Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans

Abstract

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. NF1 patients display diverse clinical manifestations, including vascular disease, which results from neointima formation and vessel occlusion. However, the pathogenesis of NF1 vascular disease remains unclear. Vessel wall homeostasis is maintained by complex interactions between vascular and bone marrow-derived cells (BMDCs), and neurofibromin regulates the function of each cell type. Therefore, utilizing cre/lox techniques and hematopoietic stem cell transplantation to delete 1 allele of Nf1 in endothelial cells, vascular smooth muscle cells, and BMDCs alone, we determined which cell lineage is critical for neointima formation in vivo in mice. Here we demonstrate that heterozygous inactivation of Nf1 in BMDCs alone was necessary and sufficient for neointima formation after vascular injury and provide evidence of vascular inflammation in Nf1+/- mice. Further, analysis of peripheral blood from NF1 patients without overt vascular disease revealed increased concentrations of inflammatory cells and cytokines previously linked to vascular inflammation and vasoocclusive disease. These data provide genetic and cellular evidence of vascular inflammation in NF1 patients and Nf1+/- mice and provide a framework for understanding the pathogenesis of NF1 vasculopathy and potential therapeutic and diagnostic interventions.

Figure 1. Histological and morphometric analysis of WT, Nf1^+/–, Nf1^fl/+;Tie2cre, and Nf1^fl/+;SM22cre mice.

(A) Representative H&E-stained cross sections of uninjured and injured carotid arteries from WT, Nf1^+/–, Nf1^fl/+;Tie2cre, and Nf1^fl/+;SM22cre mice. Red arrows indicate neointima boundaries. Scale bars: 50 μm. (B and C) Quantification of neointima area (B) and I/M ratio (C) of uninjured (white bars) and injured (black bars) carotid arteries from WT, Nf1^+/–, Nf1^fl/+;Tie2cre, and Nf1^fl/+;SM22cre mice. Data represent the mean of 3 arterial cross sections (400, 800, and 1,200 μm proximal to the ligation) ± SEM, n = 4–7. *P < 0.001 for Nf1^+/– uninjured versus Nf1^+/– injured and for Nf1^+/– injured versus WT injured, Nf1^fl/+;Tie2cre injured, and Nf1^fl/+;SM22cre injured, by 1-way ANOVA with Tukey’s post-hoc test.

Figure 2. Histological and morphometric analysis of WT and Nf1^+/– mice transplanted with WT and Nf1^+/– BM.

(A) Representative H&E-stained cross sections of uninjured and injured carotid arteries from WT and Nf1^+/– mice transplanted with WT or Nf1^+/– BM. Red arrows indicate boundaries of neointima. Scale bars: 50 μm. (B and C) Quantification of neointima area (B) and I/M ratio (C) of uninjured and injured carotid arteries from WT or Nf1^+/– recipients transplanted with WT or Nf1^+/– BM. Data represent the mean neointima area of 3 arterial cross sections (400, 800, and 1200 μm distal to the ligation) ± SEM, n = 5–8. *P < 0.001 for WT mice transplanted with Nf1^+/– BM uninjured versus WT mice transplanted with Nf1^+/– BM injured and for Nf1^+/– mice transplanted with Nf1^+/– BM uninjured versus Nf1^+/– mice transplanted with Nf1^+/– BM injured; **P < 0.001 for WT mice transplanted with Nf1^+/– BM and Nf1^+/– mice transplanted with Nf1^+/– BM versus WT mice transplanted with WT BM and Nf1^+/– mice transplanted with WT BM injured, by 1-way ANOVA with Tukey’s post-hoc test.

Figure 3. Identification of VSMCs and BMDCs within the neointima of WT and Nf1^+/– mice transplanted with WT and Nf1^+/– BM.

(A) Representative photomicrographs of uninjured and injured carotid arteries from WT and Nf1^+/– mice transplanted with either WT or Nf1^+/– BM stained with anti–α-SMA (red) antibody to identify VSMCs and anti-GFP (green) antibody to identify BMDCs. Cell nuclei are visible by DAPI stain (blue), and some tissue autofluorescence is visible (green). White arrows indicate neointima boundaries. Yellow boxes identify area of injured WT and Nf1^+/– mice transplanted with Nf1^+/– BM magnified in the far-right panels. Scale bars: 50 μm; far-right panels, 250 μm. (B) Quantification of the total number of GFP-positive cells within the neointima of WT and Nf1^+/– recipient mice after carotid ligation. Data represent the mean GFP-positive cells within the neointima 600 μm proximal to the ligation ± SEM, n = 6. *P < 0.05 for injured WT mice transplanted with Nf1^+/– BM versus injured WT mice transplanted with WT BM and for injured Nf1^+/– mice transplanted with Nf1^+/– BM versus injured Nf1^+/– mice transplanted with WT BM; **P < 0.05 for injured Nf1^+/– mice transplanted with Nf1^+/– BM versus injured WT mice transplanted with Nf1^+/– BM, by 1-way ANOVA with Tukey’s post-hoc test.

Figure 4. Identification of macrophage accumulation within the neointima of WT and Nf1^+/– mice transplanted with WT and Nf1^+/– BM.

(A) Representative photomicrographs of injured carotid artery cross sections from Nf1^+/– mice transplanted with Nf1^+/– BM stained with anti-GFP (green) and anti-CD45 (red). Colocalization is shown in the right panel. Arrows indicate double staining for GFP and CD45. DAPI nuclear dye is shown in blue. Scale bars: 250 μm. (B) Representative photomicrographs of uninjured and injured carotid artery cross sections from WT and Nf1^+/– mice transplanted with WT or Nf1^+/– BM stained with anti-Mac3 antibody (brown) and counterstained with hematoxylin (blue). Red arrows indicate neointima boundaries. Black boxes identify area of injured artery that is magnified in the far-right lower panels. Black arrows represent positive Mac3 staining. Scale bars: 50 μm. Original magnification of far-right panels, ×400.

Figure 5. In vitro function of Nf1^+/– macrophages.

(A) Percent monocytes in peripheral blood of WT and Nf1^+/– mice. Data represent mean percentage ± SEM, n = 8. *P = 0.02 for Nf1^+/– versus WT by Student’s unpaired t test. (B) WT (white bars) and Nf1^+/– (black bars) macrophage proliferation in response to M-CSF. Data represent mean cpm ± SEM, n = 4. *P < 0.001 for unstimulated WT versus M-CSF–stimulated WT macrophages; **P < 0.001 for M-CSF–stimulated Nf1^+/– versus M-CSF–stimulated WT macrophages, by 1-way ANOVA. (C) WT (white bars) and Nf1^+/– (black bars) macrophage haptotaxis in response to M-CSF. Data represent mean number of migrated cells ± SEM, n = 4. *P < 0.001 for unstimulated WT versus M-CSF–stimulated WT macrophages; **P < 0.001 for unstimulated WT versus unstimulated Nf1^+/– macrophages; ^#P < 0.001 for unstimulated Nf1^+/– versus M-CSF–stimulated Nf1^+/– macrophages, by 1-way ANOVA. (D) WT (white bars) and Nf1^+/– (black bars) macrophage adhesion to fibronectin. Data represent mean optical density ± SEM, n = 4. *P < 0.001 for WT versus Nf1^+/– macrophages; **P < 0.01 for Nf1^+/– macrophages at 60 minutes versus Nf1^+/– macrophages at all other time points, by 1-way ANOVA. (E) Macrophage stimulation of WT and Nf1^+/– VSMC proliferation. Data represent mean cpm ± SEM, n = 3. *P < 0.05 for Nf1^+/– VSMCs cocultured with WT or Nf1^+/– macrophages versus WT VSMCs; ^#P < 0.05 for WT VSMCs cocultured with Nf1^+/– macrophages versus WT macrophages and for Nf1^+/– VSMCs cocultured with Nf1^+/– macrophages versus WT macrophages; ^†P < 0.05 for WT and Nf1^+/– VSMCs treated with MEK inhibitor versus no inhibition, by 1-way ANOVA.

Figure 6. Identification of circulating monocytes from peripheral blood of healthy adult controls and NF1 patients.

(A) Representative multiparameter flow cytometry analysis plot of peripheral blood monocytes identified by CD14 and CD16 cell surface expression in healthy adult controls and NF1 patients. Black box identifies the CD14^dimCD16^bright monocyte population. Isolated CD14^dimCD16^bright monocytes identified by May-Grünwald-Giemsa staining are shown in the far-right panel. Arrows indicate monocytes. Data represent 10 independent observations. (B) Quantification of the frequency of CD45⁺CD14⁺ monocytes in circulation from healthy adult control and NF1 patient peripheral blood MNCs. Data represent the mean frequency ± SEM, n = 5. *P = 0.024 by Student’s unpaired t test with Welch correction. (C) Quantification of the frequency of CD45⁺CD14^dimCD16^bright (CD16⁺⁺) monocytes in circulation from healthy adult control and NF1 patient peripheral blood MNCs. Data represent the mean frequency ± SEM, n = 5. *P = 0.0012 by Student’s unpaired t test.

Figure 7. Analysis of inflammatory cytokines and chemokines from NF1 patient and healthy control plasma.

Determination of cytokine levels in plasma isolated from control peripheral blood and NF1 patient peripheral blood. (A) IL-1β levels. Data represent the mean pg/ml ± SEM, n = 6. *P = 0.014 by Mann-Whitney U test. (B) IL-6 levels. Data represent the mean pg/ml ± SEM, n = 6. *P = 0.046 by Student’s unpaired t test of log-transformed data. (C) Fractalkine levels. Data represent the mean pg/ml ± SEM, n = 6–8. *P = 0.0393 by Student’s unpaired t test with Welch correction.