Physiological and pathophysiological mechanisms of the molecular and cellular biology of angiogenesis and inflammation in moyamoya angiopathy and related vascular diseases

Rationale The etiology and pathophysiological mechanisms of moyamoya angiopathy (MMA) remain largely unknown. MMA is a progressive, occlusive cerebrovascular disorder characterized by recurrent ischemic and hemorrhagic strokes; with compensatory formation of an abnormal network of perforating blood vessels that creates a collateral circulation; and by aberrant angiogenesis at the base of the brain. Imbalance of angiogenic and vasculogenic mechanisms has been proposed as a potential cause of MMA. Moyamoya vessels suggest that aberrant angiogenic, arteriogenic, and vasculogenic processes may be involved in the pathophysiology of MMA. Circulating endothelial progenitor cells have been hypothesized to contribute to vascular remodeling in MMA. MMA is associated with increased expression of angiogenic factors and proinflammatory molecules. Systemic inflammation may be related to MMA pathogenesis. Objective This literature review describes the molecular mechanisms associated with cerebrovascular dysfunction, aberrant angiogenesis, and inflammation in MMA and related cerebrovascular diseases along with treatment strategies and future research perspectives. Methods and results References were identified through a systematic computerized search of the medical literature from January 1, 1983, through July 29, 2022, using the PubMed, EMBASE, BIOSIS Previews, CNKI, ISI web of science, and Medline databases and various combinations of the keywords “moyamoya,” “angiogenesis,” “anastomotic network,” “molecular mechanism,” “physiology,” “pathophysiology,” “pathogenesis,” “biomarker,” “genetics,” “signaling pathway,” “blood-brain barrier,” “endothelial progenitor cells,” “endothelial function,” “inflammation,” “intracranial hemorrhage,” and “stroke.” Relevant articles and supplemental basic science articles almost exclusively published in English were included. Review of the reference lists of relevant publications for additional sources resulted in 350 publications which met the study inclusion criteria. Detection of growth factors, chemokines, and cytokines in MMA patients suggests the hypothesis of aberrant angiogenesis being involved in MMA pathogenesis. It remains to be ascertained whether these findings are consequences of MMA or are etiological factors of MMA. Conclusions MMA is a heterogeneous disorder, comprising various genotypes and phenotypes, with a complex pathophysiology. Additional research may advance our understanding of the pathophysiology involved in aberrant angiogenesis, arterial stenosis, and the formation of moyamoya collaterals and anastomotic networks. Future research will benefit from researching molecular pathophysiologic mechanisms and the correlation of clinical and basic research results.


Introduction
Moyamoya angiopathy (MMA) is an angiopathy unique to the cerebrovasculature that is characterized by chronically progredient stenosis of the bilateral intracranial internal carotid artery (ICA) and its proximal bifurcations and development of a network of aberrant collateral arteries to compensate for the stenosed vessels. MMA pathophysiology may include a consecutive secondary response of compensatory collateral circulation development by means of vasculogenesis and alteration of cerebral hemodynamics as a result of a primary narrowing of distinct intracranial vessels (1-6) (Figures 1-3) (see the Supplementary Table 1 for definitions of gene symbols, proteins, and additional terminology).
The purpose of this review article is to describe the physiological and pathophysiological mechanisms of signaling pathways, cells, and genes relevant to angiogenesis and inflammation in MMA and MMS along with future moyamoya research perspectives and treatment strategies implemented into clinical practice ( Figure 4). This article discusses if these mechanisms may be regarded as causative of the angiopathy or if they may be viewed as a consequence of ischemic processes observed in MMA. We also aim to further specify proposed therapeutic and diagnostic targets related to angiogenesis and inflammation in MMA, that may lead to disease-modifying treatment strategies (4,6,9,(43)(44)(45)(46).

Methods
References were identified by use of a systematic, comprehensive computerized literature search from January 1, 1983, through July 29, 2022, performed by both authors, using the PubMed, Embase, BIOSIS Previews, CNKI, ISI Web of Science, and Medline databases and the key words "moyamoya, " "angiogenesis, " "anastomotic network, " "moyamoya syndrome, " "molecular mechanism, " "signaling pathway, " "genetics, " "biomarker, " "physiology, " "pathophysiology" "blood-brain barrier, " "endothelial function, " "endothelial progenitor cells, " "intracranial hemorrhage, " "inflammation, " and "stroke" in various combinations. Relevant articles on MMA and supplemental basic science articles almost exclusively published in English were included. References of included publications have been searched for supplementary sources, and 350 publications have consequently been cited in the manuscript. After being reviewed by a member of the panel, the manuscript has been reviewed by five expert peer reviewers. Even though several basic research results about physiologic characteristics of angiogenesis, arteriogenesis, vasculogenesis (13,14), and associated signaling pathways  as well as knowledge regarding inflammation in pediatric ischemic stroke (47)(48)(49)(50)(51)(52)(53)(54)(55)(56) have been included for the convenience of readers who may be unfamiliar with these topics, this article emphasizes MMA basic, laboratory and clinical research results, future research perspectives, treatment strategies, and their implementation in clinical practice. As several aspects of MMA have been studied in greater detail in comparison to others, distinct topics receive additional attention. Despite substantial progress in the MMA field of research in recent years, the literature in great part remains descriptive. Continued basic and clinical research is essential to further elucidate the pathogenesis of MMA, and to obtain significant results. stroke (5). Masuda et al. demonstrated the infiltration of T cells and macrophages into vascular sections without stenosis, indicating that microthrombi may result from chronic inflammation instead of causing this process (5,61). Presence of microthrombi may not be specific for MMA (5). Inflammation may cause hyperplasia of intimal SMCs and neovascularization through endothelial cell proliferation, leading to lumen stenosis and formation of collaterals (9). In 2006, Takagi et al. demonstrated that apoptosis, evidenced through activated caspase-3, may occur in the MCA media in MMA patients. Consequently, MCA specimens from MMA patients showed vascular wall/medial thinning compared to controls (62). In their 2008 study in 19 adult MMA patients, Kwag et al. suggested that linear and/or non-linear mean blood flow velocity (MBFV) changes in the posterior and anterior cerebral circulation, related to distinct intracranial vessels, may be helpful in both follow-up and initial evaluation of distinct angiographic Suzuki stages of MMA, and may provide results to further ascertain hemodynamic changes related to the disappearance of the bilateral anterior circulation. The research group stated that the MBFV in the ACA, terminal ICA, and the MCA showed a non-linear increase up to Suzuki stage III, and subsequently progressively decreased as far as Suzuki stage VI. Moreover, the ophthalmic artery showed non-linear changes of blood flow velocity, with an MBFV increase as far as Suzuki stage IV, followed by an MBFV decrease as far as Suzuki stage VI. The MBFV of the basilar artery showed a linear increase from a normal velocity at an early MMA stage to a stenotic velocity at a late MMA stage. No statistically significant regression model for the relationship between the angiographic Suzuki stage of MMA and the MBFV in the PCA was evident (63). In their 2011 study in 292 MMA or MMS patients, Lee et al. stated that, in response to superficial temporal artery (STA)-middle cerebral artery (MCA) bypass surgery, flow rates at the vascular anastomosis increased 5 fold to a mean of 22.2 ± 0.8 mL/min. In comparison to adult MMA or MMS patients (23.9 ± 1.0 mL/min; P < 0.0001), MCA flow rates were significantly decreased in pediatric MMA or MMS patients (16.2 ± 1.3 mL/min) (64). The research group hypothesized that increased local flow rates may be related to improvement of clinical symptoms. Persistent post-operative complications were low (<5%) (64). Also, the group suggested that eminently increased post-operative MCA flow rates, in comparison to controls, may be related to transient neurologic deficits (28.6 ± 5.6 mL/min; P = 0.047), hemorrhage (32.1 ± 10.2 mL/min; P = 0.045), and post-operative stroke (31.2 ± 6.8 mL/min; P = 0.045) (64). In their 2013 study in 13 MMA patients and 10 healthy, age-matched controls, Chen et al. ascertained the beginning of dynamic cerebral autoregulation impairment at an early MMA stage (65). Every autoregulatory parameter correlated well with the angiographic MMA stage (65). The research group suggested that cerebral autoregulation impairment may progress with MMA progression toward complete vascular occlusion (65). Due to an increased risk of intracranial hemorrhage and ischemia, blood pressure intervention may be warranted (65-67). In 2013, Schubert et al. referred to a characteristic proximal pattern of collaterals (68). In 2015, Baltsavias et al. stated that the previously imprecisely described "moyamoya abnormal network" in pediatric MMA may be specified as a composition of four anastomotic networks with a readily distinguishable vascular structure (69). Accordingly, in their 2015 retrospective study in newly diagnosed 14 pediatric MMA and 11 pediatric MMS patients, Baltsavias et al. described four types of anastomotic networks in pediatric MMA, two deepparenchymal networks and two superficial-meningeal networks (69). As deep-parenchymal networks the research group detailed the previously undescribed subependymal network and the inner striatal and inner thalamic networks. The subependymal network may be fed by the intraventricular branches of the choroidal system and diencephalic perforators, which, at the level of the periventricular subependymal zone, anastomose with medullary-cortical arteries and also with striatal arteries (69). The inner striatal and thalamic networks may be comprised of intrastriatal connections among striatal arteries and intrathalamic connections among thalamic arteries when MMA compromises the origin of one or additional of their supply sources (69). As superficial-meningeal networks, the research group specified the leptomeningeal and the durocortical networks (69). Apart from the previously described leptomeningeal network observed in the convexial watershed zones, the group described the basal temporo-orbitofrontal leptomeningeal network. The second superficial-meningeal network was detailed as the durocortical network, with a calvarian or a basal location (69). In their 2015 study, Karunanithi et al., using computational fluid dynamics (CFD), evaluated 8 adult hemorrhagic MMA patients treated with encephaloduroarteriosynangiosis (EDAS) revascularization surgery, through analysis of pressure reduction in the right and left ICA before and after EDAS surgery, to ascertain how hemodynamic parameters including pressure reduction and flow rates may be the decisive factor for treatment outcome. The research group stated that pressure drop indicator (PDI), defined as the difference in pressure reduction in the ICA bilaterally, which, by use of patient-specific inflow rates, may be calculated post-operatively and at follow-up, may assist clinicians in reliable risk stratification of MMA patients regarding long-term follow-up (70). Also, PDI may further elucidate the hemodynamic mechanism associated with intracranial hemorrhage in MMA, including recurrent hemorrhage (70). In their 2016 retrospective, 1:2 matched case-control study in 180 MMA patients with or without Type 2 diabetes mellitus (T2DM), Ren et al. suggested that EDAS surgery may be an effective treatment for adult MMA, stating that T2DM patients may gain improvement of symptomatology as well as a more favorable collateral circulation post-operatively. Whereas T2DM was related to a favorable clinical outcome, PCA involvement and late post-operative stroke were identified as predictors of an unfavorable clinical outcome in both study groups (71). In 2016, Story et al. performed a study consisting of a single-institution case series of 204 MMA patients, with an average age at surgery of 9.5 years, who underwent pial synangiosis between 2005 and 2013. Transdural collaterals were present in almost half of all pre-operative arteriograms in MMA patients. These collaterals were demonstrated to be more common in advanced MMA, are associated with stroke as a perioperative complication, and may suggest an increased capacity to produce surgical collaterals post-operatively. Consequently, the research group supports the utility of pre-operative arteriography  (100). In their 2019 retrospective study, Yu et al. stated that, in comparison to patients with acute idiopathic primary intraventricular hemorrhage (PIVH), patients with acute MMA-associated PIVH may exhibit a lower short-term mortality, be of a younger age, may have a more favorable renal function, and a lower admission blood pressure (101). In their 2020 study, Zhang et al. compared the five-year prognosis in 123 adult hemorrhagic MMA patients who underwent either combined superficial temporal artery to middle cerebral artery (STA-MCA) bypass and EDAS, or EDAS alone. The research group stated that both combined revascularization and EDAS alone may reduce the risk of recurrent hemorrhage in hemorrhagic MMA patients (102). Combined revascularization was found to be superior to EDAS alone regarding the prevention of recurrent hemorrhage (102). In Kaplan-Meier survival analysis, combined revascularization was demonstrated to have a more favorable prognosis compared to EDAS alone, and multivariate regression analysis demonstrated that the combined revascularization procedure may be related to a more favorable outcome (102

Pathophysiologic characteristics of inflammation in pediatric ischemic stroke
The significance of inflammation in pediatric stroke has become noticeably evident (47). Ischemia may trigger various cascades of inflammatory reactions, both alleviating and aggravating ischemia, including inhibition and activation of inflammation through chemokines, proteases, adhesion molecules, and cytokines (47,48). Furthermore, it has been demonstrated that the pathophysiology of pediatric stroke may be associated with inflammation (47), as evident in transient cerebral arteriopathy (47,49) and post-varicella angiopathy (47,50). Consequently, in pediatric stroke, ischemia may cause inflammation, and inflammation may equally lead to ischemia (47). In comparison to the adult brain, significant differences are evident in the neonatal brain (47). In neonatal stroke, ischemia may be the predominant pathophysiologic mechanism, with inflammation and infection having a significant effect on the degree and course of tissue damage (47). In childhood, ischemia may be caused by an associated inflammatory pathophysiologic mechanism, as evident in MMA, sickle cell anemia, dissection, transient focal arteriopathy, and, increasingly generalized, in generalized vasculitis, meningitides, and genetic arteriopathies such as Deficiency of Adenosine deaminase 2 (DADA2) (47). Focal inflammation is prone to be located in the distal ICA or the proximal medial cerebral artery (MCA), whereas generalized inflammation predominantly affects small arteries (47).
Various genes may be associated with MMA (47,51). Whether these genes are dysfunctional due to ischemia or inflammation or whether these genes are dysfunctional as such remains to be elucidated (47,51). The Ring finger protein 213 (RNF213) (17q25.3) genetic variant has been demonstrated to be expressed at an increased level in mature lymphocytes in comparison to lymphoid progenitor cells (47,51). In MMA, blood levels of circulating endothelial progenitor cells (EPC) may be increased, suggesting that the RNF213 genetic variant may alter the function of EPCs in the spleen (47,51). C3, IgG, and IgM have been demonstrated in the vascular wall of MMA patients (47,52). Fujimura et al. (47,104) and Young et al. (5,47) have reviewed signaling cascades and the histology associated with MMA, suggesting an increase in transforming growth factor (TGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in MMA patients (47). These growth factors may be related to angiogenesis and inflammation (47). Extracellular inflammatory biomarkers including matrix metalloproteinase (MMP)-9, interleukin (IL)-8, and prostaglandin may be increased in MMA patients (47). If disease progression may be affected through stimulation or blockade of particular sequences of a signaling cascade remains to be ascertained (47). Blockade of several of such elements may reduce perioperative surgical risk (47,53).
Cerebral ischemia may initiate an inflammatory signaling cascade causing cell death, which subsequently may initiate inflammation (47,48). Hypoperfusion may initiate anaerobic glycolysis which may catalyze two main metabolic pathways causing inflammation: sodium-potassium pump failure may cause excitotoxic glutamate release and membrane destabilization. Activation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate and N-methyl-D-aspartate receptors or signaling pathways may lead to both necrosis, and, through intracellular increase of sodium and potassium, to inflammation, oxidative stress, and mitochondrial failure (47,48). Through blood-brain barrier (BBB) disturbance and membrane degradation, anaerobic glycolysis may lead to inflammation, cyclooxygenase (COX) activation, leukocyte infiltration, and cell adhesion molecule expression. Inflammation may cause both necrosis and apoptosis (47,48). Inflammation may cause the release of various distinct proteases, chemokines, cytokines, and adhesion molecules which may affect the inflammation process (47,48). Inflammation may as well be related to coagulation, leading to a procoagulant state through its impact on fibrin formation (47,48). Also, endothelial inflammation may affect coagulation and lead to blood clot formation (47,48). The postischemic inflammation pathway in the adult is complex, yet increasingly ascertained (47). An extensive network of anti-inflammatory and pro-inflammatory chemokines, proteases, adhesion molecules, and cytokines exists (47,48). Initial substrate release predominantly stimulates inflammation within the initial hours and minutes (47,48). Subsequently, predominantly anti-inflammatory substrates, leading to angiogenesis and recovery, are released (47,48). Post-ischemic necrotic neurons may release damage associated molecular patterns (DAMPs), thus activating macrophages (47,48). Macrophages are associated with proinflammatory cytokine release, including IL-1β and TNF-α, which may induce inflammation (47,48). Also, macrophage IL-23 release may cause T-cell recruitment which, through IL-17 release, may induce inflammation (47,48). Such an inflammatory reaction may be induced within hours and minutes after ischemia onset (47,48). Over weeks and days after ischemia, immune cells are associated with anti-inflammatory substance production, such as TGF, insulin-like growth factor (IGF), and IL-20 (47,48). Purine release may assist in cleaning necrotic cells of debris, and VEGF release may lead to angiogenesis (47,48). Secretion of anti-inflammatory and inflammatory biomarkers, such as proteases, chemokines, and cytokines, may be ascertainable in the cerebrospinal fluid (CSF) during acute stroke (47,54,55). These biomarkers may be related to stroke severity and stroke subtypes, and may further elucidate stroke pathogenesis (47,54). Various research has been designed for further ascertainment of these distinctive signaling cascades, aiming at modifying factors relating to the post-ischemic disease process (47). Comparison of adult rodents to preterm and/or neonatal rodents demonstrated that, while signaling cascades may be similar, there may be differences between the adult system and the immature prenatal system (47). Various experimental models for age-related ischemia may be warranted to further ascertain these signaling cascades in addition to encourage research into interventions to improve patient outcome (47). The pathophysiology of pediatric stroke may be caused through inflammation, which may exert a specific effect on the inflammatory signaling cascade related to ischemia (47,56

Physiologic characteristics of angiogenesis, arteriogenesis, and vasculogenesis
Moyamoya vessels suggest that aberrant angiogenic, arteriogenic, and vasculogenic processes may be involved in the pathophysiology of the arteriopathy (105).
Physiologic angiogenesis comprises six steps.
Step three comprises lumen formation. Endothelial cell thinning or intercalation and fusion of pre-existing vessels may increase vessel diameter and length (13). VEGF121, VEGF165 and their receptors increase lumen formation and vessel length. VEGF189 decreases the luminal diameter, VEGF in combination with ANG1 increases the luminal diameter (13). αvβ3 or α5 integrins influence lumen formation. Thrombospondin (TSP)-1 inhibits lumen formation (13).
Step five comprises endothelial differentiation. Specialized endothelial cells are partly determined by their host tissue. Interaction between VEGF and the ECM, causes endothelial cells to become discontinuous and fenestrated (13).
Physiologic arteriogenesis comprises three steps. Regarding step one, in SMC migration and growth, aberrant arteriogenesis causes enlargement of preexisting collaterals after occlusion of the supplying artery. Consequently, endothelial cells express MCP-1 as well as intercellular adhesion molecule 1 (ICAM-1). Vascular wall infiltration and media disruption by monocytes is associated with TNF-α and proteases. Subsequently, endothelial cells upregulate PDGF-BB, bFGF, and TGF-β1, thus inducing SMC growth and vessel enlargement (13). In step two, a lack of fibrillin-1, fibrillin-2, collagen and elastin causes vessel wall weakening and aneurysmal dilatation. Elastin decreases SMC growth, and thereby prevents intimal hyperplasia (13,107). In atherosclerosis or restenosis SMCs dedifferentiate from a contractile to an embryonic, synthetic phenotype (13). Regarding step three, in remodeling a sustained imbalance between NO and endothelin-1 may induce vasospasms and progress to vascular loss (13).

Physiologic characteristics of angiogenesis signaling pathways
Signaling pathways associated with a condition may provide an interface of genetic and environmental interaction. Integration and crosstalk between signaling pathways may occur at intracellular nodes where signaling cascades intersect (15), and also at the level of receptor activation (20). ANG1-Tie receptor tyrosine kinases (Tie)2 binding leads to crossphosphorylation of cytoplasmic Tie2 tyrosine residues, which recruit adaptor proteins that activate PI3K/Akt, MAPK, Erk, and docking protein 2 (Dok-R) signaling pathways. These pathways are involved in recruiting and sustaining periendothelial support cells (e.g., pericytes, SMCs) that relate to stabilization and maturation of newly formed vessels (18,19). In quiescent vessels, ANG1 recruits Tie2 to cell-cell contacts, forming complexes with Tie2 from adjoining cells, thus activating PI3K/Akt signaling (16,18,19,23). Migrating endothelial cells cause ANG1 to recruit Tie2 into contact with the ECM, which causes the formation of focal adhesion complexes and activation of PTK2/FAK, MAPK-1/ERK2, and MAPK3/ERK1; this, in turn, causes sprouting angiogenesis (16)(17)(18)(19)(20)(21)(22). Activation of Tie1-Tie2 heterodimers depends on β1 integrin (21). In ischemia, ANG1 causes synchronous activation of Tie2 and integrin signaling, which is related to angiogenic remodeling and tightening of endothelial cell junctions ( Figure 5) (18). Tie1 deficiency impairs ANG1-induced Tie2 and Akt phosphorylation and FOXO1 inactivation, leading to FOXO1 nuclear translocation (21). Inflammation causes cleavage of the Tie1 ectodomain, which results in a switch of ANG2 from a Tie2 agonist to a Tie2 antagonist, linked to a positive feedback loop of FOXO1-driven ANG2 expression, causing endothelial cell destabilization via β1 integrin, vascular remodeling, and leakage . /fneur. . (21, 24, 25). Autocrine secretion of ANG2 disrupts connections between endothelium and perivascular cells, causing cell death and vascular regression (16) that lead to impaired barrier properties of brain endothelial cells and intracranial hemorrhage and ischemia (16,26). ANG2 and VEGF block ANG1-mediated stabilization and maturation, resulting in endothelial cell migration and proliferation and, then, angiogenic neovascularization (18). ANG1-Tie2 activation stimulates recruitment of ABIN-2 that, in turn, creates suppression of NF-κB, a pro-inflammatory transcription factor, and protection of endothelial cells from apoptosis. Truncated ABIN-2 inhibits ANG1 from preventing endothelial cell death (27). The erythropoietin (Epo)/Epo receptor (EpoR) signaling pathway induces proliferation, migration, chemotaxis, and angiogenesis, and inhibits apoptosis (28,29). EPO signaling potentially inhibits apoptotic pathways triggered by ischemia and may reduce hypoxic injury by promoting or facilitating angiogenesis (28). Cytokines and growth factors associated with hematopoiesis may also be involved in angiogenesis (31). Endothelial cells expressing EPO-R respond to EPO by differentiation into vascular structures, associated with JAK2 phosphorylation, cell proliferation, and MMP-2 production (30, 31).
The erythropoietin-producing human hepatocellular receptor (Eph)/Eph receptor-interacting protein (ephrin) signaling pathway is involved in vasculogenesis and tissue homeostasis (32,35). Ephephrin bidirectional signaling affects both receptor-and ephrinexpressing cells and segregates Eph-expressing cells from ephrinexpressing cells (33-35). Eph and ephrin may contribute to vascular development by restricting arterial and venous endothelial mixing thus stimulating the production of capillary sprouts and also by stimulating mesenchymal differentiation into perivascular support cells (32). EphA receptor activation may be involved in VEGFinduced angiogenesis (32). Cooperation between ephrin-A1 and Slit2 regulates a balance between pro-and antiangiogenic functions of Slit2, suggesting Slit2 may differentially regulate angiogenesis in the context of ephrin-A1 (36). The Eph family transmembrane ligand ephrin-B2 marks arterial but not venous endothelial cells. The ephrin-B2 receptor Eph-B4 marks veins but not arteries. Differences between arteries and veins may be in part genetically determined, suggesting that reciprocal signaling between arterial and venous endothelial cells is essential for morphogenesis of the capillary beds (37). Interaction of ephrin-B2 on arterial endothelial cells and Eph-B3 and Eph-B4 on venous endothelial cells may define the boundary between arterial and venous domains (37). EphB2 and ephrin-B2 expression on mesenchymal cells suggests involvement in vessel wall development via endothelial-mesenchymal interaction (38). Absent ephrin-B2 expression in mice disrupts embryonic development of the vasculature due to a deficient restructuring of the primary network (39).
The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) signaling pathway includes cytoplasmic signal transducer and activator of transcription (STAT)1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. STATs are activated by tyrosine phosphorylation in response to external stimuli, including cytokines, growth factors, and hormones. Ischemia leads to estradiol-, IL-6-, EPO-, and G-CSF-mediated tyrosine phosphorylation and activation of STAT3. STAT3 dimerization and translocation to the nucleus stimulate binding of DNA regions in STAT-inducible elements, which leads to transcription of neuroprotective genes linked to estradiolmediated neuroprotection and neuronal survival. Endothelial STAT3 activation causes endothelial cell migration and proliferation, leading to angiogenesis and ECM remodeling that are important in long-term post-stroke recovery (40)(41)(42).

Moyamoya angiopathy related angiogenesis and inflammation signaling pathways
The pathogenesis of MMA and MMS may be associated with infection and inflammation (5,108). Imbalance of angiogenic and vasculogenic mechanisms has been suggested to be a potential cause of MMA (105). Aberrant expression of angiogenic factors, adhesion molecules, and mitogens, and/or an aberration of the cellular immune response to cytokines and growth factors may indicate an association of hematopoietic as well as inflammatory signaling pathways with cells of the vasculature, which has been hypothesized to constitute an essential pathophysiologic mechanism in MMA pathogenesis ( Figure 4) (5,(109)(110)(111)(112)(113).
/fneur. . of endothelial cells activates the PI3K pathway and leads to cell migration. Endothelial activation of Akt1 is associated with structurally abnormal blood vessels. PI3K/Akt/mTOR pathway inhibition decreases VEGF and angiogenesis (114). Increases in caveolin-1 lead to decreases in ceramide synthesis, inhibiting the Akt signaling pathway, cell proliferation, migration, and invasion, thus inhibiting PI3K activity (116). Hypoxia-inducible factor (HIF)-1 expression is associated with the PI3K/Akt signaling pathway in MMA (9,117). The PI3K/Akt signaling pathway in endothelial cells may lead to transcriptional activation of Ring finger protein 213 (RNF213) (17q25.3). Inhibition of the PI3K/Akt signaling pathway has been demonstrated to decrease inflammation in autoimmune diseases (9,118). MMA, if associated with inflammation, may be related to the PI3K/Akt pathway (9 125). S-nitrosylation means posttranslational modification through addition of a nitrosyl group to the reactive thiol group of cysteine, forming S-nitrosothiol, which constitutes a pivotal mechanism in NO-mediated signal transfer (119). Ubiquitin ligase S-nitrosylation may lead to its auto-ubiquitination, thus increasing its substrate levels. NFAT, through cGMP-dependent protein kinase (PKG) activation, which leads to GSK-3β phosphorylation, may be regulated through sGC (119, 126). Furthermore, caveolin may be associated with NO signaling regulation (119). Caveolin-1, an ∼ 21-24 kDa integral membrane protein, is present predominantly in plasma membrane caveolae, 50-100-nm flask-shaped invaginations, where it functions as a scaffold to arrange a multitude of molecular complexes which regulate diverse cellular functions. Caveolin-1 may be regulated through the Ca2 + /calcineurin/NFAT signaling cascade (119, 127). Caveolin-1 has been stated to be related to pulmonary arterial hypertension, coronary artery disease, and MMA (119). Caveolin-1 functioning may be sufficiently studied in pulmonary arterial hypertension (119, 128-130). In comparison to healthy controls or cerebral atherosclerotic stroke patients, caveolin-1 levels have been demonstrated to be decreased in MMA, and were shown to be distinctly decreased in patients with the RNF213 R4810K genetic variant (119, 131). If RNF213 bears an indirect or a direct relation to caveolin-1, remains to be ascertained, e.g., caveolin-1 may be a target object for ubiquitination through RNF213. eNOS release is related to caveolin-1. eNOS release produces NO, which may be metabolized through sGC. eNOS binding to the caveolin-1 scaffolding domain has been associated with eNOS inactivation (119, 132). Caveolin-1 absence may lead to dysfunction of eNOS, which has been related to cerebrovascular diseases (119). NF-κB and HIF-1 are involved in inflammation regulation (9,133). RNF213 genetic variants may cause NF-κB-associated inflammation, leading to VSMC damage, which is characteristic of MMA pathophysiology (119). RNF213 may lead to lipotoxicity-mediated protection of cells against inflammation and endoplasmic reticulum (ER) stress (119, 134). RNF213 depletion may cause NF-κB pathway inhibition during exposure to palmitate, may reestablish the cellular lipidome, and may stabilize the expression of the ER stress gene (119, 134). Recent research has demonstrated that RNF213 may concur with Ubc13, the E2 enzyme, leading to K63-linked polyubiquitin chain generation (119, 135,136). K63 linkages have been associated with protein sorting, removal of defective mitochondria, innate immune responses, DNA repair, and with regulation of NF-κB transcription factor activation (119, 137). Deletion of Lys-63-specific deubiquitinase BRCC36 (BRCC3), an E3 ligase cleaving K63-linked polyubiquitin chains specifically, has been related to X-linked MMS (119, 138). BRCC3 may be associated with DNA damage response, and may regulate an abundance of such polyubiquitin chains in chromatin (119). The majority of genetic changes in the RNF213 RING finger domain proven in MMA patients may diminish E3 ligase activity, and various of these genetic changes may induce NF-κB activation (119, 136). Such genetic changes, which may lead to NF-κB activation, may include both Caucasian cysteine/histidine mutations and proline mutations, such as P4033L in a Caucasian patient as well as p.P4007R in a Chinese patient (119). In association with NF-κB, these genetic changes may lead to apoptosis (119). The p.D4013N genetic variant may neither affect NF-κB activation nor E3 ligase activity (119). Point mutations in both the Walker B and A motif of the AAA domains, may fully eliminate NF-κB activation through genetic changes in the RNF213 RING finger domain (119). Consequently, NF-κB signaling pathwaymediated inflammation may be suppressed in absence of RNF213, while inflammation may be augmented through RNF213 genetic variants in MMA patients (119). With respect to NF-κB activation, RNF213 genetic variants may be associated with gain of function (119). RNF213 may as well regulate immune cell maturation and differentiation, the cell cycle, and mitochondrial function. These characteristics may be associated with MMA pathogenesis (119). Caveolin-1 is related to inflammation, and may be associated with MMA (9,139,140). Caveolin-1 serum levels were shown to be decreased in MMA, and demonstrated to be significantly decreased in MMA patients with the RNF213 genetic variant (9, 131). Caveolin-1 has been associated with angiogenesis (9, 141, 142), along with a bidirectional interaction between the Caveolin-1/ERK and the Wnt/β-catenin pathway (9,143).
In 2000, Galbiati et al. hypothesized that caveolin-1 expression may control Wnt/β-catenin/Lef-1 signaling through modulating the intracellular β-catenin localization (144). The Wnt signaling pathway may be related to angiogenesis (9). In 2016, Scholz et al. demonstrated that the endothelial RSPO3-driven non-canonical Wnt/Ca2 + /NFAT signaling pathway may be associated with vascular stability maintenance, providing insight into vascular remodeling mechanisms (120). Furthermore, the research group stated that RNF213 in vascular endothelial cells may be associated with the Wnt signaling pathway and angiogenesis regulation (9,120). Under physiologic conditions, stimulation of endothelial cells through shear stress or growth factors may induce the Ca2 + -calmodulin  (10). Various signaling pathways related to smooth muscle contraction, vasculogenesis, and immune response may be associated with the regulatory mechanism of lncRNAs (10). Mitogen-activated protein kinase (MAPK) signaling pathway was found to have a central function in this regulatory network of signaling pathways (10). In 2021, Sarkar and Thirumurugan demonstrated the regulation of RNF213 through the TNFα/PTP1B signaling pathway and PPARγ, suggesting that RNF213, similar to TNFα, may constitute an additional connection between MMA, inflammation, insulin resistance, and obesity (11). Toll-like receptors (TLR) have been ascertained to be essential in activating the innate immunity through recognition of distinct patterns of microbial constituents. Toll-interleukin-1 receptor (TIR) homology domaincontaining adapter protein Myeloid differentiation primary response 88 (MYD88) may be indispensable for the induction of proinflammatory cytokines induced by all TLRs (145). In 2020, Key et al. stated that in MMA, the low penetrance of RNF213 mutations may be modified through dysfunctions in the TLR3 signaling pathway or the mitochondria (Figure 4) (146). Due to infections or autoimmune diseases and induced by inflammatory cytokines, every signal transduction pathway involved in MMA may be reciprocally activated by RNF213 (9).

Moyamoya angiopathy cell-based biomarkers
Derived from the bone marrow, circulating endothelial progenitor cells (EPC) are involved in postnatal physiological and pathological neovascularization (9,147,148). Circulating EPCs have become objects of moyamoya research, referring to the hypothesis that MMA is associated with constant vascular remodeling, involving both the subsequent angiogenesis from collateral development as well as the primary arteriopathy. SMC proliferation in the vascular wall of affected arteries has frequently been demonstrated in MMA (2,61). Analysis of smooth muscle progenitor cells (SPCs) isolated from the blood of MMA patients demonstrated a differential expression exceeding 200 genes, including a decreased CD31 expression, and irregular tube formation in assays in comparison to matched controls (2,149). Studies have indicated the migration of endothelial cells into the ICA intima in stenotic sections in moyamoya, hypothesizing that these cells might be involved in both distal collateral development and proximal arterial narrowing (2,150). CD34+ cells, a subpopulation of endothelial progenitor cells, have been reported to be increased in the blood of MMA patients compared to healthy controls and also when compared to patients with non-MMA intracranial arterial stenosis (2,151,152). Inconsistent results have been obtained from research into CD34+ cells in pediatric MMA. Kim et al. performed a study in 28 pediatric MMA patients, demonstrating decreased levels as well as a defective function of CD34+ cells compared to 12 healthy volunteers (2,9,153). Rafat et al. performed a study in 20 adult MMA patients, demonstrating an enhancement of circulating EPCs. The research group suggested an involvement of circulating EPCs in angiogenesis and arteriogenesis in MMA (9,154). A decrease in EPCs following revascularization surgery in MMA has also been reported (9,155). EPCs secrete angiogenetic factors including ANG1, hepatocyte growth factor (HGF), VEGF, stromal-derived factor-1a (SDF-1a), bFGF, PDGF, and IGF-1 (9,152,154,(156)(157)(158). Tinelli et al. morphologically, phenotypically, and functionally characterized circulating EPCs from the peripheral blood of a homogeneous group of adult Caucasian, non-operated MMA patients and healthy controls, suggesting that a significantly reduced circulating EPC level may be a potential marker of MMA (105). Analyzing the function of circulating EPCs in vitro, as measured by assays of colony formation and tube formation, may indicate a significantly decreased function of these cells in MMA (2,9,159). Choi et al. suggested an impaired functional recovery of EPCs in vivo in moyamoya patients in comparison to controls (9, 160). In 2008, Jung et al. stated that distinct characteristics of circulating EPCs (CFU numbers and tube formation were found to be lower in advanced MMA cases than in those with early stage disease, and outgrowth cells were more frequently detected in those with early MMA and moyamoya vessels than in those with advanced MMA) may reflect mixed conditions of aberrant vasculogenesis and vasculars occlusion in MMA pathogenesis (159). Regarding their 2008 study results, Yoshihara et al. suggested that an increased level of CD34+ cells, related to ischemia, may be correlated with neovascularization of the human arterial cerebral circulation at sites of ischemic brain injury (151). In their 2010 study, Kim et

Moyamoya angiopathy molecular biomarkers
Treatment of an underlying inflammatory disease may lead to remission of MMA symptomatology (9). An immune response related to angiogenesis may be facilitated through M2 macrophages which may be induced through anti-inflammatory cytokines such as TGF-β, interferon (IFN)-α, IL-13, IL-10, and IL-4. Fujimura et al. demonstrated that CXCL5 and CD163 serum levels of MMA patients were significantly increased compared to controls, hypothesizing that MMA pathophysiology may be related to M2 macrophages (9,174). Anti-inflammatory cytokines may induce angiogenetic markers. TGF-β of Treg/Th17 cells with distinct CD4+ T-helper cell subsets has been demonstrated to be associated with aberrant angiogenesis in MMA by means of VEGF signaling regulation (9,175). Increase in angiogenetic markers including VEGF, HGF, PDGF, bFGF, cellular retinoic acid-binding protein-1 (CRABP-1), HIF-1 and MMPs may be associated with MMA ( Figure 4) (9,109,154,(176)(177)(178)(179)(180)(181)(182)(183)(184)(185). These markers have been hypothesized to be associated with proliferation of the intima as well as angiogenesis through their influence on endothelial cells, and with progression or initiation of MMA (9). Pro-inflammatory cytokines, including IL-6, IL-1, TNF-α, IFN-γ, and IFN-β, which may induce the pro-inflammatory, RNF213dependent cytokine pathway, may have a different mechanism of action compared to cytokines involved in anti-inflammatory cytokine pathways (9).  (190). In their 2018 prospective study in 11 MMA patients, Ishii et al. observed changes in biomarkers associated with tight junctions in the blood-brain barrier (BBB). The research group stated that their preliminary results may indicate that significant hemodynamic change and transient neurologic symptoms (TNS) in some patients may be related to BBB disruption after direct MMA bypass surgery (191). In 2018, Yokoyama et al. demonstrated that CSF proenkephalin 143-183 may be a useful diagnostic biomarker in pediatric MMA. The effect of enkephalin peptides by means of delta opioid receptor or opioid growth factor receptor may be related to MMA pathophysiology, suggesting an association between temporal changes in moyamoya collateral vessels and concentration of proenkephalin (192). In 2020, Surmak et al. showed that a [ 11 C]-PiB PET signal related to intracranial inflammation in MMA patients and a single relapsing-remitting multiple sclerosis (RRMS) patient may be corresponding to functional cerebral imaging of SULT1E1, suggesting that significant focal [ 11 C]-PiB PET signals may be received from the inflamed living human brain (193). In 2021, Han et al. suggested that elevated CSF and serum sortilin levels may be associated with MMA onset, and, in addition to levels of proinflammatory cytokines, may be effective markers in clinical practice. The research group hypothesized, that sortilin may break through a compromised blood brain barrier (BBB), may consecutively induce inflammation, and thus induce MMA (194). In their 2021 study, Ren et al. demonstrated that cortical astrocytic neogenin (NEO1) deficiency may be associated with MMA pathogenesis. NEO1, a member of deleted in colorectal cancer (DCC) family netrin receptors, was reduced in brain specimens of MMA patients. Astrocytic Neo1-loss resulted in an increase of small blood vessels, selectively in the cortex. These blood vessels were dysfunctional, with a leaky blood-brain barrier (BBB), thin arteries, and accelerated hyperplasia in veins and capillaries, resembling the symptomatology of a moyamoya disease-like vasculopathy. Additionally, the research group found that both MMA patients and Neo1 mutant mice exhibited altered gene expression in the cerebral cortex in proteins critical for both angiogenesis [e.g., an increase in vascular endothelial growth factor A (VEGFA)], and axon guidance (e.g., netrin family proteins) and inflammation. In aggregates, these results suggest a critical role of astrocytic NEO1loss in the development of a moyamoya disease-like vasculopathy, providing a mouse model for investigating mechanisms of a moyamoya disease-like vasculopathy (195). In 2021, Sesen et al. described urinary biomarkers that may identify MMA presence to a high degree of accuracy and sensitivity. These markers may be detected from the CNS to the urine, and may correlate with response to treatment, such as radiographic verification of revascularization. Urinary MMP-2 showed an accuracy of 91.3%, a specificity of 100%, and a sensitivity of 87.5%. The research group hypothesized that urinary proteins may constitute a new, non-invasive device which may assist in treatment, follow-up, prognosis, and diagnosis of MMA (196). In their 2021 study, Dei Cas et al. carried out a complete lipidomic analysis of MMA patient plasma through mass spectrometry and measured inflammatory and angiogenic protein levels through enzyme-linked immunosorbent assay (ELISA). ELISA showed an MMP-9 decrease in MMA patient plasma. Lipidomic analysis demonstrated a cumulative depletion of lipid asset in MMA patient plasma in comparison to healthy controls. The research group noted a decrease in peripherally circulating membrane complex glycosphingolipids, observed in MMA patient plasma, compared to healthy controls, indicative of cerebral cellular recruitment. This quantitative targeted approach showed increased free sphingoid .
bases, which may be related to aberrant angiogenesis. The results of the group may suggest that the lipid signature/plasma lipid profile of MMA patients may be closely associated with the condition and that a comprehensive biomarker profile may help to further elucidate the complexity of MMA pathogenesis (197). In their 2021 study, Lu et al., using plasma samples from 84 MMA patients, demonstrated that MMP-9 may serve as a biomarker for prediction of intracranial hemorrhage in MMA. The research group showed that a serum MMP-9 level >1,011 ng/ml may be an independent risk factor for hemorrhagic stroke in MMA. Also, the research group demonstrated that adult MMA patients, in comparison to pediatric MMA patients, showed an increased blood-brain barrier (BBB) permeability and MMP-9 serum level elevation. Furthermore, the group demonstrated that hemorrhagic MMA patients, in comparison to ischemic MMA patients, showed an increased BBB permeability and an elevated MMP-9 serum level ( Figure 4) (198).  (9). Moreover, the research group hypothesized that additional factors, including immune response and inflammation, may be associated with MMA onset (9,201). The frequency of the p.R4810K genetic variant has been demonstrated to be significantly increased in MMS compared to controls (9,202,203), suggesting an involvement of the RNF213 p.R4810K genetic variant in MMS. In contrast, in 2015, Miyawaki et al., based on their results from a small sample size, stated that the RNF213 c.14576G>A genetic variant may not be related to MMS (9,204). Pro-inflammatory cytokines such as IFN-γ, IFN-β, and TNF-α may synergistically activate RNF213 transcription both in vivo and in vitro (9,124,205). Pro-inflammatory cytokines may decrease angiogenic activity through RNF213 induction (Figure 4) 3p25.2). These genes may be related to signaling pathways that are associated with inflammation as well as angiogenesis (5,9). Changes in protein folding and gene transcription may be associated with aberrant expression of ICAM-1, VCAM-1 and E-selectin, induced through pro-inflammatory cytokines TNF-α and IL-1β, by means of NF-κB activation (5,207). A variety of genes involved in MMA may be associated with inflammation. Whether these genes are causative factors of MMA or a result of MMA pathogenesis, remains to be elucidated (4,5,119,205,(208)(209)(210)

Moyamoya angiopathy non-coding ribonucleic acids
High-throughput sequencing has established a large quantity of distinct ribonucleic acids (RNAs) created from non-coding DNA (245,246). Similar to protein-coding RNAs, non-coding RNAs appear to be linear molecules with 3 ′ and 5 ′ termini, which constitute defined end and start points of the RNA polymerase on the DNA template (245). Non-coding RNAs vary in length (245). Long non-coding RNAs (lncRNAs) exceed 200 nucleotides, lack protein-coding capacity, and are associated with post-transcriptional .
processing, transcriptional control, and chromatin remodeling (9,247,248). Regulation of lncRNAs may be associated with inflammation (9,249,250). Moreover, lncRNAs may be related to MMA pathophysiology by means of an inflammatory signaling cascade comprising the MAPK signaling pathway (9,10,251). In their 2017 study, Wang et al. demonstrated that an integrated analysis of lncRNA-mRNA coexpression networks may be associated with the MAPK signaling pathway, the Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction, and inflammation. The research group indicated that differentially expressed genes may help to ascertain crucial components in MMA pathophysiology (251). In their 2020 study, Gu et  Gene Ontology (GO) analyses of potential proteincoding genes, the transcription of which may be regulated in cis through ascertained differentially expressed lncRNAs, indicated an association with branching related to blood vessel morphogenesis, positive regulation of cytokine production, the T-cell receptor signaling pathway, and antibacterial humoral response (254). MicroRNAs (miRNAs) are endogenous, short non-coding ∼23 nucleotide RNAs, which may regulate gene expression through pairing to the mRNAs of protein-coding genes to control their posttranscriptional repression or cleavage (9,245,255,256). miRNAs may be of vital significance regarding the control of cell aging, differentiation, survival, and proliferation (9,255). Additionally, miRNAs may be related to angiogenesis, neurogenesis, and inflammation (9,257). miRNAs may regulate TLR signaling through reduction of inflammation, enhanced tissue repair, and regaining of homeostasis following tissue injury and infection (9,258). MiR-126, miR-155, and miR-21 may be associated with inflammation and vascular disorders. To do research into the network involving miRNAs and their targets leading to a coordinated gene expression pattern may lead to results which may help establish new treatment strategies to approach both aberrant vascular remodeling and to induce neovascularization after ischemia (259). Increased expression of miRNA Let-7c and miRNA-196a2 may be used as MMA biomarkers (9,260,261). In their 2019 study, Lee et al. analyzed the impact of RNF213 mutations and MMA on the profiles of cell-free miRNA and protein in patient plasma samples. Levels of selected MMA-affected miRNAs in EV-depleted plasma, extracellular vesicles (EVs), and whole plasma have been confirmed through real-time quantitative polymerase chain reaction (qPCR). The research group showed that EV-encapsulated miRNA may be utilized as non-invasive biomarkers to evaluate MMA progression (262). The changes of proteins and miRNAs ascertained may be related to signaling processes such as immune activation and angiogenesis which may further elucidate MMA pathogenesis (262). Ischemic conditioning may be used to decrease the stroke risk in asymptomatic intracranial atherosclerotic arterial stenosis (263,264). Ischemic preconditioning involves inducing moderate ischemia to exert protective functions against following severe ischemic events. Epigenetics may be associated with the outcome and the pathophysiology of stroke. Recent research has demonstrated miRNA expression following ischemic preconditioning; miRNA profiling 3 h following ischemic preconditioning demonstrated upregulation of miRNA families miR-182 and miR-200 that have been associated with neuroprotective effects of the HIF-1 and prolyl hydroxylase 2 signaling pathways (264)(265)(266). Furthermore, ischemic preconditioning has been shown to promote anti-inflammatory mechanisms by modifying the expression of cytokines during ischemic insults, suggesting a critical role of the vasculature and endothelial cells during ischemic conditioning stimuli (264,267). Also, ischemic post-conditioning may represent a promising neuroprotective strategy in ischemic insults by means of antiinflammatory, anti-apoptotic, and CBF-based mechanisms (264,268,269). In 2014, Dai et al. using real-time PCR, identified a serum miRNA signature in MMA. The research group demonstrated in an independent MMA cohort that serum miR-125a-3p was significantly decreased, whereas serum miR-126, miR-130a, and miR-106b were significantly increased. Gene Ontology (GO) analysis demonstrated that differentially expressed serum miRNAs may be enriched in signal transduction, transcription, and metabolic processes. Pathway analysis demonstrated that the most enriched pathway may be the mTOR signaling pathway. Also, the research group demonstrated that 13 and 16 aberrant serum miRNAs coordinately inhibited BRCC3 and RNF213 protein expression at the posttranscriptional level, associated with MMA pathogenesis and aberrant angiogenesis (270). In 2015, Zhao et al. showed that increased serum miRNA let-7c expression in MMA patients may be associated with MMA pathogenesis through its influence on RNF213, suggesting that let-7c may be a potential biomarker of MMA ( Figure 4)  In 2017, Zhao et al. demonstrated that various circRNAs may be involved in MMA pathogenesis, and may be associated with modulation of the MAPK signaling pathway. Besides providing a set of potential diagnostic biomarkers for MMA, the results of the research group suggest that therapeutic strategies targeting the MAPK signaling pathway or these circRNAs may be effective MMA treatment strategies (272). Recent research may provide evidence that regulatory RNAs including miRNAs or lncRNAs may be associated with MMA pathogenesis. In comparison with other kinds of miRNA sponges, circRNAs have higher expression levels and an increased amount of binding sites and, compared to linear RNAs, are viewed as more efficient regarding gene expression regulation and sequestering miRNAs (245,273). CircRNAs have been associated with various disorders that involve various neurological disease, and are correlated with miRNA expression (273,274). In 2017, Zhao et al. demonstrated that 146 circRNAs may be expressed in MMA patients, and these circRNAs may contribute to MMA pathogenesis (272,273). Of these 146 circRNAs, 29 circRNAs were upregulated, and 117 circRNAs were downregulated (272,273). Hsa_circRNA_067130, hsa_circRNA_067209, and hsa_circRNA_062557 were upregulated, while hsa_circRNA_089763, hsa_circRNA_089761, and hsa_circRNA_100914 were downregulated with highest fold variations, providing sufficient evidence to state that these circRNAs may be potential MMA biomarkers (272,273). In their 2019 pilot study of neutrophil samples from asymptomatic MMA patients and an aberrant circRNA profile obtained through highthroughput microarray analysis, Ma et al. demonstrated a critical function of circRNAs and neutrophils in the differentiation of asymptomatic MMA patients compared to healthy controls, suggesting a relation of angiogenic and anti-inflammatory markers to asymptomatic MMA (264,275). The research group carried out Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment (KEGG) analyses to both predict functioning and for the annotation of differentially expressed circRNAs, stating that differentially expressed circRNAs may be associated with metabolism, angiogenesis and immune response in asymptomatic MMA. Also, the research group suggested an association of the HIF-1α signaling pathway with increased VEGF and angiogenesis in MMA pathogenesis (264,275). Moreover, Ma et al. suggested that anti-inflammatory mechanisms and neutrophils may be associated with MMA progression (264,275). Research results may indicate a HIF-1/VEGF mechanism associated with angiogenesis (264,267). In their 2021 study, Li et al. conducted neutrophilic tsRNA profiling in asymptomatic MMA patients and healthy controls (276). Pathophysiological mechanisms, including immune response, angiogenesis, axon guidance, and metabolism adjustment, were highlighted through differentially expressed (DE)-tsRNAs and DE-mRNA in asymptomatic MMA patients, which may support the potential receptivity of asymptomatic MMA to medical therapeutics, such as immune-modifying drugs (Figure 4) (276). disease and atherosclerosis were found to be associated with both adult and pediatric MMA. Adult-onset autoimmune disease was associated with pediatric MMA but not with adult MMA. The research group suggested that both adult and pediatric MMA may be associated with inflammation, hypothesizing that inflammation may be associated with MMA pathogenesis (287). In 2017, Uchino et al. stated that failure to notice non-focal physical symptoms, suggestive of orthostatic intolerance, including headache, motion sickness, difficulty getting out of bed, fatigue, and vertigo/dizziness, may significantly impair the quality of life in pediatric MMA patients up to 5 years after revascularization surgery, resulting in 57% of patients being unable to attend school. These symptoms, inversely associated with the number of years after surgery, may serve as independent clinical markers to monitor disease outcome (288). In  (294). Additional compensatory collaterals, including extracranial arterial collateral circulation anastomoses from the middle meningeal, maxillary and facial arteries to the ophthalmic artery, and dural arteriolar anastomoses from the occipital artery and middle meningeal artery through the parietal foramen and mastoid foramen, may as well correlate with the clinical outcome postoperatively ( Figure 1) (294, 295). Most recently, the research group observed different hemodynamic sources of the recipient parasylvian continental arteries (PSCAs) among the parietal, temporal, and frontal PSCAs in MMA hemispheres (80,294), suggesting that the recipient vessel in STA-MCA bypass surgery may not necessarily originate from the MCA (294). Consequently, neurosurgeons may be advised to rely predominantly on digital subtraction angiography (DSA) to ascertain the hemodynamic source of recipient vessels (294

Moyamoya angiopathy, moyamoya syndrome, and inflammation
Two predominant pathways have been suggested to be associated with inflammation and initiation or progression of MMA. A proinflammatory cytokine pathway, leading to RNF213 activation, as well as an anti-inflammatory cytokine pathway. The proinflammatory pathway is associated with increased inflammatory cytokines in inflammatory diseases which affect RNF213, leading to MMS onset. This hypothesis may be supported by the evidence of an increased frequency of the RNF213 p.R4810K variant in MMS. Late-onset MMA may be associated with this variant (9,(298)(299)(300)(301). Pro-inflammatory cytokines may be involved in fulminant MMA progression (9,302). This particularly applies to MMS associated with hyperthyroidism (9,(303)(304)(305)(306). The pro-inflammatory pathway may function as an initiator of MMA (9). The anti-inflammatory cytokine pathway involves anti-inflammatory mediators in the CSF or the blood that may affect acceleration or acute aggravation of MMS. Antiinflammatory cytokines may be involved in autoregulation as well as vascular reactivity, leading to MMA progression ( Figure 4) (9).
In their 2002 study, Soriano et al. using dual-antibody enzymelinked immunoassays, demonstrated increased CSF levels of soluble endothelial adhesion molecules, vascular cell adhesion molecule Type 1 (VCAM-1), intercellular adhesion molecule Type 1 (ICAM-1), and E-selectin, suggesting that pediatric MMS patients may have persistent central nervous system inflammation, with marginal blood-brain barrier (BBB) impairment. The research group suggested that these soluble adhesion molecules may be of use in clinical practice as markers of this central nervous system inflammation process. Moreover, the research group stated that their results may not completely ascertain an association of these adhesion molecules with vascular pathological processes related to MMS, since cerebral ischemia as well may lead to expression of these adhesion molecules (110  (3,312). DOCK8 deficiency may be associated with MMA (312,313), potentially due to ischemia (312). Recent reports have described an underlying autoimmune disease mechanism related to T cell dysregulation in these patients, particularly in unilateral MMA, as evident in her patient (312,314). Knowledge of therapy management and revascularization procedures for patients like the one described in this report remain to be ascertained (312).
Prescribing antiplatelet drugs to patients affected with DOCK8 deficiency should be done with caution, due to hemorrhage being an ascertained symptomatology of MMA, particularly of hemorrhagic MMA (312,315 (317). In their 2019 moyamoya multicenter study, Bonasia et al. ascertained three types of anastomoses between the anterior and posterior cerebral circulation, consisting of collaterals from the posterior choroidal arteries (20%), the posterior callosal artery (20%), in addition to a potential pio-pial anastomosis between cortical collaterals of the posterior cerebral artery (PCA) and the anterior cerebral artery (ACA) (15%), with a distinct capacity for retrograde compensation of the anterior circulation. In advanced Suzuki stages from IV to VI in particular, collaterals are frequently observed in MMA. Collaterals may develop due to their ability to compensate the leptomeningeal anastomosis, duro-pial anastomosis, and the ophthalmic-ACA anastomosis collateral systems and due to a diminished blood supply to the ACA territory. The research group suggested a 4-grade classification based on the capability of the three types of PCA-ACA anastomoses to provide retrograde supply to the ACA territory (Figures 2, 3) (Figure 4) (329).

Conclusion, treatment strategies, and future research perspectives in moyamoya angiopathy
We have reviewed the physiological and pathophysiological mechanisms of signaling pathways, cells, and genes involved in MMA and MMS and their association with aberrant angiogenesis and inflammation ( Figure 4). If mediators involved in these mechanisms are associated with signaling pathway activation or if they constitute downstream mediators remains to be elucidated (5). To do research into the effects of signaling molecules involved in MMA and the part of a signaling pathway they act, may be advocated (5). Moyamoya collateral vessel formation seems to be subsequent to ICA stenosis (5). Thus, prevention of the above-mentioned stenotic process may help avoid the subsequent formation of fragile moyamoya collaterals (5). Angiogenesis in MMA may be either decreased or facilitated (9). Research results indicate that aberrant angiogenesis, decreased or facilitated, may be associated with MMA pathogenesis. These findings seem to be validated by revascularization surgery for MMA, by which increased angiogenesis and an improved formation of a collateral circulation is achieved by restoration of blood flow to the brain (9). Despite a limitation of the number of cases involved, consensual evidence of inflammation in MMA appears to be present (5). Inflammation in pediatric stroke is critically important, both due to the inflammatory signaling cascades activated through ischemia and because of inflammatory baseline pathologies causing stroke (47). Reciprocal action of such fundamental pathophysiologic mechanisms may be of substantial importance, warranting further research (47). Focal pathophysiology may be associated with proximal vessels, such as the circulus arteriosus cerebri, the MCA (M1), ACA (A1), and the distal ICA, whereas generalized pathologies may affect small arteries or peripheral vessels (47,330). Considerable differences in inflammatory signaling cascades in the neonatal and the adult brain are evident (47). Developmental trajectories of inflammatory signaling cascades from the neonate to the adult remain to be ascertained. Therapeutic interference with such an inflammatory pathology might be feasible by means of additional studies (47). Animal models are warranted to ascertain if these findings may be involved in MMA pathogenesis (5). Regardless whether these processes may induce MMA or result from the arteriopathy, there is growing evidence of a reversible inflammatory process being present in the vascular wall which may contribute to lumen stenosis (5). Inflammation, although not a direct cause of MMA and MMS, may influence RNF213, and thus result in aberrant angiogenesis (9).

Moyamoya angiopathy treatment strategies
Enhanced interaction between neurons and cells of the vasculature, increased angiogenic activity, induced curative angiogenesis, and increased formation of a collateral circulation may be Research Topics fundamental to establishing future treatment strategies (9).
In susceptibility genes to be associated with homocysteine metabolism. Furthermore, due to enrichment of the expression of these susceptibility genes in the immune system, the research group suggested that therapeutic interventions directed at those pathways could be efficient MMA treatment approaches (208). In 2018, Ishii et al. stated that, in case the post-operative serum level of matrix metalloproteinase (MMP)-9 and Occludin (OCLN) may be significantly elevated, systolic blood pressure should be continuously controlled to avoid post-operative intracranial hemorrhage and/or epilepsy. Particularly regarding MMP-9, the administration of minocycline may be considered (191,332

Moyamoya angiopathy future research perspectives
Recent MMA research may concentrate on the three main sectors therapy, prognosis, and diagnosis (6, 337). MMA therapeutic innovation research has remained behind the significant achievements in the diagnostic and prognostic area of MMA research (6, 337). Prognosis of MMA has been advanced through non-invasive biomarkers and new imaging methodologies (2,6,64,196,290,338). MMA diagnosis has profited greatly from the latest advancements in molecular genetics, with significant progress in the identification of specific genetic variants related to clinical phenotypes and radiographic presentations (6, 51, 230, 236,339).
Genetic analysis of familial MMA may help to ascertain the pathogenesis of MMA (4). In case of identification of relevant genes, development of novel gene therapies and prevention of MMA occurrence in genetically susceptible individuals may be possible (4). Also, the Japan Adult Moyamoya Trial (4, 99, 340-345) may contribute to ascertain the advantages of combined or direct bypass surgery for the prevention of recurrent hemorrhage in MMA (4). Additional follow-up and epidemiological studies are warranted to ascertain the pathogenesis of asymptomatic MMA (4). These results will be important to refine the guidelines for surgical and medical MMA treatment, in particular for asymptomatic or hemorrhagic MMA patients (4).
In  (194). In 2021, Li et al. stated that additional studies may be warranted to help clarify the pathophysiologic mechanism associated with neutrophilic tsRNAs and their associated signaling pathways in asymptomatic MMA patients, to further elucidate MMA pathogenesis (276). In 2021, Lu et al. suggested that the serum levels of BBB-related proteins and MMP-9, in addition to their comparison between MMA subgroups, should be compared to healthy controls (198). Also, the research group indicated that the pharmaceutical significance of a strengthened impact of MMP-9 on surgery and the predictive value of intracranial hemorrhage prediction should be subject to validation in future research (198). Moreover, the group stated that additional research may be warranted to further ascertain the function of MMP-9 and BBB impairment in MMA pathophysiology (198). In 2021, Mineharu et al. stated that the functions of GUCY1A3 and RNF213 have been intensively studied in VSMCs and vascular ECs. However, with the distinct mechanism of fibrosis and intimal thickening in MMA remaining to be elucidated, research into the function of GUCY1A3 and RNF213 in immune cells, especially in dendritic cells, neutrophils, B cells, and T cells may be warranted. Additional vascular components, including the extracellular matrix (ECM), platelets, and inflammatory cells, should be subject of future research (119). In 2021, Sarkar et al. stated that further RNF213 knockdown studies may be warranted to confirm both the function of RNF213 in TNFα/PTP1B mediated obesity and insulin resistance and detailed pathophysiologic mechanisms related to this signaling pathway (11). In 2021, Sarkar et al. suggested that doing research on the effect of iron-binding in MMA and on the pathophysiologic mechanism of RNF213 in cancer and obesity may be warranted (237). In 2021, Wu et al. stated that additional prospective studies may be warranted to further ascertain the association between bleeding spots and aberrant MMA collaterals (103). Relating to their 2022 study results, Jin and Duan stated that, due to the complex functions and molecular genetic mechanisms, their bioinformatics results may warrant verification experiments. Jin and Duan hypothesized that the slow progression of MMA may be associated with a distinct gene expression at each MMA stage, that the genetics of adult MMA and pediatric MMA may be different, warranting additional clarification of such potential variations (241). Referring to the results of their 2022 transcriptome-wide analysis, Xu et al.
suggested that the sex difference should be considered in future MMA research (243).
Continued research into MMA pathophysiology and associated signaling pathways may identify new treatment strategies, therapeutic applications, and mechanism-tailored interventions that may halt MMA progression. Research into EPCs, endothelial cells, and pericytes may further elucidate the function of vasculogenic, angiogenic, and anti-angiogenic markers and associated signaling pathways (3,348). Reduction of interlaboratory variations and methodological differences may facilitate cooperation between laboratories (3,348,349). Constant evaluation of novel prognostic and diagnostic resources obtained through research may help to effectively and safely transfer research results into practice (2). Ongoing collaborative, prospective basic laboratory, and large-scale, large cohort clinical research on pathophysiologic mechanisms, a multi-professional, multi-center, international collaboration between vascular and stroke physicians, and clinician-scientists pursuing translational research are essential to establish large biorepository, imaging, and clinical data sets, which may be required if we are to better understand the complex etiology of MMA, potentially leading to increasingly differentiated diagnoses and disease-modifying treatment strategies (2,6,44,92,337,350).

Author contributions
KD contributed to developing the concept of the review, developing the figures, and writing and editing the manuscript. JW oversaw the project and contributed to developing the concept of the review, developing the figures, and editing the manuscript. Both authors contributed to the article and approved the submitted version.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material
The