Comprehensive Profiling of Secreted Factors in the Cerebrospinal Fluid of Moyamoya Disease Patients

Abstract

Moyamoya disease (MMD) is characterized by progressive occlusion of the intracranial internal carotid arteries, leading to ischemic and hemorrhagic events. Significant clinical differences exist between ischemic and hemorrhagic MMD. To understand the molecular profiles in the cerebrospinal fluid (CSF) of MMD patients, we investigated 62 secreted factors in both MMD subtypes (ischemic and hemorrhagic) and examined their relationship with preoperative perfusion status, the extent of postoperative angiographic revascularization, and functional outcomes. Intraoperative CSF was collected from 32 control and 71 MMD patients (37 ischemic and 34 hemorrhagic). Multiplex Luminex assay analysis showed that 41 molecules were significantly elevated in both MMD subtypes when compared to controls, including platelet-derived growth factor-BB (PDGF-BB), plasminogen activator inhibitor 1 (PAI-1), and intercellular adhesion molecule 1 (ICAM1) (p < 0.001). Many of these secreted proteins have not been previously reported in MMD, including interleukins (IL-2, IL-4, IL-5, IL-7, IL-8, IL-9, IL-17, IL-18, IL-22, and IL-23) and C-X-C motif chemokines (CXCL1 and CXCL9). Pathway analysis indicated that both MMD subtypes exhibited similar cellular/molecular functions and pathways, including cellular activation, migration, and inflammatory response. While neuroinflammation and dendritic cell pathways were activated in MMD patients, lipid signaling pathways involving nuclear receptors, peroxisome proliferator-activated receptor (PPAR), and liver X receptors (LXR)/retinoid X receptors (RXR) signaling were inhibited. IL-13 and IL-2 were negatively correlated with preoperative cerebral perfusion status, while 7 factors were positively correlated with the extent of postoperative revascularization. These elevated cytokines, chemokines, and growth factors in CSF may contribute to the pathogenesis of MMD and represent potential future therapeutic targets.

Keywords: Cerebrospinal fluid; Moyamoya disease; Neuroinflammation; Revascularization; Stroke; Surgical outcome.

Fig. 1

Relationship between angiograde and perfusion status in moyamoya patients. A, B The clinical variables examined included age at treatment, sex, side of disease, baseline perfusion status, side of treatment (unilateral versus bilateral), initial mRS, final mRS, and race. We found that postoperative angiograde was significantly associated with preoperative perfusion status and side of treatment. C Stack plot shows the frequency of perfusion grade in each angiograde score. Note that patients with poor perfusion status at baseline had better postoperative angiograde

Fig. 2

Top CSF secreted factors (A) in MMD ischemic (MMD-I) and MMD hemorrhagic (MMD-H) groups. A graph shows the top 10 factors in MMD-I and MMD-H (B) groups. Data are plotted as log2FC normalized over the control group. Bar graphs on the right depict the levels of top proteins (PDGF-BB, RETN, IL-7, and PAI-1) expressed as log2 MFI (mean fluorescence intensity). Data indicates the least square mean. Error bars are the 95% confidence of the LS mean. **p < 0.01, ***p < 0.001, and ****p < 0.0001, Tukey corrected. N = 32 for control, n = 37 for MMD-I, and n = 34 for MMD-H. C C-X-C motif chemokine 10 (CXCL10/IP10) was the only factor showing significant levels between MMD-I and MMD-H. D Bar graphs indicate mean protein levels of cellular retinoic acid binding protein 1 (CRABP1) in each group quantitated by ELISA. Dots represent the levels of CRABP1 in individual patients. ***p < 0.001 and ****p < 0.0001. One-way ANOVA followed by Bonferroni post hoc test

Fig. 3

Top molecular functions and signaling pathways in MMD patients. A Table indicates the top molecular and cellular functions in the MMD-I and MMD-H groups. B Graphs show the top 10 canonical pathways from IPA® pathway analysis in MMD-I and MMD-H groups. P-values for the significance of each pathway were computed based on the number of molecules involved in the pathway by using Fisher’s exact test and negatively log10 transformed (represented in the upper x-axis). The ratio expresses the number of differentially expressed molecules over the total number of molecules in each canonical pathway based on the IPA knowledge database (represented in the lower x-axis). C Comparison analysis of common pathways between ischemic and hemorrhagic MMD. The color bar shows z-scores, where > 2 and < 2 indicate activation (red) and inhibition (blue) of each predicted pathway, respectively

Fig. 4

Relationship of secreted factors with preop perfusion status and postop angiograde. A Ordered probit regression displaying odds ratio assessing CSF factors in relation to preop perfusion status and postop degree of revascularization. A higher expression of IL-13 and IL-27 increases the odds of having worse preop perfusion status. B Higher expression of these 7 CSF factors increases the odds of having better postop angiograde outcome. Note that while an increase in these CSF factors increases the probability of better angiograde scores, it was the reverse for perfusion grade