# Study on Abnormal Angiogenesis in Moyamoya Disease via Mitochondrial D‐Loop Methylation

## TL;DR

This study finds that lower mitochondrial DNA methylation in MMD patients helps distinguish them from non-MMD patients and healthy individuals, suggesting a new path for understanding the disease.

## Contribution

The study identifies a novel link between mitochondrial D-loop methylation and MMD, proposing a methylation-metabolism-angiogenesis axis for disease mechanisms.

## Key findings

- MMD patients had significantly lower D-loop methylation than non-MMD ICASO patients and healthy controls.
- RNF213 knockout in endothelial cells reduced methylation and ROS while increasing ATP and mitochondrial potential.
- A diagnostic model combining methylation and clinical data achieved an AUC of 0.891 for distinguishing MMD and non-MMD ICASO.

## Abstract

Mitochondrial D‐loop methylation leads to abnormal cerebral angiogenesis. This study examines its role in vascular phenotypes of moyamoya disease (MMD).

Blood samples from 96 intracranial artery stenosis/occlusion (ICASO) patients (35 MMD, 61 non‐MMD) and healthy controls underwent methylation analysis. D‐loop methylation levels were analyzed using chi‐square, T‐test, and Wilcoxon tests. Propensity score matching (PSM) adjusted for age and gender disparities. Combine clinical information with methylation data to build a diagnostic model. Further studies included the methylation levels in MMD patients carrying the RNF213 p.R4810K mutation and the methylation levels and possible mechanisms of human brain microvascular endothelial cells (hCMEC/D3) with RNF213 knockdown.

Healthy controls showed higher D‐loop methylation than MMD (p < 0.05). Post‐PSM, non‐MMD ICASO patients showed higher D‐loop methylation than MMD (p < 0.05). The AUC of the prediction model was 0.891 (95% CI, 0.821–0.961) after combining clinical information with methylation data. MMD patients with the RNF213 mutation exhibited reduced methylation at most sites, though not statistically significant (p > 0.05). RNF213 knockout in hCMEC/D3 enhanced proliferation, migration, and tube formation, while reducing apoptosis and DNMT1 expression, leading to decreased D‐loop methylation and ROS level, increased ATP production and mitochondrial membrane potential.

There are differences in the methylation levels in the mitochondrial D‐loop region between MMD and non‐MMD ICASO. The methylation–metabolism–angiogenesis axis may represent a promising research direction for elucidating MMD pathogenesis.

Analysis of blood samples showed that mitochondrial DNA methylation was significantly lower in MMD patients than in non‐MMD patients with intracranial artery stenosis/occlusion and healthy controls. Combined with clinical data, this difference effectively distinguished the two groups of patients, and the RNF213 gene was associated with reduced methylation levels and altered endothelial cell function.