Persistent infiltration and pro-inflammatory differentiation of monocytes cause unresolved inflammation in brain arteriovenous malformation

Abstract

An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68(+) cells at 2 weeks (P = 0.02), similar numbers at 4 weeks (P = 0.97), and more at 8 weeks (P = 0.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8 weeks (P = 0.064) after angiogenic stimulation and more RFP(+) bone marrow-derived macrophages than WT mice (P = 0.01). More CD34(+) cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (P < 0.001). These data indicate that persistent infiltration and pro-inflammatory differentiation of monocytes might contribute to macrophage accumulation in bAVM. Blocking macrophage homing to bAVM lesions should be tested as a strategy to reduce the severity of bAVM.

Keywords: Angiogenesis; Animal models; Arteriovenous malformations; Cerebrovascular disease; Macrophages; Microglia.

Fig. 1. Animal models

a Model 1: bAVMs were in induced in R26^CreER/+;Eng^2f/2f mouse line that has a Rose promoter driving and estrogen inducible cre recombinase and an Eng gene with exons 5 and 6 flanked by loxP sites [23] by injection of AAV1-VEGF stereotactically into the basal ganglia to induce brain focal angiogenesis and i.p. injection of 3 doses of tamoxifen (TM) on 3 consecutive days to globally delete the Eng gene. Brain samples were collected 2, 4 and 8 weeks after AAV1-VEGF injection. b Model 2: bAVMs were induced in Alk1^1f/2f;Ccr2^RFP/+/Cx3cr1^GFP/+ mice that have Alk1 gene deleted in one allele and floxed in the other allele [25], RFP gene knocked into one allele of Ccr2 gene and GFP knocked into one allele of Cx3cr1 gene [26] through co-injection of Ad-Cre and AAV1-VEGF stereotactically into the cortex to induce brain focal deletion of Alk1 floxed allele and angiogenesis. Brain samples were collected 8 weeks after the vector injection.

Fig. 2. VEGF expression and chaotically assembled vessels in the angiogenic region of Eng-deficient mice (Model 1)

a Quantification of human VEGF levels in the AAV1-VEGF-injected region. N=6. b Representative image of chaotically assembled bAVM vessels in AAV1-VEGF injection site. Scale bar: 1 mm.

Fig. 3. More CD68⁺ cells in bAVM lesion of Eng-deficient brain (Model 1) 8-weeks after AAV1-VEGF injection

a Representative images of sections stained with antibodies specific to CD68 (red, macrophages) and CD31 (green, brain microvasculatures). Nuclei were counterstained with DAPI (blue). The animals were perfused with heparinized PBS before sample collection. Arrows indicate CD68⁺ cells outside vessels or on vessel wall. Scale bar: 100 μm. b Bar graph shows quantifications. *: P<0.05; N=6

Fig. 4. Slow onset and persistent CD68⁺ cells homed to the brain angiogenic region of Eng-deficient mice (Model 1)

a, b Representative images of sections stained with antibodies specific to CD68 (red, macrophages) and CD31 (green, brain microvasculature). Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm c Bar graph shows quantifications. *: P<0.05; N=6

Fig. 5. BM-derived macrophages and microglia clustered around bAVM vessels in the Alk1-deficient brain 8 weeks after angiogenic stimulation (Model 2)

a Representative images show RFP⁺ BM-derived macrophage. b Quantification of RFP⁺ cells. * P<0.05 c Representative images show GFP⁺ microglia. d Quantification of GFP⁺ microglia. Scale bar: 100 μm. Nuclei were counterstained with DAPI. N=5

Fig. 6. More HHT CD34⁺ cells differentiated into macrophages

a Quantification of surviving cells. b Percentage of cells that migrated to bottom chamber. *: P=0.001

Fig. 7. ENG and ALK1 expression in HHT monocytes

a ENG expression decreased in both HHT1 and HHT2 monocytes. ***: P<0.001 vs. control. b ALK1 expression decreased only in HHT2 monocytes. *: P=0.017 vs. control.