The role of the RING finger protein 213 gene in Moyamoya disease

Abstract

Moyamoya Disease (MMD) represents a chronic and progressive cerebrovascular disorder characterized by the gradual occlusion of the terminal portions of the bilateral internal carotid arteries and their major branches, accompanied by the formation of abnormal vascular networks at the base of the skull. In adolescents, particularly in pediatric populations, MMD is a significant cause of stroke, posing a severe challenge to human health and imposing a heavy burden on healthcare systems. Ring Finger Protein 213 (RNF213), as the primary susceptibility gene for MMD, plays a crucial regulatory role in the initiation, progression, and prognosis of the disease. Despite extensive research on the role of RNF213 in the pathogenesis of MMD, the underlying molecular mechanisms remain incompletely understood and represent a pressing scientific challenge requiring further exploration. This review aims to synthesize the latest research findings and systematically elucidate the multifaceted roles of RNF213 in MMD, including genetic susceptibility, immune-inflammatory responses, blood-brain barrier(BBB) disruption, and angiogenesis. By integrating these findings, this study seeks to provide new insights and theoretical support for a comprehensive and in-depth understanding of the pathophysiological processes of MMD. This research not only contributes to further unraveling the complex pathogenesis of MMD but also lays a solid theoretical foundation for the development of targeted preventive and therapeutic strategies.

Introduction

Moyamoya Disease (MMD) is a rare cerebrovascular disorder characterized by the progressive stenosis or occlusion of the terminal portions of the bilateral internal carotid arteries, along with stenotic-occlusive changes in the Willis circle and abnormal proliferation of vascular networks at the base of the skull. These features collectively constitute the pathological foundation of MMD [1,2,3]. As one of the common causes of stroke in children and young adults, MMD poses a high risk of disability and mortality, severely threatening patients’ lives and health. In pediatric MMD cases, ischemic symptoms are particularly prominent, especially transient ischemic attacks, which have emerged as one of the primary inducements of stroke in children [1, 4, 5]. In contrast, adult MMD patients more frequently exhibit intracranial hemorrhage [6]. The definitive diagnosis of MMD primarily relies on Digital Subtraction Angiography (DSA) [7], and with the continuous advancement and popularization of imaging technologies, the detection rate of MMD has been increasing annually. In China, the average incidence of MMD is 3.92 per 100,000, with a morbidity rate of approximately 0.43 per 100,000. Although the incidence of MMD in East Asia is relatively low, ranging from 0.5 to 1.5 per 100,000, the large population base in this region results in a significant number of MMD patients. MMD not only severely impacts patients’ quality of life but also imposes a substantial medical burden [8,9,10]. Currently, extracranial-intracranial bypass surgery is the primary treatment for MMD, among which the superficial temporal artery-middle cerebral artery (STA-MCA) branch anastomosis is the most widely used and has a significant effect on preventing ischemic stroke [11]. However, due to the incomplete elucidation of the pathogenesis of MMD, clinically effective strategies to mitigate disease progression or reverse its course are still lacking.

In recent years, the RNF213 gene has received extensive attention as an important susceptibility gene for MMD. RNF213 is a highly conserved giant protein in chordates, with a molecular weight of 591 kDa. Its unique structure tightly integrates a dynein-like ATPase core with a ubiquitin E3 ligase module [12, 13]. In 2011, Liu et al. conducted a Genome-Wide Association Study (GWAS) on eight pedigrees with three generations of MMD and first identified the novel MMD susceptibility gene variant RNF213 p. R4810K(rs112735431), which significantly increases the risk of developing MMD [14]. In the same year, the GWAS study by Kamada et al. also confirmed the susceptibility of the RNF213 gene in 72 MMD patients [12]. Subsequently, multiple studies in populations from different countries have found a close association between RNF213 p. R4810K and MMD, further establishing the status of RNF213 as an important susceptibility gene for Asian MMD patients [12, 14, 15]. Notably, follow-up studies have also revealed associations between the RNF213 gene and various intracranial and extracranial vascular diseases, suggesting that RNF213 gene variants may be a common cause of a series of vascular diseases [12]. However, despite the widespread recognition of the crucial role of RNF213 in MMD, researchers have not yet fully elucidated how RNF213 mutations participate in the initiation and progression of MMD [3]. Therefore, in-depth exploration of the mechanism of action of the RNF213 gene in MMD is of great significance for determining the optimal treatment for MMD and improving patient prognosis.

Fundamental functions of RNF213

The RNF213 gene is located on the q25.3 region of human chromosome 17, characterized by its intricate molecular structure and diverse functionalities, making it a focal point of recent research endeavors. The protein encoded by this gene comprises multiple crucial domains, including an N-terminal structural motif, six ATPase-active AAA (ATPases associated with a variety of cellular activities) units, and an E3 ubiquitin ligase domain featuring ring finger and zinc finger characteristics [13]. These domains collectively endow RNF213 with a range of biological functions.

As a major susceptibility gene for MMD, the discovery of RNF213 has significantly propelled advancements in related research fields [12]. In 2020, Ahel et al. utilized cryo-electron microscopy to elucidate the high-resolution structure of RNF213, revealing its ATPase core comprising six AAA units. Notably, unlike other AAA ATPases, the ATPase of RNF213 does not generate movement. Although the precise mechanism of action of the ATPase core remains incompletely understood, studies suggest that it may regulate the oligomerization state of RNF213 through ATP binding and hydrolysis [16]. It is worth noting that the oligomerization process of RNF213 is closely associated with lipid droplet (LD) transport [17], and RNF213 mutants defective in ATP binding or hydrolysis exhibit significant functional deficiencies in targeting LDs and bacteria [18]. These findings strongly imply that oligomerization is essential for RNF213 to perform its normal biological functions, particularly in targeting LDs and bacteria. Besides, the E3 ligase module of RNF213 is another critical functional region. This module is responsible for the ubiquitin ligase activity of RNF213 and can synergistically interact with E2 enzymes such as UBE2L3 and UBE2D2, at least in vitro, participating in the ubiquitination process [13, 18,19,20]. Intriguingly, mutants associated with MMD predominantly cluster near the E3 module of RNF213, suggesting that the pathogenesis of MMD may be linked to disruptions in ubiquitination activities [21]. In 2022, Bhardwaj et al. further corroborated this notion, discovering that RNF213 variants in MMD not only result in decreased E3 activity and ubiquitination levels but also produce a dominant-negative effect within the RNF213 oligomer structure [20]. These research findings indicate that reduced RNF213 E3 ubiquitin ligase activity may be one of the core pathogenic mechanisms underlying MMD [20, 21].

In summary, the RNF213 gene plays a pivotal role in cellular biological processes through its complex molecular structure and diverse functional domains. As the primary susceptibility gene for MMD, mutations in RNF213 may lead to abnormalities in its biological functions, thereby triggering the occurrence and progression of MMD. Therefore, an in-depth exploration of the functions of RNF213 and its relationship with MMD is of great significance for revealing the pathogenesis of MMD and developing effective therapeutic strategies.

RNF213 and genetic susceptibility

Since the late 20th century, researchers have gradually unveiled the intimate connection between MMD and genetic factors. In 1999, Ikeda et al., in a groundbreaking study involving 16 Japanese MMD families (encompassing 77 patients), discovered the linkage between MMD and genetic markers located within the 3p24.2-26 region of the chromosome, marking the identification of the first genetic locus associated with the pathogenesis of familial moyamoya disease [22]. Subsequently, Zafeiriou et al. also validated the presence of this locus in their study of Greek MMD twins. Beyond this locus, several other genetic loci have been confirmed to be associated with MMD, including 3p24–p26, 6q25, 8q23, 12p12, and 17q25 [23,24,25]. Notably, rare alleles at 17q25 are more prevalent in Asian populations, such as Japanese, Korean, and Chinese [26]. In the Chinese population, specific variations in the RNF213 gene, including p.A4399T, p.A5021V, and rs9916351, have been identified as susceptibility variants for MMD [27,28,29].

Studies on pediatric MMD patients have also revealed various RNF213 gene variations, such as p.K4115del, S4118F, C4017S, H4014N, and V4146A, which may be associated with the risk of MMD [30,31,32,33,34]. Furthermore, Guey et al.‘s research indicates that, while the RNF213 p.R4810K variant is rare in Caucasian moyamoya patients, other rare variants in RNF213, particularly those in the C-terminal region, are significantly associated with moyamoya disease, especially in familial cases [30, 33]. In addition to the RNF213 gene, human leukocyte antigen (HLA) is also linked to the genetic susceptibility of MMD. In Japanese and Korean populations, specific HLA alleles, such as HLA-DQB10502, HLA-B51, HLA-DR4, as well as HLA-DRB11302, HLA-DRB10609, and HLA-B35, are significantly associated with the risk of MMD [35,36,37,38]. Moreover, Kang et al.‘s study suggests that the G/C heterozygous genotype at position − 418 of the TIMP2 promoter may be a genetic susceptibility factor for familial MMD [39]. Notably, the RNF213 c.14576G > A variant is prevalent in MMD patients, with a detection rate of 95% in familial MMD cases and approximately 79% in sporadic MMD cases [12, 40]. However, a subset of MMD patients does not carry this variant, and this subset is more prevalent in Western countries, suggesting that the pathogenesis of MMD may involve a combination of multiple genetic and environmental factors [40]. Further research has found that the RNF213 p.R4810K variant is more common in familial MMD patients and is associated with ischemic MMD, while non-R4810K variants (such as A4399T) are associated with hemorrhagic MMD [29, 30]. In Japanese and Chinese familial MMD patients, the carrier rates of the RNF213 p.R4810K variant are 95.1% and 67.5%, respectively, while in sporadic cases, they are 79.2% and 17.2% [41, 42]. In recent years, various non-R4810K variants, such as rs148731719 and rs397514563, have been discovered in Caucasian, as well as East and South Asian MMD cases, and these variants may also contribute to the pathogenesis of MMD [14, 29, 30, 43]. Additionally, RNF213 variations are not only associated with MMD but also with intracranial atherosclerosis and systemic vascular diseases, such as peripheral pulmonary artery stenosis and renal artery stenosis [2, 44,45,46]. Fukushima et al.‘s study indicates that the R4810K variant, in its heterozygous state, may cause typical MMD, while in its homozygous state, it may lead to MMD and systemic vascular diseases in a gene dose-dependent manner [45]. Finally, the role of multigene interplay in MMD is increasingly gaining attention. Grangeon et al.‘s research revealed the synergistic effect of rare variants in the RNF213 and PALD1 genes in a European familial MMD case, suggesting that variants in these two genes may jointly contribute to the pathogenesis of MMD [47]. Therefore, the interaction between RNF213 variants and other genes or environmental factors may be an important cause of phenotypic heterogeneity in RNF213 [47]. In conclusion, the RNF213 gene plays a crucial role in the genetic susceptibility of MMD, but its pathogenesis involves the combined effects of multiple genetic and environmental factors, warranting further in-depth research.

RNF213 and immune-inflammatory responses

In recent years, clinical reports have frequently described the progression of moyamoya syndrome (MMS), which is closely associated with infections or autoimmune diseases. Among these, immune-inflammatory responses have been widely recognized as a core element in the pathogenesis of MMS [48]. Various pathogens, including but not limited to Haemophilus influenzae [49, 50], Streptococcus pneumoniae [51], Mycobacterium tuberculosis [52], Propionibacterium acnes [53], and Leptospira, may cause the emergence of MMS while inducing bacterial meningitis [49,50,51,52, 54]. In 2020, a case of vasculopathy similar to MMD following enterovirus infection causing hand, foot, and mouth disease was first reported. The patient carried the ancestral variant of RNF213 (p.R4810K), suggesting that RNF213 polymorphism may increase the risk of developing MMD after viral infection [55]. These findings imply that post-infectious vasculopathy may be mediated by infection-triggered autoimmune responses, thereby elevating the risk of MMD/MMS [56].

Beyond MMS, an increasing number of studies have uncovered direct links between infections and MMD. Case reports of MMD following viral infections are also rising, with varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus infections sometimes being implicated in the occurrence of MMD [56,57,58]. Although genetic studies have identified RNF213 as the primary susceptibility gene for MMD, its penetrance in genetically susceptible individuals is relatively low. These accumulating molecular and clinical evidences strongly suggest that immune-related responses may serve as a second hit in the onset of MMD, closely related to its pathogenesis [21]. This perspective aligns with the theory that inflammatory and immune signals may act as environmental triggers for MMD [59,60,61,62].

The study by Asselman et al. further emphasizes the characteristics of MMD as an immune-related vasculopathy [21]. In 1996, Aoyagi et al. conducted histological examinations of superficial temporal arteries from MMD patients and found significantly thickened intima with notable accumulations of macrophages and T cells in the intimal surface layer compared to controls [63]. In 2017, Weng et al. revealed that the proportions of Treg cells and Th17 cells in the circulation of MMD patients, as well as the levels of their mainly secreted cytokines, were significantly higher than those in controls. However, the increased Treg cells in MMD patients exhibited defective suppressive functions. Additionally, Treg cells or transforming growth factor-β (TGF-β) positively correlated with the Suzuki staging of MMD, and the level of circulating Treg cells was an independent factor associated with MMD staging [48]. Nevertheless, a clear understanding of how circulating Treg/Th17 cells specifically participate in the pathogenesis of MMD patients remains lacking. Notably, the complex relationship between the RNF213 gene and immune inflammation is gradually being elucidated [60]. RNF213 is highly expressed in immune cells, particularly peripheral natural killer (NK) cells and T cells, and plays a crucial role in resisting bacterial infections through ubiquitination of lipopolysaccharides (LPS) [18]. The study by Yang et al. revealed that the RNF213 gene regulates the differentiation process of Treg cells by ubiquitinating FOXO1 at the K63 site, promoting FOXO1 nuclear entry, and enhancing Foxp3 transcription, thereby promoting Treg cell differentiation and inhibiting the development of multiple sclerosis [64]. However, unfortunately, due to various reasons such as the lack of animal models, the validity of this mechanism in MMD has not been fully validated. The results of Roy et al.‘s study indicated that RNF213-deficient cerebral endothelial cells (ECs) secrete more proinflammatory cytokines, suggesting that RNF213 mutations may play a central role in initiating immune responses in the pathogenesis of MMD. The study by Ohkubo et al. also supports the notion of an association between RNF213 and immune responses in ECs [60]. RNF213 is constitutively expressed in various tissues [14], but its expression level is upregulated under the stimulation of LPS, tumor necrosis factor-α (TNF-α), inflammation, or interferon (IFN) [60]. The study by Ohkubo et al. further showed that the administration of TNF-α and costimulation with other proinflammatory cytokines can induce the transcription of RNF213, thereby closely linking environmental factors to RNF213 responses. Interferon-β (IFN-β) increases the expression of the RNF213 gene through STAT binding sites in the promoter region and downregulates angiogenic capacity. Meanwhile, the R4810K polymorphism of RNF213 may result in reduced tube-forming capacity in response to environmental stimuli [59]. Several molecular studies have deeply uncovered the crucial role of RNF213 as a key antibacterial protein, which eliminates pathogens by binding to the LPS of bacterial outer membranes and thus plays an indispensable role in the immune system [17, 18]. The study by Otten et al. demonstrated that RNF213-mediated ubiquitination of Salmonella in the cytosol is a key step in degrading bacteria through xenophagy. RNF213 acts directly on the LPS shell of Salmonella using its RING structure and zinc finger domains to ubiquitinate it [18]. Subsequently, a research endeavor conducted by Thery and colleagues extended the understanding of the antimicrobial spectrum of RNF213, demonstrating its inhibitory efficacy against Listeria monocytogenes as an additional bacterial species, and further validating its activity against herpes simplex virus type 1, human respiratory syncytial virus, and coxsackievirus B3.The level of RNF213 expression positively correlates with anti-infectious capacity [17]. Furthermore, the study by Houzelstein et al. found that animals with RNF213 gene knockout are more susceptible to Rift Valley fever virus, and they speculated that this susceptibility may be mediated through the role of RNF213 in lipid metabolism [65]. The study by Ahmed et al. revealed that Rnf213 gene knockout alleviates diabetes symptoms in the Akita mouse model induced by endoplasmic reticulum (ER) stress. They speculated that RNF213 depletion may inhibit ER stress by increasing the levels of the SEL1L-HRD1 complex, thereby promoting ER-associated degradation processes both in vitro and in vivo [66]. In summary, the RNF213 gene is closely associated with immune-inflammatory responses in the pathogenesis of MMD (Fig. 1), and its complex regulatory mechanisms and functions warrant further in-depth research and exploration.

Fig. 1

The role of RNF213 and immune inflammation in MMD. The RNF213 gene plays a multifaceted role in MMD by facilitating Treg cell activation, contributing to endothelial cell damage, inducing endoplasmic reticulum stress, and promoting the release of various inflammatory factors. It also supports immune cell infiltration into the vascular intima and mediates anti-infective effects through ubiquitination pathways. Furthermore, the activation of diverse immune cells can reciprocally enhance RNF213 expression, highlighting its complex involvement in immune regulation and disease pathogenesis

RNF213 and the blood-brain barrier (BBB)

The BBB, serving as a pivotal component of the neurovascular unit (NVU), constitutes a sophisticated barrier co-constructed by the capillary walls of the brain and glial cells. Positioned between the plasma and brain cells, it effectively prevents the intrusion of harmful substances from the bloodstream into brain tissue while selectively regulating the passage of small molecules from the circulating blood into the central nervous system [67]. The structural foundation of this barrier primarily encompasses the continuous capillary endothelium with its intercellular tight junctions, an intact basement membrane, pericytes, and a glial membrane formed by the endfeet of astrocytes [68]. Among these, the continuous capillary endothelium constitutes the primary structure of the BBB, exhibiting extremely low paracellular and transcellular permeability [67, 69,70,71,72,73]. Pericytes are also crucial for maintaining the integrity of the BBB [70, 74].

However, damage to the BBB frequently occurs in various cerebrovascular diseases, particularly MMD [68, 75, 76]. A groundbreaking study by Narducci et al. in 2019 first demonstrated in vivo BBB impairment in MMD patients, based on a retrospective analysis of BBB integrity in 11 patients undergoing bypass surgery for MMD. They assessed the extravasation of fluorescein sodium (NaFL) using video angiography and quantified the degree of extravasation using a grading system, comparing the frequency and intensity of extravasation between different groups. This finding not only revealed the compromised state of the BBB in MMD patients but also suggested that the unique pathological mechanisms of MMD may intrinsically lead to BBB disruption [77]. Further research, such as the work by Lu et al. in 2020, similarly demonstrated significant BBB damage in MMD patients and found that the blood flow index (BFI) in damaged cortex was significantly reduced compared to cortex with intact BBB. Notably, after STA-MCA bypass surgery, cortical perfusion in these BBB-injured areas improved significantly, further confirming the pivotal role of BBB damage in MMD [78]. MMD, as an intimal progressive disease, may be closely related to endothelial dysfunction in its early stages [79]. Abnormalities in the BBB often follow endothelial injury [68, 80], particularly in MMD patients carrying RNF213 mutations, where EC dysfunction is particularly prominent [81, 82]. Research by Roy et al. revealed that brain ECs with RNF213 knockout exhibited significant morphological changes, downregulation and delocalization of key endothelial junction proteins involved in BBB maintenance, such as platelet endothelial cell adhesion molecule-1 (PECAM-1), and increased BBB permeability. Additionally, RNF213-deficient cerebrovascular ECs demonstrated abnormal leukocyte migration potential and secreted abundant pro-inflammatory cytokines, such as IL-6 [83]. IL-6 not only affects BBB function by promoting plasmacytes survival and stimulating the production of aquaporin-4 antibodies but may also directly disrupt BBB integrity [84]. Long-term exposure to IL-6 also affects connexins, further exacerbating BBB damage [85]. Furthermore, IL-8 levels are significantly elevated in MMD patients [86, 87], and studies by Bahrani et al. have shown that IL-8 is associated with acute stroke and BBB dysfunction [88], although the specific mechanisms by which IL-8 regulates the BBB in MMD are not fully understood. These findings collectively suggest that RNF213 may be a crucial regulator of cerebral endothelial integrity, and its disruption may be one of the important causes of BBB damage in MMD patients [83] (Fig. 2). Finally, research by Wang et al. revealed elevated levels of bradykinin (BK) in the serum and superficial temporal artery tissue of MMD patients. BK not only has the ability to inhibit angiogenesis and promote cell migration and proliferation but also disrupts BBB integrity [89]. Therefore, modulating BK levels in the serum and brain may emerge as a potential strategy to improve BBB permeability in MMD patients.

Fig. 2

BBB Disruption in MMD. In MMD, RNF213 contributes to the disruption of the BBB through multiple mechanisms. It directly impacts the tight junctions of ECs and mediates the production of the pro-inflammatory cytokine IL-6 by ECs. This, in turn, activates plasma cells and regulates the expression of aquaporin-4 antibodies, ultimately exacerbating the breakdown of the BBB

RNF213 and angiogenesis

Abnormal vascular proliferation, particularly the hyperplasia of arteries and arterioles, as well as the formation of moyamoya vessels, constitutes a prominent feature of MMD [1]. Among the pathological characteristics of MMD, Ye et al.‘s research has unveiled intimal thickening and medial thinning as two core manifestations, further emphasizing the pivotal role of vascular pathology in the pathogenesis of MMD [3]. Notably, extracranial involvement in MMD has also been reported in some pediatric patients, including cases of coronary artery [90, 91], pulmonary artery (27375007), and renal artery stenosis, broadening the understanding of the scope of vascular pathology in MMD [92]. As a star molecule in MMD research, the RNF213 gene exhibits a close association with the vascular pathology of MMD [1, 40, 93]. Numerous studies have indicated that mutations in the RNF213 gene may impact a series of signaling processes related to angiogenesis and immune activity, which may underlie the pathological progression of MMD [94]. However, studies in mouse models have yielded complex results. Sonobe et al. observed no changes in angiogenesis in mouse with the RNF213 gene knocked out, and subsequent studies with mouse carrying the RNF213 R4859K mutation yielded similar conclusions to previous research [93, 95, 96]. Maeda et al.‘s research demonstrated a reduced angiogenic capacity in carriers of the RNF213 c.14576G > A variant [97], suggesting that the decline in normal angiogenic capacity in MMD may be a potential cause of moyamoya vessel formation. Ye et al.‘s latest research found that the knockout of the RNF213 gene activates the Hippo pathway effector molecules Yes-associated protein (YAP)/tafazzin (TAZ) and promotes the expression of the downstream effector VEGFR2, significantly enhancing the proliferation, migration, and tubulogenesis of cerebrovascular ECs. This effect can be effectively reversed by inhibiting YAP/TAZ, providing a new potential therapeutic target for MMD patients [3]. Consistent with this, Ohkubo et al.‘s research found that overexpressing RNF213 mediated the PI3K-Akt signaling pathway, significantly inhibiting lumen formation [60, 98]. Furthermore, Rnf213-/- mouse exhibited enlarged vessel size and increased pathological angiogenesis in the cerebral cortex, hippocampus, and retina, further confirming the role of RNF213 loss of function in promoting pathological angiogenesis in arterioles [3]. From the perspective of post-ischemic injury processing in mouse, Ito et al.‘s research found that mouse lacking Rnf213 exhibited significantly enhanced angiogenesis after ischemia, indicating that RNF213 deficiency may play an important role in the development of abnormal vascular networks in chronic ischemia, providing new insights into the potential mechanism of moyamoya vessel formation [96]. Additionally, Roy et al.‘s research in cellular culture models found that the knockout of the RNF213 gene in ECs disrupts intercellular connections and promotes EC migration by activating inflammatory pathways (34991336). Using Rnf213 gene-knockout zebrafish, similar vascular sprouting phenomena were observed in intersegmental arteries 72 h post-fertilization, along with the detection of mulberry-like clusters of disordered-flow vascular channels [99, 100]. These studies reveal the complex role of RNF213 in angiogenesis, potentially inhibiting normal vessel formation while promoting the neovascularization of pathological vessels, which may be related to the type of RNF213 gene mutation.

Regarding the impact of RNF213 on smooth muscle cells (SMCs), current research results are controversial. Tokairin et al.‘s in vitro study using induced pluripotent stem cell (iPSC)-derived SMCs showed that RNF213 mutations did not impair the proliferation, migration, and contraction capabilities of SMCs from MMD patients [101]. This suggests that the pathological characteristics of MMD may be primarily driven by cerebrovascular ECs, while SMCs may require specific environmental factors to play a role in MMD, providing new insights into the pathophysiological mechanisms of MMD. However, Phi et al.‘s research found that functionally defective endothelial progenitor cells abnormally recruit smooth muscle progenitor cells to critical vascular sites through the action of CCL5 [102]. Liu et al.‘s research revealed higher levels of circZXDC in the plasma of MMD patients, especially in those carrying RNF213 mutations. Overexpression of circZXDC leads to a shift in the VSMC phenotype towards a synthetic state, increasing proliferation and migration activity. This research once again confirms the involvement of vascular SMCs and ECs in the formation of new intima in MMD and reveals the pathophysiological mechanisms of MMD [103]. Furthermore, the circZXDC-miR-125a-3p-ABCC6 axis plays a crucial role in the progression of MMD, with circZXDC acting as a sponge to adsorb miR-125a-3p, thereby increasing the expression of ABCC6, inducing endoplasmic reticulum stress (ERS), and regulating the transdifferentiation of VSMCs from a contractile phenotype to a synthetic phenotype. CircZXDC may serve as a diagnostic biomarker for MMD and a specific inhibitor of ABCC6, providing a potential target for the development of new drug therapies in the future [103]. However, Hitomi et al.‘s research drew different conclusions. They used fibroblasts-derived iPSCs from MMD patients and healthy controls to assess related angiogenic activity and found that the proliferative capacity of patient and carrier cells was reduced compared to control cells. This may be due to the complex pathological environment of MMD, where the proliferative capacity of different cell types is regulated by multiple factors [98].

Despite this, increasing evidence indicates that the RNF213 gene plays a significant role in the vascular abnormalities of MMD. Xue et al.‘s research found that patients carrying the RNF213 p.R4810K heterozygote and other rare variants in the C-terminal region exhibited different moyamoya angiographic phenotypes and significantly higher periventricular anastomotic scores compared to wild-type patients [104]. Ge et al.‘s research showed that the RNF213 p.R4810K heterozygote variant is associated with the progression of unilateral MMD to bilateral MMD and postoperative collateral vessel formation [105, 106]. Ishigami et al.‘s research further indicated that patients heterozygous for the RNF213 p.Arg4810Lys variant are more likely to experience cerebrovascular events in both hemispheres and present symptoms at a younger age compared to wild-type patients [107]. Finally, Ito et al.‘s research observed that good indirect collateral circulation development was more common after STA-MCA anastomosis combined with indirect encephalo-myo-synangiosis in adult MMD patients with RNF213 polymorphisms [108], suggesting that the p.R4810K variant plays a role in the significant growth of indirect collateral circulation and emphasizing the importance of preoperative genetic analysis. In summary, the RNF213 gene plays a complex and crucial role in angiogenesis and abnormal vessel formation in MMD (Fig. 3), and its different variants may have varying effects on angiogenesis. Future research needs to further explore the specific mechanisms of the RNF213 gene in the pathogenesis of MMD, providing new ideas and methods for the diagnosis and treatment of MMD.

Fig. 3

The role of angiogenesis in MMD. The RNF213 gene plays a critical role in MMD by regulating both EC proliferation and migration, as well as angiogenesis. Dysfunctional endothelial cells can recruit SMCs via the chemokine CCL5. Elevated levels of circZXDC in the bloodstream contribute to endoplasmic reticulum stress, driving the phenotypic transformation and migration of SMCs. These processes may ultimately result in intimal thickening and medial thinning, potentially underlying the mechanisms of pathological angiogenesis in MMD

Fig. 4

The interplay between immune inflammation, BBB disruption, and angiogenesis in MMD. Exogenous infections and RNF213 gene mutations can directly or indirectly compromise the integrity of the BBB. Additionally, both infections and RNF213 mutations activate the immune system and promote the release of various inflammatory cytokines, further exacerbating BBB damage and creating a vicious cycle of mutual reinforcement. Aberrant immune infiltration into the central nervous system may enhance pathological angiogenesis, and individuals with RNF213 mutations appear to be more susceptible to this pathological process

Outlook and conclusion

In 2019, Narducci et al. first demonstrated BBB disruption in patients with MMD using in vivo NaFL extravasation experiments [77]. This finding was further supported by Lu et al. [78]. Additionally, Eduardo et al. highlighted that imaging techniques, such as magnetic resonance imaging, provide compelling evidence of BBB damage [109], though direct imaging evidence of BBB disruption in MMD remains lacking. These preliminary findings suggest that BBB damage may be one of the initiating factors in MMD pathogenesis. While the precise mechanisms underlying BBB disruption in MMD are not fully elucidated, existing studies implicate chronic ischemic injury as a critical mediator. Xi et al. demonstrated that ischemia and hypoxia can compromise the BBB, with neuroinflammation playing a central role. Activated microglia secrete various cytokines, chemokines, and inflammatory mediators—such as TNF-α, interleukin-1β (IL-1β), and IL-6—which directly or indirectly impair tight junction proteins and ECs, ultimately resulting in BBB disruption [110]. However, Narducci et al. also noted that chronic ischemic injury alone does not fully account for the severe BBB damage observed in MMD, which is more pronounced compared to atherosclerotic cerebrovascular disease (ACVD) [77]. This suggests the involvement of additional pathological mechanisms beyond ischemia in MMD-related BBB disruption.

In recent years, increasing attention has been directed toward environmental factors, particularly immune-mediated inflammation, in the pathogenesis of MMD [18, 21, 53, 111]. Immune inflammation is not only closely associated with MMD onset but also directly linked to BBB disruption. For example, Yamada et al. identified Propionibacterium acnes infection and related immune factors as potential contributors to MMD pathogenesis [53]. Wang et al. reported elevated BK levels in the serum and superficial temporal arteries of MMD patients, which may lead to BBB disruption [89]. Moreover, Shi et al. demonstrated that CD4₊ T cells release interleukin-17 (IL-17), compromising BBB integrity in hemorrhagic brain injury [112]. The significant accumulation of macrophages and T cells in the intimal layers of the superficial temporal artery, as well as the altered ratios and cytokine profiles of Treg and Th17 cells in MMD patients, further underscores the importance of immune inflammation in MMD pathogenesis [48, 63].

The role of the RNF213 gene, a pivotal factor in MMD, in regulating the immune landscape remains poorly understood. Current studies suggest that RNF213 may contribute to MMD pathogenesis by modulating inflammatory responses, angiogenesis and BBB integrity [113]. For instance, Roy et al. found that RNF213-knockout endothelial cells secreted higher levels of inflammatory mediators [83], potentially contributing to BBB disruption. Similarly, Bean et al. demonstrated that T-cell activity suppression effectively reduced circulating TNF-α and IL-17 levels in pregnant rats, improving BBB permeability [114]. Additionally, Li et al. revealed that RNF213 might promote Treg cell differentiation through FOXO1 ubiquitination, thereby inhibiting multiple sclerosis development [64]. These findings suggest that the role of RNF213 in MMD involves intricate regulatory mechanisms.

ECs, as critical components of the BBB, maintain its integrity and function through tight junctions. Disruptions in tight junctions directly compromise the BBB, exacerbating MMD pathology [115,116,117]. Various bacterial and viral infections have been shown to indirectly degrade or dysregulate tight junctions, further emphasizing the role of immune inflammation in MMD pathogenesis. Notably, cytokines such as IL-1β, IL-6, IL-9, IL-17, IFN-γ, TNF-α, and CCL2 have been implicated in reducing tight junction expression or causing their mislocalization, thereby disrupting BBB integrity [118,119,120,121,122,123]. Astrocytes play a crucial role in maintaining BBB integrity and regulating its permeability by producing pro- or anti-inflammatory mediators depending on the immune triggers or inflammation stages [124]. Microglia, another key immune cell type in the central nervous system, also significantly influence BBB integrity through their M1 (pro-inflammatory) and M2 (anti-inflammatory) activation states [125]. The M1 phenotype exacerbates BBB dysfunction through the release of inflammatory mediators and oxidative stress, while the M2 phenotype promotes BBB repair and immune regulation [126, 127].

Peripheral immune cells, through the secretion of inflammatory mediators such as reactive oxygen species (ROS) and matrix metalloproteinases (MMP-1 and MMP-2), further compromise BBB permeability, creating a destructive feedback loop [128, 129]. Infection, a well-documented trigger for MMD, can directly disrupt the BBB and exacerbate BBB damage in individuals with RNF213 mutations, potentially triggering a cascade of pathological responses [130, 131]. The core pathological feature of MMD lies in the abnormalities of blood vessels, which not only impair patients’ quality of life but also constitute the primary cause of their symptoms. Pathological changes such as vascular stenosis and rupture of moyamoya vessels originate from the progressive stenosis within the internal carotid artery system, particularly the two main pathological characteristics of intimal thickening and medial thinning [3]. The proliferation of SMCs may also play a pivotal role in this process; however, existing studies have not observed a significant effect when exploring the influence of the RNF213 gene on SMC proliferation [101]. This finding may imply that the proliferation of SMCs and intimal thickening are not solely driven by the RNF213 gene alone but may involve more complex regulatory mechanisms. Although attempts to model MMD phenotypes through RNF213 gene editing in animal models have been unsuccessful [93], these findings underscore the complexity of MMD pathogenesis, which likely involves interactions between multiple genes and signaling pathways. Therefore, future research needs to focus on developing models that more closely mimic the pathological features of MMD, in order to comprehensively elucidate the specific mechanisms of action of the RNF213 gene in the pathogenesis of MMD and explore more effective therapeutic strategies.

However, this review also has certain limitations. Firstly, despite the widely acknowledged importance of the RNF213 gene in MMD, the results of animal experiments have not observed the imaging manifestations of MMD. This finding suggests that the use of RNF213 gene-edited animal models may not accurately reflect the true pathological changes in MMD in some aspects. Secondly, the pathogenesis of MMD cannot be fully explained by a single gene. An increasing number of studies have indicated that, besides RNF213, various other genes are also closely related to the onset of MMD [23,24,25, 27,28,29,30,31,32,33,34]. Unfortunately, due to page limitations, this review failed to comprehensively discuss the roles of these genes. This fact implies that in understanding the complete pathogenesis of MMD, it needed a broader and more comprehensive perspective, not limited solely to the RNF213 gene.

Therefore, in future research, it is important to delve deeper into the pathogenesis of MMD, focusing not only on the RNF213 gene but also on other related genes and their interactions. At the same time, it is imperative to strive to develop more accurate and comprehensive MMD disease models to better simulate the real pathological environment of MMD, thereby providing more effective strategies and methods for the treatment of MMD.

In summary, the pathogenesis of MMD represents a complex model of multifactorial interactions, where genetic variations—particularly RNF213 mutations—play a central role (Fig. 4). However, the actual onset of the disease often requires additional environmental factors, referred to as the “second hit.” In this process, BBB disruption potentially mediated by RNF213 mutations may provide a pathological basis for inflammatory storms triggered by environmental factors to infiltrate the central nervous system. This intricate interplay between genetic predisposition and environmental triggers may collectively mediate the development of MMD. Therefore, the triggering mechanism of MMD should be comprehensively understood as the result of the combined influence of genetic and environmental factors.