Endothelial oncogenic KRAS mutation drives the dynamics of microglia and macrophages in brain arteriovenous malformation
Hyejin Park, Jung-Eun Park, Bridger H. Freeman, Bosco Seong Kyu Yang, Shun-Ming Ting, Alexander K. Suh, Jude P.J. Savarraj, Shuning Huang, Jakob Körbelin, Huimahn Alex Choi, Sean P. Marrelli, Jaroslaw Aronowski, Peng Roc Chen, Eunhee Kim, Eun S. Park
Hyejin Park, Jung-Eun Park, Bridger H. Freeman, Bosco Seong Kyu Yang, Shun-Ming Ting, Alexander K. Suh, Jude P.J. Savarraj, Shuning Huang, Jakob Körbelin, Huimahn Alex Choi, Sean P. Marrelli, Jaroslaw Aronowski, Peng Roc Chen, Eunhee Kim, Eun S. Park
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Research Article Neuroscience Vascular biology

Endothelial oncogenic KRAS mutation drives the dynamics of microglia and macrophages in brain arteriovenous malformation

Abstract

Mutation of KRAS in endothelial cells (KRAS-ECs) leads to intracerebral hemorrhage (ICH) in brain arteriovenous malformation (bAVM), resulting in severe disabilities or even death. However, it is unclear what causes this hemorrhagic conversion of bAVMs. Here, using a locally established, clinically relevant sporadic bAVM mouse model, created by overexpressing mutant KRAS (KRASG12V) in brain ECs, we demonstrate that KRAS-ECs act as trigger for activation of microglia (MG) and infiltration of macrophages (Mϕ). Using a 3-dimensional immunostaining approach with cleared human and mouse bAVM tissues, we demonstrate an abundance of MG/Mϕ around the bAVM nidus. The presence of MG/Mϕ was correlated to the blood-brain barrier leakage in bAVM areas. Time-lapsed intravital imaging in Cx3cr1-gfp;Ccr2-rfp reporter mice demonstrated the dynamic activation of MG and infiltration of Mϕ toward mutant KRASG12V–modified dysplastic vessels. Importantly, a time-course analysis showed that these activated MG and infiltrated Mϕ are present around the bAVMs prior to hemorrhagic conversion, and controlled depletion of MG/Mϕ reduced ICH incidence in bAVMs. Inhibition of MG/Mϕ with long-term minocycline treatment attenuated the incidence of ICHs around bAVMs. Our study indicates that MG/Mϕ are involved in destabilization of KRASG12V-induced bAVM, leading to hemorrhagic conversion/ICH. Thus, modulation of MG/Mϕ may reduce ICH risk in patients with bAVM.

Authors

Hyejin Park, Jung-Eun Park, Bridger H. Freeman, Bosco Seong Kyu Yang, Shun-Ming Ting, Alexander K. Suh, Jude P.J. Savarraj, Shuning Huang, Jakob Körbelin, Huimahn Alex Choi, Sean P. Marrelli, Jaroslaw Aronowski, Peng Roc Chen, Eunhee Kim, Eun S. Park

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Figure 3

Intravital tracing of MG/Mϕ in bAVM territories in KRASG12V/bEC mice.

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Intravital tracing of MG/Mϕ in bAVM territories in KRASG12V/bEC mice. (A...
(A) Representative immunofluorescence images showing the dense accumulation of both MG and Mϕ around bAVM vasculature. The dual-reporter Cx3cr1-gfp;Ccr2-rfp mice at 6 weeks after AAV-BR1-KRASG12V injection were used to visualize the spatial relationship between MG (CX3cr1-GFP, green), Mϕ (Ccr2-RFP, red), and vasculature (CD31; cyan) in bAVM territories. Note the enlarged lumens (asterisk) of vessels in the bAVM. Cx3cr1-GFP+ (green) microglia and Ccr2-RFP+ (red, arrow) Mϕ were more clustered around the bAVM territory, as compared with intact vessels. The arrows indicate colocalized activated Cx3cr1-GFP+ MG and infiltrated Ccr2-RFP+ blood-derived Mϕ at the enlarged bAVM vessels. Open arrow indicates Ccr2-RFP+ alone. Scale bar: 50 μm. (B and C) Bar graphs quantifying numbers of Cx3cr1-GFP+ MG or Ccr2-RFP+ Mϕ (B) and the coexpressed Cx3cr1-GFP+/Ccr2-RFP+ cells (C) around intact vessels versus bAVM-containing territories. Unpaired, 2-tailed t test. ***P < 0.001. Each dot indicates a randomly selected ROI (n = 8–9) obtained from mice (n = 3 per group). (D) A glass cranial window was implanted in the skull 1 week after AAV-BR1-KRASG12V injection in Cx3cr1-gfp;Ccr2-rfp mice. Four weeks later, intravital imaging was performed. (E) Representative immunofluorescence images indicating Cx3cr1-GFP+ MG and CCR2-RFP+ Mϕ around WGA-CF405+ dysplastic/malformed vessels in Cx3cr1-gfp;Ccr2-rfp mice. Scale bar: 100 μm. (F) Representative magnified images of white insets in E show the dense clustering of Cx3cr1-GFP+ MG and CCR2-RFP+ Mϕ in malformed vessels compared with intact. Scale bar: 50 μm. (G) Bar graphs quantifying numbers of Cx3cr1-GFP+ MG (GFP fluorescence pixel area) and Ccr2-RFP+ Mϕ (number of RFP+ cells) around dysplastic bAVM vessels compared to intact. All images were acquired by maximal projection. Z-stacks = 33 μm. Unpaired, 2-tailed t test. ***P < 0.001. Each dot indicates a randomly selected ROI (n = 6–8) obtained from mice (n = 3 per group). (H) Intravital time-lapse imaging captured from the inset (yellow box) in E shows the dynamic infiltration of Ccr2-RFP+ Mϕ from the dysplastic vessel lumen into the parenchyma. White arrows indicate the dynamic position of Ccr2-RFP+ cells (red). Dotted lines indicate the boundary between blood vessels and parenchyma. Scale bar: 20 μm.