however, the role of RNF213 in the development of ICAS including MMD is unclear. The constitutive expression of the RNF213 gene is relatively weak in brain tissue, while information regarding the expression patterns of the RNF213 gene under cerebral ischemia, which is one of characteristic pathologies associated with ICAS, is currently limited. Our objective was to address this critical issue, and we investigated Rnf213 mRNA expression in rat brains after 5 minutes of transient global cerebral ischemia (tGCI) by occluding the common carotid arteries coupled with severe hypotension. Rnf213 gene expression patterns were investigated with in situ RNA hybridization and a real-time polymerase chain reaction (PCR) from 1 to 72 hours after tGCI. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the hippocampus CA1 sub-region 48 hours after tGCI. The significant induction of the Rnf213 gene was also evident in the ischemic cortex. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression exclusively in neurons. The real-time PCR analysis confirmed the induction of the Rnf213 gene after tGCI. The up-regulation of the Rnf213 gene in vulnerable neurons in the hippocampus CA1 after tGCI suggests its involvement in forebrain ischemia, which is an underlying pathology of MMD. Further investigations are needed to elucidate its exact role in the pathophysiology of ICAS including MMD.

Hemodynamic contribution of transdural collateral flow in adult patients with moyamoya disease.

To evaluate the hemodynamic contributions of collateral flow in adult patients with moyamoya disease, neurological deterioration or fluctuation during admission, Suzuki grade, various collateral routes, lesion volume, cerebral blood flow (CBF), and their associations were analyzed. Thirty patients (60 cerebral hemispheres, mean age 45 ± 25 years, and 73.3 % female) who were diagnosed with moyamoya disease or syndrome were enrolled over 3 years. Moyamoya stages from each hemisphere were stratified according to the Suzuki's criteria through six-vessel angiography into internal carotid arteries (ICAs), external carotid arteries (ECAs), and vertebral arteries (VAs). Collateral routes were categorized into the circle of Willis, leptomeningeal, and transdural. The volume of ipsilateral infarction was analyzed by magnetic resonance imaging. CBF volume was measured using color-coded duplex sonography. Suzuki's grade was inversely correlated with flow volume of the ICAs (p < 0.001), whereas no association was found with that of the ECAs (p = 0.445) or VAs (p = 0.096). Among hemispheres with ≥ grade 3 (n = 36), patients with transdural ECA collateral flow had less neurological deterioration or fluctuation (0.0 vs. 30.8 %, p = 0.047), smaller lesion volume (2.4 ± 3.6 vs. 27.6 ± 59.3 mL, p = 0.041), lower ICA flow (88.4 ± 45.9 vs. 146.2 ± 121.7 mL/min, p = 0.022), higher ECA flow (205.7 ± 77.7 vs. 135.9 ± 52.7 mL/min, p = 0.046), and a higher ECA/ICA flow volume ratio (31.8 ± 92.8 vs. 1.7 ± 1.9, p = 0.024). Our results suggest that ICA flow volume is inversely correlated with Suzuki grade, and that transdural ECA collaterals appear to be an important detour in adult patients with advanced stage moyamoya disease, suggesting a protector against an impending ischemic attack.

Reversible striatal hypermetabolism in chorea associated with moyamoya disease: a report of two cases.

BACKGROUND: The pathophysiological mechanism of chorea as a presentation of pediatric moyamoya disease remains unknown, although ischemia is suspected as a likely cause. The authors describe two cases of pediatric moyamoya disease, both of which presented with hemichorea in the stable phase after successful bypass surgery. CLINICAL PRESENTATION: Cerebral blood flow was almost normal in one case and decreased in the basal ganglia and watershed area in the other case due to infarcts occurring before surgery. In both cases, 18F-fluorodeoxyglucose positron emission tomography revealed elevated glucose metabolism in the corresponding side of the striatum, which reverted to normal after recovery from chorea. Magnetic resonance angiography revealed a dilated and extended lenticulostriate artery at the exact site of the hypermetabolic lesion.

Germline mutation of CBL is associated with moyamoya disease in a child with juvenile myelomonocytic leukemia and Noonan syndrome-like disorder.

Germline mutations in CBL have been identified in patients with Noonan syndrome-like phenotypes, while juvenile myelomonocytic leukemia (JMML) harbors duplication of a germline CBL, resulting in acquired isodisomy. The association between moyamoya disease and Noonan syndrome carrying a PTPN11 mutation has recently been reported. We present a patient with JMML who developed moyamoya disease and neovascular glaucoma. Our patient exhibited a Noonan syndrome-like phenotype. Genetic analysis revealed acquired isodisomy and a germline heterozygous mutation in CBL. This is a rare case of CBL mutation associated with moyamoya disease. Prolonged RAS pathway signaling may cause disruption of cerebrovascular development.

Molecular structure and function of mysterin/RNF213.

Mysterin is a large intracellular protein harboring a RING finger ubiquitin ligase domain and is also referred to as RING finger protein 213 (RNF213). The author performed the first molecular cloning of the mysterin gene as the final step in genetic exploration of cerebrovascular moyamoya disease (MMD) and initiated the next round of exploration to understand its molecular and cellular functions. Although much remains unknown, accumulating findings suggest that mysterin functions in cells by targeting massive intracellular structures, such as lipid droplets (LDs) and various invasive pathogens. In the latter case, mysterin appears to directly surround and ubiquitylate the surface of pathogens and stimulate cell-autonomous antimicrobial reactions, such as xenophagy and inflammatory response. To date, multiple mutations causing MMD have been identified within and near the RING finger domain of mysterin; however, their functional relevance remains largely unknown. Besides the RING finger, mysterin harbors a dynein-like ATPase core and an RZ finger, another ubiquitin ligase domain unique to mysterin, while functional exploration of these domains has also just commenced. In this review, the author attempts to summarize the core findings regarding the molecular structure and function of the mysterin protein, with an emphasis on the perspective of MMD research.

Circulating immune cell landscape and T-cell abnormalities in patients with moyamoya disease.

BACKGROUND: Moyamoya disease (MMD) stands as a prominent cause of stroke among children and adolescents in East Asian populations. Although a growing body of evidence suggests that dysregulated inflammation and autoimmune responses might contribute to the development of MMD, a comprehensive and detailed understanding of the alterations in circulating immune cells associated with MMD remains elusive. METHODS: In this study, we employed a combination of single-cell RNA sequencing (scRNA-seq), mass cytometry and RNA-sequencing techniques to compare immune cell profiles in peripheral blood samples obtained from patients with MMD and age-matched healthy controls. RESULTS: Our investigation unveiled immune dysfunction in MMD patients, primarily characterized by perturbations in T-cell (TC) subpopulations, including a reduction in effector TCs and an increase in regulatory TCs (Tregs). Additionally, we observed diminished natural killer cells and dendritic cells alongside heightened B cells and monocytes in MMD patients. Notably, within the MMD group, there was an augmented proportion of fragile Tregs, whereas the stable Treg fraction decreased. MMD was also linked to heightened immune activation, as evidenced by elevated expression levels of HLA-DR and p-STAT3. CONCLUSIONS: Our findings offer a comprehensive view of the circulating immune cell landscape in MMD patients. Immune dysregulation in patients with MMD was characterized by alterations in T-cell populations, including a decrease in effector T-cells and an increase in regulatory T-cells (Tregs), suggest a potential role for disrupted circulating immunity in the aetiology of MMD.

Downregulation of Securin by the variant RNF213 R4810K (rs112735431, G>A) reduces angiogenic activity of induced pluripotent stem cell-derived vascular endothelial cells from moyamoya patients.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p<0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p<0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.

Data-Independent Acquisition-Based Serum Proteomic Profiling of Adult Moyamoya Disease Patients Reveals the Potential Pathogenesis of Vascular Changes.

Moyamoya disease (MMD) is a chronic cerebrovascular disease with unknown etiology. The pathogenesis of vascular changes remains unclear. Ischemic and hemorrhagic adult MMD patients and healthy volunteers were enrolled to collect serum for data-independent acquisition (DIA)-based proteomic analysis and ELISA validation. DIA serum proteomic revealed that apolipoprotein C-I (APOC1), apolipoprotein D (APOD), and apolipoprotein A-IV (APOA4) were decreased. The reductases glutathione S-transferase omega-1 (GSTO1) and peptidyl-prolyl cis-trans isomerase A (PPIA) were upregulated, and ADAMTS-like protein 4 (ADAMTSL4) was downregulated in both ischemic and hemorrhagic MMD. Afamin (AFM) and transforming growth factor-beta-induced protein ig-h3 (TGFBI) increased in ischemic patients but decreased in hemorrhagic patients. Serum ELISA results confirmed that APOA4, APOC1, and APOD were decreased compared to controls. Then, we retrospectively analyzed biochemical indexes of 200 MMD patients. A total of 54 enrolled MMD patients showed decreased total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-c). APOA4, APOC1, and APOD were vital factors in the HDL decrease in MMD patients. Lipoprotein dysfunction in MMD patients is involved in MMD. Intimal thickening by enhanced adhesion, middle layer vascular smooth muscle cell migration, and decreased lipid antioxidant function represented by HDL are potential pathogeneses of vascular changes in MMD.

CircZXDC Promotes Vascular Smooth Muscle Cell Transdifferentiation via Regulating miRNA-125a-3p/ABCC6 in Moyamoya Disease.

Moyamoya disease (MMD) is an occlusive, chronic cerebrovascular disease affected by genetic mutation and the immune response. Furthermore, vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) participate in the neointima of MMD, but the etiology and pathophysiological changes in MMD vessels remain largely unknown. Therefore, we established the circZXDC (ZXD family zinc finger C)-miR-125a-3p-ABCC6 (ATP-binding cassette subfamily C member 6) axis from public datasets and online tools based on "sponge-like" interaction mechanisms to investigate its possible role in VSMCs. The results from a series of in vitro experiments, such as dual luciferase reporter assays, cell transfection, CCK-8 assays, Transwell assays, and Western blotting, indicate a higher level of circZXDC in the MMD plasma, especially in those MMD patients with the RNF213 mutation. Moreover, circZXDC overexpression results in a VSMC phenotype switching toward a synthetic status, with increased proliferation and migration activity. CircZXDC sponges miR-125a-3p to increase ABCC6 expression, which induces ERS (endoplasmic reticulum stress), and subsequently regulates VSMC transdifferentiation from the contractive phenotype to the synthetic phenotype, contributing to the intima thickness of MMD vessels. Our findings provide insight into the pathophysiological mechanisms of MMD and indicate that the circZXDC-miR-125a-3p-ABCC6 axis plays a pivotal role in the progression of MMD. Furthermore, circZXDC might be a diagnostic biomarker and an ABCC6-specific inhibitor and has the potential to become a promising therapeutic option for MMD.