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 1

KRAS-ECs drive the activation of MG and infiltration of Mϕ toward bAVM territories in KRASG12V/bEC mice.

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KRAS-ECs drive the activation of MG and infiltration of Mϕ toward bAVM t...
(A) T2*-weighted MRI revealed the presence of ICH 8 weeks after AAV-BR1-KRASG12V injection. For labeling, whole brains were cleared using the tissue rapid clearing technique before being processed for immunostaining. Confocal microscopy visualized the bAVMs (tissue corresponding to the red square of the MRI image) as a dense network of CD31+ (EC marker) vasculature. A nidus shape of bAVM vessels (CD31, green) is distinguished from intact vessels. Dotted blue lines with arrows indicate the bAVM nidus. An abundant population of MG/Mϕ (Iba1, red) surrounding bAVMs was clearly detected. Scale bar: 100 μm. The large hypointense bleeding spot on the MRI (arrow) was not analyzed due to tissue disintegration following ICH from the bAVM nidus. (B) High magnification of the dotted area in A representing the intact brain (yellow-filled black dotted) or bAVM-containing area (yellow-filled, red dotted). Scale bar: 25 μm. Note the less dense contact between MG/Mϕ and the vasculature (CD31+) in the intact brain area (dotted arrows), while bAVM vessels exhibit an enhanced contact between Mg/Mϕ and bAVM vessels (arrows). (C) Representative 3D images reconstructing stacked images of cleared/immunofluorescently stained brain tissues. The 3D images clearly show a higher density and intensity of myeloid (Iba1+) cells attached to CD31+ dysplastic bAVM vessels, compared with the intact vessels in KRASG12V/bEC mice. Units indicate scales (149 μm × 149 μm × 67 μm). Sample thickness: 2 mm. Z-stacks: 67 μm. (D) Representative immunofluorescence images showing the increased clustering of Iba1+ cells in the bAVM territory compared with intact vessels 6 weeks after AAV-BR1-KRASG12V injection. Arrows indicate the contact of Iba1+ cells to the CD31+ ECs. Scale bar: 25 μm. (E and F) Bar graphs quantifying volume of Iba1+ cells (E) or contact area of Iba1+ cells to the CD31+ ECs (F) around intact vessels versus bAVM-containing territories. Unpaired, 2-tailed t test. ****P < 0.0001. Each dot indicates a randomly selected ROI (n = 15) obtained from mice (n = 6 per group). (G and I) Representative immunofluorescence images showing the enhanced production of IL-1β or IL-6 by Iba1+ cells in the bAVM territory compared with intact vessels 6 weeks after AAV-BR1-KRASG12V injection. Arrows indicate IL-1β+Iba1+ cells (G) or IL-6+Iba1+ cells (I). Open arrows indicate IL-1β+CD31+ cells (G) or IL-6+CD31+ cells (I). Scale bar: 50 μm. (H and J) Bar graphs quantifying numbers of IL-1β+Iba1+ (E) or IL-6+Iba1+ cells (G) around intact vessels versus bAVM-containing territories. Unpaired, 2-tailed t test. **P < 0.01. Each dot indicates a randomly selected ROI (n = 8) obtained from mice (n = 3 per group).