Nf1+/- mice have increased neointima formation via hyperactivation of a Gleevec sensitive molecular pathway

Abstract

Neurofibromatosis type I (NF1) is a genetic disorder caused by mutations in the NF1 tumor suppressor gene. Neurofibromin is encoded by NF1 and functions as a negative regulator of Ras activity. Somatic mutations in the residual normal NF1 allele within cancers of NF1 patients is consistent with NF1 functioning as a tumor-suppressor. However, the prevalent non-malignant manifestations of NF1, including learning and bone disorders emphasize the importance of dissecting the cellular and biochemical effects of NF1 haploinsufficiency in multiple cell lineages. One of the least studied complications of NF1 involves cardiovascular disorders, including arterial occlusions that result in cerebral and visceral infarcts. NF1 vasculopathy is characterized by vascular smooth muscle cell (VSMC) accumulation in the intima area of vessels resulting in lumen occlusion. We recently showed that Nf1 haploinsufficiency increases VSMC proliferation and migration via hyperactivation of the Ras-Erk pathway, which is a signaling axis directly linked to neointima formation in diverse animal models of vasculopathy. Given this observation, we tested whether heterozygosity of Nf1 would lead to vaso-occlusive disease in genetically engineered mice in vivo. Strikingly, Nf1+/- mice have increased neointima formation, excessive vessel wall cell proliferation and Erk activation after vascular injury in vivo. Further, this effect is directly dependent on a Gleevec sensitive molecular pathway. Therefore, these studies establish an Nf1 model of vasculopathy, which mirrors features of human NF1 vaso-occlusive disease, identifies a potential therapeutic target and provides a platform to further dissect the effect of Nf1 haploinsufficiency in cardiovascular disease.

Figure 1.

Histological and morphometric analysis of injured carotid arteries from WT and Nf1+/− mice. (A) Representative photomicrographs of carotid arteries from WT (top panels) and Nf1+/− (bottom panels) mice stained with H&E 21 days following no injury (left panels) or injury (right panels). Red arrows indicate boundary of neointima. Scale bars represent 50 µm. Results are representative of five independent experiments. (B) Neointima area of uninjured and injured carotid artery cross-sections from WT and Nf1+/− mice. Data represent mean neointima area of five cross-sections±SEM, n = 5. For Nf1+/− uninjured versus injured, *P < 0.05, and for WT injured versus Nf1+/− injured, **P < 0.05 by one-way ANOVA. (C) I/M ratio of uninjured and injured carotid artery cross-sections from WT and Nf1+/− mice. Data represent mean I/M ratio of five cross-sections±SEM, n = 5. For Nf1+/− uninjured versus injured, *P < 0.01, and for WT injured versus Nf1+/− injured, **P < 0.05 by one-way ANOVA. (D) Percentage of carotid artery stenosis 21 days following injury in WT and Nf1+/− mice. Data represent mean percent stenosis of five cross-sections±SEM, n = 5, *P < 0.04 by Student's unpaired t-test with Welch correction.

Figure 2.

α-SMA analysis of injured carotid arteries from WT and Nf1+/− mice. Representative photomicrographs of uninjured (left panels) and injured (right panels) carotid arteries from WT (top panels) and Nf1+/− (bottom panels) mice stained with α-SMA (red). Cell nuclei are counterstained with DAPI (blue) and autofluorescence of murine tissue is visible (green). White boxes indicate area magnified in inset. White arrowheads indicate neointima boundary. Scale bars represent 100 µm. Results are representative of five independent experiments.

Figure 3.

Analysis of cellular proliferation within the neointima of injured carotid arteries from WT and Nf1+/− mice. (A) Representative photomicrographs of uninjured (left panels) and injured (right panels) carotid arteries from WT (top panels) and Nf1+/− (bottom panels) mice stained with anti-Ki67 (brown) antibody and hematoxylin (blue). Black boxes in middle panels indicate areas that are magnified in the far right panels. Red arrows indicate neointima boundary. Black arrowheads represent positive Ki67 staining of proliferating cells. Results are representative of five independent experiments. Scale bars represent 50 µm. (B) Quantification of percent Ki67 positive cells within the neointima of injured carotid arteries of WT and Nf1+/− mice. Data represent mean percentage of Ki67 positive cells in the neointima±SEM, n = 3, *P < 0.005 by Student's unpaired t-test with Welch correction.

Figure 4.

Analysis of Erk phosphorylation within the neointima of injured carotid arteries of WT and Nf1+/− mice. (A) Representative photomicrographs of uninjured (left panels) and injured (right panels) carotid arteries from WT (top panels) and Nf1+/− (bottom panels) mice stained with anti-phosphorylated-Erk (brown) antibody and counterstained with hematoxylin (blue). Black boxes in middle panels indicate areas that are magnified in the far right panels. Red arrows indicate neointima boundary. Black arrowheads represent positive phosphorylated-Erk staining. Results are representative of five independent experiments. Scale bars represent 50 µm. (B) Quantification of percent phosphorylated-Erk positive cells within the neointima of injured carotid arteries of WT and Nf1+/− mice. Data represent mean percentage of phosphorylated-Erk positive cells in the neointima±SEM, n = 3, *P < 0.04 by Student's unpaired t-test.

Figure 5.

Histological and morphometric analysis of injured carotid arteries from Gleevec-treated WT and Nf1+/− mice. (A) Representative photomicrographs of H&E-stained carotid arteries from WT (top panels) and Nf1+/− (bottom panels) mice 21 days following no injury and PBS treatment (left panels), injury and PBS treatment (middle panels) or injury and Gleevec treatment (right panels). Red arrows indicate boundary of neointima. Scale bars represent 50 µm. Results are representative of five independent experiments. (B) I/M ratio of injured carotid artery cross-sections from PBS and Gleevec-treated WT and Nf1+/− mice. Data represent mean I/M ratio of five cross-sections±SEM, n = 4–6 mice. For Nf1+/− uninjured versus injured with PBS treatment, *P < 0.001; for Nf1+/− injured with PBS versus injured with Gleevec treatment, **P < 0.001; and for WT injured with PBS treatment versus Nf1+/− injured with PBS treatment, ***P < 0.05 by one-way ANOVA.

Figure 6.

Analysis of α-SMA, Ki67 and Erk phosphorylation within the neointima of injured carotid arteries from Gleevec-treated Nf1+/− mice. Representative photomicrographs of α-SMA staining (left panels) of carotid artery cross-sections from injured Nf1+/− PBS-treated (top panels) and Nf1+/− Gleevec-treated (bottom panels) mice. α-SMA staining is seen in red, DAPI nuclear dye is blue and murine tissue autofluorescence is green. White arrows indicate neointima boundary. White boxes indicate area magnified in inset. Representative images of Ki67 (middle panels) and phoshophorylated-Erk (right panels) staining of carotid artery cross-sections counter-stained with hematoxylin (blue). Red arrows indicate neointima boundary. Black arrowheads represent positive Ki67 or phosphorylated-Erk staining (brown). Black boxes indicate areas magnified in insets. Results are representative of five independent experiments. Scale bars represent 50 µm.