Abstract
Mutations in the NF1 tumor suppressor gene cause Neurofibromatosis type 1 (NF1). Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity. Some NF1 patients develop cardiovascular disease, which represents an underrecognized disease complication and contributes to excess morbidity and mortality. Specifically, NF1 patients develop arterial occlusion resulting in tissue ischemia and sudden death. Murine studies demonstrate that heterozygous inactivation of Nf1 (Nf1+/−) in bone marrow cells enhances neointima formation following arterial injury. Macrophages infiltrate Nf1+/− neointimas, and NF1 patients have increased circulating inflammatory monocytes in their peripheral blood. Therefore, we tested the hypothesis that heterozygous inactivation of Nf1 in myeloid cells is sufficient for neointima formation. Specific ablation of a single copy of the Nf1 gene in myeloid cells alone mobilizes a discrete pro-inflammatory murine monocyte population via a cell autonomous and gene-dosage dependent mechanism. Furthermore, lineage-restricted heterozygous inactivation of Nf1 in myeloid cells is sufficient to reproduce the enhanced neointima formation observed in Nf1+/− mice when compared with wild-type controls, and homozygous inactivation of Nf1 in myeloid cells amplified the degree of arterial stenosis after arterial injury. Treatment of Nf1+/− mice with rosuvastatin, a stain with anti-inflammatory properties, significantly reduced neointima formation when compared with control. These studies identify neurofibromin-deficient myeloid cells as critical cellular effectors of Nf1+/− neointima formation and propose a potential therapeutic for NF1 cardiovascular disease.
INTRODUCTION
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease affecting 1 in 3500 individuals. NF1 results from mutations in the NF1 tumor suppressor gene that encodes the protein neurofibromin (1). Neurofibromin is a GTPase-activating protein for Ras and negatively regulates Ras signaling (2,3). Whereas loss of NF1 heterozygosity is described in primary tumor samples, germline mutations that cause NF1 affect only one copy of the NF1 gene. Haploinsufficiency of NF1 results in disease with complete penetrance and a range of neoplastic and non-neoplastic manifestations.
Common non-neoplastic manifestations of NF1 include skeletal abnormalities, hearing loss and cognitive deficits. One of the least studied manifestations of NF1 involves disorders of the cardiovascular system, which represent an underrecognized disease complication and contribute to excess morbidity and mortality, particularly among younger patients (4–9). Specifically, NF1 patients develop intimal hyperplasia, characterized by vascular smooth muscle cell (VSMC) accumulation and vessel lumen stenosis resulting in cerebral and visceral ischemia and infarction (4–9).
Neurofibromin is a regulator of endothelial cell (EC), VSMC and hematopoietic cell function in response to multiple growth factors implicated in neointima formation (10–13). However, the lineage-specific regulatory function of neurofibromin in the coordination of ECs, VSMCs and circulating hematopoietic cells to maintain blood vessel wall homeostasis in vivo remains poorly understood. Previous data demonstrate that Nf1+/− mice form enlarged neointimas characterized by proliferating VSMCs, infiltration of inflammatory macrophages and amplification of Ras-ERK signaling in response to arterial injury (14).
Utilizing cre/lox technology, we previously demonstrated that cell-specific loss of a single Nf1 allele in ECs and/or VSMCs failed to induce neointima formation after arterial injury (15). However, lethally irradiated wild-type (WT) mice reconstituted with Nf1+/− hematopoietic stem cells uniformly developed enlarged neointimas after arterial injury similar to Nf1+/− mice with germline heterozygous inactivation of Nf1(15). These data provide genetic and cellular evidence that bone marrow-derived Nf1+/− hematopoietic cells are sufficient for neointima formation after vessel injury. However, the hematopoietic cell lineage responsible for this NF1 vascular phenotype remains unknown. Identification of this cell population is important for understanding disease pathogenesis and development of experimental therapeutics and potential biomarkers of disease progression.
Although multiple hematopoietic cell lineages contribute to neointima formation, several lines of experimental evidence suggest that Nf1+/− myeloid cells are potentially important for neointima formation in vivo. Nf1+/− macrophages are abundant in Nf1+/− murine neointimas and neurofibromin is a critical regulator of Ras-ERK signaling in multiple myeloid cell subtypes, including myeloid progenitors, osteoclasts and mature differentiated macrophages (15,16). Furthermore, in human corollary studies, analysis of peripheral blood from NF1 patients without overt signs of cardiovascular disease shows a significant increase in the number of circulating CD14dimCD16hi monocytes and the levels of pro-inflammatory cytokines IL-1β, IL-6 and fractalkine, which have been previously linked to cardiovascular disease in other patient populations (15).
Based on these observations, we employed cre/lox technology to generate mice that were either heterozygous or homozygous for Nf1 in myeloid cells alone to directly test the hypothesis that neurofibromin-deficient myeloid cells are the critical hematopoietic cell effectors of neointima formation after vessel injury in vivo. We further tested whether rosuvastatin, a statin with anti-inflammatory properties (17–19), could prevent or diminish neointima formation in Nf1+/− mice.
RESULTS
Genetic ablation of Nf1 in myeloid cells increases circulating Ly6Chi monocytes via a cell autonomous and gene dosage-dependent mechanism
NF1 patients demonstrate increased mobilization of CD14dimCD16hi monocytes in their peripheral blood, a cell type linked to vessel occlusion and vascular disease (20–22). To determine if inactivation of Nf1 in myeloid cells would be sufficient to mobilize Ly6Chi cells, which are the murine correlate of human pro-inflammatory monocytes (23,24), we generated mice that were heterozygous or homozygous for Nf1 in myeloid cells. Briefly, Nf1fl/fl mice, which contain conditional Nf1 alleles susceptible to Cre-mediated recombination, were intercrossed with LysMcre mice to generate Nf1fl/+;LysMcre (heterozygous) or Nf1fl/fl;LysMcre (homozygous) progeny.
Similar to NF1 patients, Nf1+/− and Nf1fl/+;LysMcre mice had increased absolute numbers of circulating Ly6Chi cells in their peripheral blood when compared with WT controls at baseline as assessed by polychromatic flow cytometry (Fig. 1A and B). Genetic ablation of the second Nf1 allele in myeloid cells alone (Nf1fl/fl;LysMcre) resulted in a 2.5-fold increase in circulating Ly6Chi monocytes when compared with mice harboring a single Nf1 allele in myeloid cells (Fig. 1B). Peripheral blood neutrophil populations were unchanged in Nf1+/−, Nf1fl/+;LysMcre or Nf1fl/fl;LysMcre when compared with WT mice (data not shown). These data provide genetic evidence that mobilization of this discrete monocyte population is cell autonomous and not dependent on growth factor secretion by other cell types in vivo.
Figure 1.
Ly6Chi monocytes are mobilized in a cell-autonomous manner. (A) Representative gating strategy: within the CD115-positive population (red box), monocytes are distinguished by positive staining for Ly6C (x-axis) and negative staining for CD11c (y-axis). Pro-inflammatory Ly6Chi monocytes are identified within the green box for representative WT, Nf1fl/+;LysMcre and Nf1fl/fl;LysMcre mice. (B) Absolute number of Ly6Chi monocytes in peripheral blood of WT, Nf1+/–, Nf1fl/+;LysMcre and Nf1fl/fl;LysMcre. Data represent mean percentage ± SEM, n = 10. *P < 0.02 for WT versus Nf1+/– and Nf1fl/+;LysMcre. **P < 0.0001 for WT, Nf1+/– and Nf1fl/+;LysMcre versus Nf1fl/fl;LysMcre. Analysis by one-way ANOVA with Tukey's post-hoc test. There was no statistical difference between Nf1+/− and Nf1fl/+;LysMcre mice.
Heterozygous or homozygous inactivation of Nf1 in myeloid cells is sufficient for enhanced neointima formation following arterial injury in vivo
To determine the effect of heterozygous inactivation of Nf1 in myeloid cells on neointima formation after arterial injury, Nf1fl/+;LysMcre mice underwent carotid artery ligation and analysis for neointima formation along with WT and Nf1+/– experimental controls. Carotid artery ligation is a well-established model that induces neointima formation through alterations in arterial hemodynamics (25).
Histologic examination of Van Gieson-stained sections from the uninjured carotid artery of each experimental group were morphologically similar and showed no evidence of neointima formation (Fig. 2A–C). However, in response to carotid artery ligation, Nf1+/– and Nf1fl/+;LysMcre mice had a 2-fold increase in both neointima area (Fig. 2A and B) and intima/media (I/M) ratio (Fig. 2C) when compared with WT mice. Media area was similar in each genotype, indicating that cell proliferation and accumulation is limited to the intimal layer (data not shown). There was no statistical difference in neointima size or I/M ratios between Nf1+/– and Nf1fl/+;LysMcre mice (Fig. 2A–C). Consistent with prior experiments, macrophages are the predominant hematopoietic cell within the mature neointima of Nf1fl/+;LysMcre mice as identified by anti-Mac3 (Fig. 2D). Neutrophils, as identified by anti-NIMP-R14 antibody, were recruited to the evolving neointima by day 7 and were absent from the mature neointimas (day 28) observed in all three genotypes (data not shown).
Figure 2.
Heterozygous inactivation of Nf1 in myeloid cells is sufficient for enhanced neointima formation. (A) Representative Van Gieson-stained cross sections of uninjured and injured carotid arteries from WT, Nf1+/– and Nf1fl/+;LysMcre mice. Black arrows indicate neointima boundaries. Black boxes identify area of injured artery that is magnified in the far-right panels. Scale bars: 100 μm. (B and C) Quantification of neointima area (B) and I/M ratio (C) of injured carotid arteries from WT, Nf1+/– and Nf1fl/+;LysMcre mice. Data represent the mean neointima area of three arterial cross sections (400, 800 and 1200 μm proximal to the ligation) ± SEM, n = 10–12. *P < 0.01 for WT uninjured versus WT injured, Nf1+/− uninjured versus Nf1+/− injured and Nf1fl/+;LysMcre uninjured versus Nf1fl/+;LysMcre injured. **P < 0.01 for WT injured versus Nf1+/– injured and Nf1fl/+;LysMcre injured. Analysis by one-way ANOVA. There was no statistical difference between Nf1+/− and Nf1fl/+;LysMcre injured. (D) Representative photomicrograph of injured carotid artery cross section from Nf1fl/+;LysMcre mice stained with anti-Mac3 antibody (brown) and counterstained with hematoxylin (blue). Black arrows indicate neointima boundaries. Black arrowheads represent positive Mac3 staining. Black box identifies area of injured artery that is magnified in the far-right panel. Scale bars: 100 μm.
Lysozyme M is expressed in monocytes and granulocytes (26) and, importantly, is not expressed in vascular wall cells (data not shown). To assess the contribution of neurofibromin-deficient neutrophils and monocytes/macrophages, we examined arterial cross sections of the evolving neointima in Nf1+/– and WT mice 7 days after arterial injury. Cross sections of Nf1+/– and WT carotid arteries did not significantly differ in the number of infiltrating myeloid cells to the neointima of Nf1+/– and WT mice as determined by staining for macrophages [3.2 versus 3.5 per high power field (HPF), respectively] or neutrophils (5.3 versus 4.8 per HPF, respectively) (Fig. 3).
Figure 3.
Macrophages and neutrophils are recruited to the evolving neointima by 7 days post-injury. Representative photomicrographs from injured carotid arteries from WT (top panels) and Nf1+/−(bottom panels) mice demonstrating macrophage (left column) and neutrophil (right column) infiltration into the neointima 7 days after arterial ligation. Black arrows indicate neointima boundaries. Black arrowheads represent positive macrophage (anti-Mac3) and neutrophil (anti-NIMP-R14) staining. Black box identifies area of injured artery that is magnified below. Scale bars: 100 μm. For each carotid artery, Mac-3-positive and NIMP-R14-positive neointimal cells were counted separately in five distinct 40 × images at 400, 800 and 1200 μm proximal to the bifurcation, and average cells per HPF were calculated (n = 5 per group). There was no difference in number of macrophages or neutrophils per HPF in the intimal layer between WT and Nf1+/− mice.
Finally, homozygous deletion of Nf1 in myeloid cells alone significantly increased arterial occlusion (Fig. 4A). As a result, homozygous deletion of Nf1 in myeloid cells resulted in a significant increase in the percentage of lumen stenosis as compared to Nf1 heterozygous and WT mice, thus providing genetic evidence that ablation of both Nf1 alleles in myeloid cells further enhances neointima size in vivo (Fig. 4B). These data demonstrate that heterozygous inactivation of Nf1 in myeloid cells alone reproduces the vascular phenotype observed in Nf1+/– mice and identify Nf1+/– myeloid cells as the critical hematopoietic cell effector of NF1 vasocclusive disease.
Figure 4.
Arterial occlusion and stenosis is regulated by neurofibromin. (A) Representative Van Gieson-stained cross section of uninjured and injured carotid arteries from Nf1fl/fl;LysMcre mice. Black arrows indicate neointima boundaries. Scale bar: 100 μm. (B) Quantification of lumen stenosis of injured carotid arteries from WT, Nf1+/−, Nf1fl/+;LysMcre and Nf1fl/fl;LysMcre arteries. Data represent the mean percentage of lumen stenosis of three arterial cross sections (400, 800 and 1200 μm distal to the ligation) ± SEM, n = 10–12. *P < 0.01 for WT versus Nf1+/− and Nf1fl/+;LysMcre. **P < 0.01 for WT, Nf1+/−, Nf1fl/+;LysMcre mice versus Nf1fl/fl;LysMcre. Analysis by one-way ANOVA. There was no statistical difference between Nf1+/− and Nf1fl/+;LysMcre.
Rosuvastatin reduces the enhanced function of neurofibromin-deficient macrophages and VSMCs to growth factors in vitro
Neurofibromin-deficient myeloid progenitor cells and their differentiated progeny demonstrate increased proliferation, migration and survival in response to various growth factors implicated in neointima formation (15,16,27). Rosuvastatin attenuates monocyte recruitment and adhesion to the vascular wall, reduces inflammatory cytokine and MMP production and inhibits neointima formation following arterial injury in vitro and in vivo (28–30). Based on these observations and the predominance of Nf1+/– macrophages within Nf1+/– neointimas at day 28 following arterial injury, we tested whether rosuvastatin would abrogate the enhanced proliferation, migration and adhesion observed in Nf1+/– macrophages in vitro in response to M-CSF, which is a growth factor previously linked to vascular inflammation and arterial wall remodeling (27). Incubation of Nf1+/– macrophages with rosuvastatin prior to M-CSF stimulation diminished Nf1+/– macrophage proliferation, migration and adhesion when compared with control (Fig. 5A–C).
Figure 5.
Rosuvastatin reduces proliferation, migration and adhesion in Nf1+/− macrophages. (A) WT (white bars) and Nf1+/– (black bars) macrophage proliferation in response to stimulation with M-CSF (20 ng/ml) in the presence of rosuvastatin, where indicated. Data represent thymidine incorporation reported as mean counts per minute (cpm) ± SEM, n = 6. *P < 0.001 for WT versus Nf1+/– macrophages stimulated with M-CSF. **P < 0.001 for WT macrophages without growth factor versus WT macrophages incubated with M-CSF and Nf1+/– macrophages without growth factor versus Nf1+/– macrophages incubated with M-CSF. #P < 0.01 for Nf1+/– macrophages stimulated with M-CSF versus Nf1+/– macrophages stimulated with M-CSF in the presence of 10 and 20 μm rosuvastatin. Analysis was performed by one-way ANOVA. (B) WT (white bars) and Nf1+/– (black bars) macrophage migration in response to stimulation with M-CSF (100 ng/ml) in the presence of rosuvastatin, where indicated. Data represent average number of migrated cells per HPF ± SEM, n = 6. *P < 0.01 for WT macrophages versus Nf1+/– macrophages in the absence and presence of M-CSF. **P < 0.001 for WT macrophages without M-CSF versus WT macrophages incubated with M-CSF and Nf1+/– macrophages without M-CSF versus Nf1+/– macrophages incubated with M-CSF. #P < 0.001 for WT macrophages stimulated with M-CSF versus WT macrophages stimulated with M-CSF in the presence of 10 μm rosuvastatin and Nf1+/– macrophages stimulated with M-CSF versus Nf1+/– macrophages stimulated with M-CSF in the presence of 10 μm rosuvastatin. Analysis by one-way ANOVA. (C) WT (white bars) and Nf1+/– (black bars) macrophage adhesion to fibronectin. Data represent mean optical density ± SEM, n = 4. *P < 0.01 for WT versus Nf1+/– macrophages; **P < 0.01 for Nf1+/– macrophages versus Nf1+/– macrophages incubated with 10μm rosuvastatin at 15 and 30 min. Analysis by one-way ANOVA.
VSMC proliferation, which is partially induced by growth factor secretion from infiltrating macrophages, within the intimal layer of the arterial wall is the central mechanism of Nf1+/– arterial stenosis. We previously demonstrated that Nf1+/− VSMCs have increased proliferation to various growth factors and Nf1+/− macrophages both in vitro and in vivo (12,15). Therefore, we tested whether rosuvastatin would abrogate the increased proliferation of Nf1+/− VSMCs in response to TNFα, which is a cytokine secreted by circulating macrophages, induces VSMC proliferation through Ras-Erk signaling and is critical for neointima formation (31–33). Nf1+/− VSMCs demonstrated increased proliferation when compared with WT controls in response to multiple doses of TNFα (Fig. 6A). However, 5 and 10 μm rosuvastatin reduced Nf1+/− VSMC proliferation to WT levels without growth factor stimulation (Fig. 6B). Collectively, these data demonstrate that rosuvastatin inhibits multiple cell functions of both Nf1+/− macrophages and VSMCs that are essential for the initiation and progression of neointima formation.
Figure 6.
Nf1+/− VSMCs have increased response to TNFα that is abrogated by incubation with rosuvastatin. (A) WT (white bars) and Nf1+/– (black bars) VSMC proliferation in response to stimulation with TNFα. Data represent thymidine incorporation reported as mean cpm ± SEM, n = 5. *P < 0.001 for WT versus Nf1+/– VSMCs stimulated with indicated concentrations of TNFα. **P < 0.01 for WT and Nf1+/– VSMCs versus WT and Nf1+/– VSMCs stimulated with indicated concentrations of TNFα. Analysis by one-way ANOVA with Tukey's post-hoc test. (B) WT (white bars) and Nf1+/– (black bars) VSMC proliferation in response to stimulation with TNFα in the presence of rosuvastatin, where indicated. Data represent thymidine incorporation reported as mean cpm ± SEM, n = 6. *P < 0.001 for WT VSMC stimulated with TNFα versus Nf1+/– VSMCs stimulated with TNFα. **P < 0.001 for WT and Nf1+/– VSMCs versus WT and Nf1+/– VSMCs stimulated with TNFα. #P < 0.001 for Nf1+/– VSMCs stimulated with TNFα versus Nf1+/– VSMCs stimulated with TNFα in the presence of 5 and 10 μm rosuvastatin. Analysis by one-way ANOVA.
Administration of rosuvastatin inhibits neointima formation in Nf1+/– mice, following arterial injury in vivo
The pleiotropic effects of statins are well described and function independent of their lipid-lowering properties (34–36). Although specific mechanisms for their anti-inflammatory effects remain elusive, several studies provide evidence that statins reduce acute phase reactants and inflammatory cytokines linked to cardiovascular disease and arterial remodeling (34,35). Furthermore, statin use in experimental animal models reduces neointima formation, following vascular injury (30,36,37).
Given these observations, we tested whether administration of oral rosuvastatin would inhibit neointima formation in Nf1+/− mice following mechanical arterial injury. Utilizing the carotid artery ligation model previously described, Nf1+/− and WT mice were administered either phosphate buffered saline (PBS) or 10 mg/kg rosuvastatin daily for 7 days prior to vascular injury and continued on daily dosing through tissue harvest at 28 days post-injury. In response to carotid artery injury, rosuvastatin treatment greatly reduced neointima formation in Nf1+/− mice when compared with PBS controls (Fig. 7A–C). Morphometric analysis of rosuvastatin-treated Nf1+/− mice demonstrated a 40% reduction in neointima area and a 50% reduction in I/M ratio when compared with PBS-treated Nf1+/− mice in response to arterial injury (Fig 7B and C). No significant difference in either intima area or I/M ratio in response to injury was observed in WT mice when comparing treatment groups. Importantly, rosuvastatin did not alter total cholesterol, triglyceride levels or circulating peripheral blood hematopoietic and progenitor cell populations in either genotype (data now shown).
Figure 7.
Rosuvastatin reduces neointima formation in Nf1+/− mice. (A) Representative Van Gieson-stained cross sections of uninjured and injured carotid arteries from rosuvastatin-treated WT and Nf1+/– mice. Representative photomicrographs of H&E-stained carotid arteries from WT (top panels) and Nf1+/− (bottom panels) mice 28 days, following no injury (left panels), injury and PBS treatment (middle panels) or injury and rosuvastatin treatment (right panels). Black arrows indicate neointima boundaries. Black boxes identify area of injured artery that is magnified below. Scale bars: 100 μm. (B and C) Quantification of neointima area (B) and I/M ratio (C) of injured carotid arteries from PBS and rosuvastatin-treated WT and Nf1+/– mice. Data represent the mean neointima area of three arterial cross sections (400, 800 and 1200 μm distal to the ligation) ± SEM, n = 10–12. *P < 0.01 for WT uninjured versus WT injured with PBS treatment and Nf1+/− uninjured versus Nf1+/− injured with PBS treatment. **P < 0.01 for WT injured with PBS treatment versus Nf1+/– injured with PBS treatment. #P < 0.01 for Nf1+/− injured with PBS treatment versus Nf1+/− injured with rosuvastatin treatment. Analysis by one-way ANOVA.
DISCUSSION
Cardiovascular disease is an important contributor to excess morbidity and mortality in NF1 patients (4). Currently, there are no recommendations for screening NF1 patients for cardiovascular disease and specific treatment options are limited. Thus, a true estimate of the disease burden and natural pathology of NF1-related vasculopathy remains elusive.
In this study, we demonstrate that inactivation of Nf1 in myeloid cells alone is sufficient to mobilize a discrete population of inflammatory murine monocytes and enhance neointima formation when compared with WT mice. Specifically, Nf1fl/+;LysMcre mice form neointimas similar to Nf1+/− mice with VSMC accumulation limited to the intimal layer, which completely replicates the phenotype previously observed in Nf1+/− mice. Although germline loss of both NF1 alleles in myeloid cells has not been demonstrated in NF1 vascular disease, loss of the second Nf1 allele in mice further mobilizes Ly6Chi monocytes and amplifies the degree of arterial stenosis providing genetic evidence that Nf1 is a critical regulator of myeloid cell function and neointima formation. Using isolated primary cells, we show that Nf1+/− macrophages have increased proliferation, migration and adhesion in response to M-CSF, which is significantly reduced when cells are preincubated with rosuvastatin. Furthermore, we show that preincubation of TNFα-stimulated Nf1+/− VSMCs with rosuvastatin reduces their proliferation to levels observed in unstimulated WT VSMCs. This is an important observation because VSMC proliferation is a critical cellular mechanism of arterial stenosis and neointima formation. Finally, based on these in vitro results, we show that once daily administration of oral rosuvastatin is sufficient to reduce neointima formation in Nf1+/− mice after carotid artery ligation in vivo.
Neurofibromin functions as a negative regulator of Ras activity in myeloid cells and renders them hypersensitive to growth factors (1,38). Specifically, granulocyte-macrophage progenitors, mast cells and osteoclasts are hypersensitive to multiple growth factors predisposing some NF1 patients to the development of juvenile myelomonocytic leukemia, plexiform neurofibromas and bone disorders (16,38,39). Thus, the finding that neurofibromin is a critical regulator of monocyte/macrophage function and that loss of neurofibromin in myeloid cells directly contributes to Nf1+/− neointima formation is consistent with other manifestations of NF1. Furthermore, asymptomatic NF1 patients have increased circulating levels of pro-inflammatory monocytes and cytokines linked to cardiovascular disease supporting the hypothesis that neurofibromin is a critical regulator of vascular homeostasis and that heterozygous inactivation of NF1 leads to vascular inflammation (15). In support of this hypothesis, murine Ly6Chi monocytes, an important pro-inflammatory monocyte population, are independently mobilized in mice with heterozygous and homozygous deletion of Nf1 in myeloid cells, which is similar to Nf1+/− mice. Importantly, Ly6Chi monocytes express high cell surface levels of the CCR2 receptor and are actively recruited to sites of inflammation and differentiate into macrophages and dendritic cells (40,41). Thus, the finding that Ly6Chi monocytes are increased in Nf1+/− mice and mobilized in a cell-autonomous manner indicates that CCR2 signaling may be important in Nf1+/− neointima formation. Ongoing studies are currently investigating this hypothesis in an attempt to further identify therapeutic targets to treat NF1 vascular disease because pharmaceutical agents are already available to inhibit the CCR2 signaling axis.
Emerging evidence suggests that Ras signaling tightly regulates vascular wall homeostasis at rest and in response to growth factor stimulation, and perturbations of Ras signaling can result in a variety of cardiovascular disease manifestations. In fact, patients with diverse genetic disorders that affect Ras signaling, which are now termed the ‘Rasopathies,’ display various degrees of cardiovascular disease.
Recent animal studies also demonstrate that amplification of Ras signaling significantly enhances neointima formation, whereas inhibition of the canonical Ras-Mek-Erk pathway reduces neointima formation and VSMC proliferation and migration in vivo (42,43). In support of the hypothesis that enhanced Ras signaling augments neointima formation, previous histologic examination of arterial cross sections from Nf1+/− neointimas showed increased Erk phosphorylation when compared with WT controls (14). Furthermore, treatment of Nf1+/−mice with Gleevec, an important inhibitor of the PDGF-BB-Ras-Erk signaling pathway, significantly reduced neointima formation and Erk phosphorylation in Nf1 heterozygous mice (14). Although mounting evidence suggests that Ras-Erk signaling actively participates in hematopoietic cell recruitment to the injured artery, regulates proliferation and migration of VSMCs and augments remodeling of the vascular wall, specific drug-targeted inhibition of the Ras-Mek-Erk pathway to reduce neointima formation following mechanical injury has not been demonstrated in vivo. Testing whether current MEK inhibitors, which are in clinical use for other diseases, inhibit neointima formation in our NF1 mouse model of vascular disease could provide further therapeutic insights.
Statins have been used in several NF1 patient populations and animal models of NF1-related pathologies as an investigational therapeutic agent. In murine models of NF1 skeletal dysgenesis, statin use improved bone marrow mineralization and reduced Ras activation in neurofibromin-deficient osteoblasts and osteochondroprogenitors, a subset of the myeloid cell lineage (16,44). Interestingly, statin use improves cognitive function in Nf1 heterozygous mice and regulates functional connectivity in the brain of NF1 patients, although it failed to produce significant improvement in cognitive functioning in a randomized clinical trial, which demonstrates the complex functionality of statins as a therapeutic agent in treating non-neoplastic manifestations of NF1 (45).
Statins are an important clinical therapeutic with wide-ranging pleiotropic effects that appear to extend beyond their lipid-lowering profile and may protect patients with subtle cardiovascular risk factors from developing overt cardiovascular disease (34–36,46). Although these effects may be generalizable to the entire statin group, human studies assessing the effect of rosuvastatin demonstrate a reduction in major cardiovascular events in asymptomatic patients with elevated inflammatory markers without hyperlipidemia (18,19). These studies suggest that chronic inflammation contributes to cardiovascular disease and that rosuvastatin may possess anti-inflammatory properties through inhibition of various signaling pathways in myeloid cells that control the secretion of inflammatory mediators, though the mechanism for these effects is not completely understood (18,19). Relevant to the current study, previous murine studies of primary cells and neointima formation demonstrated that rosuvastatin reduced VSMC proliferation and neointima formation in vitro and in vivo (17,30). More importantly, emerging evidence from murine models and human clinical trials suggests that statins, particularly atorvastatin and rosuvastatin, suppress the production of reactive oxygen species and cytokines that contribute to vascular inflammation (28,47,48). Of note, in contrast to prior studies, rosuvastatin (10 mg/kg/day) failed to diminish neointima formation significantly in WT mice. However, prior studies in mouse and rat neointima models showed significant neointima reduction at a higher daily dosing range of 20–40 mg/kg/day (30,49,50), which could potentially explain differences in our experimental results. Furthermore, statins display divergent effects on important signaling cascades, including Jak-STAT, Ras-Mek-Erk and PI-3K, which contribute to the various observed cardiovascular effects (51,52). Thus, the increased response of Nf1+/− myeloid cells to growth factors likely renders them more sensitive to alterations in transcellular signaling pathways and more responsive to lower doses of rosuvastatin in vivo. Given the pleiotropic effects of rosuvastatin, understanding the precise molecular mechanism of how this therapy diminishes neointima formation in Nf1+/− mice will provide more insights into the molecular mechanism of NF1 vascular disease and will potentially reveal new therapeutic options.
In this report, we showed that heterozygous inactivation of Nf1 in myeloid cells is sufficient to enhance neointima formation in Nf1+/− mice. This is the first report demonstrating that loss of the Nf1 allele in a single cell lineage is sufficient to cause cardiovascular disease and provides potential important insights into the development of cardiovascular disease in NF1 patients. Consistent with our previous observations in NF1 patients, we also observed a cell autonomous mobilization of inflammatory monocytes in Nf1+/− and Nf1fl/+;LysMcre mice, which supports our overarching hypothesis that NF1 patients experience chronic inflammation that predisposes them to cardiovascular disease. Finally, we show that rosuvastatin reduces neointima formation in Nf1+/− mice when compared with placebo, which may be a therapeutic option for the prevention and/or treatment of NF1 patients predisposed to cardiovascular disease.
MATERIALS AND METHODS
Animals
Protocols were approved by the Indiana University Laboratory Animal Research Center. Nf1+/– mice were obtained from Tyler Jacks (Massachusetts Institute of Technology, Cambridge, MA, USA) and backcrossed for 13 generations into the C57BL/6J strain. Nf1fl/fl mice were obtained from Luis Parada (University of Texas Southwestern Medical Center, Dallas, TX, USA) and backcrossed for 13 generations into the 129SvJ strain (53). LysMcre (stock 4781) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained on C57BL/6 strain. Nf1fl/fl mice were crossed with LysMcre mice to generate C57BL/6 × 129SvJ pups. Nf1fl/+;LysMcre (heterozygous loss of Nf1 in myeloid cells only) and Nf1fl/fl;LysMcre (homozygous loss of Nf1 in myeloid cells only) mice were used for experiments. LysM is expressed in neutrophils and macrophages (54). LysM is expressed in monocytes (80% efficiency) and granulocytes (100% efficiency) (26). Cre-mediated recombination was confirmed by PCR as previously described (53). Crossing Nf1fl/fl;129SvJ mice with Nf1+/– C57BL/6 mice generated Nf1 heterozygous and WT controls for experiments. Male mice, between 12 and 15 weeks of age, were used for experiments.
Carotid artery ligation
Carotid artery injury was induced by ligation of the right common carotid artery as previously described (15). Briefly, mice were anesthetized by inhalation of an isoflurane (2%)/oxygen (98%) mixture. Under a dissecting scope, the right carotid artery was exposed through a midline neck incision and ligated proximal to the bifurcation using a 6-0 silk suture. The contralateral carotid artery was sham ligated as a control. Mice were administered 15 μg of buprenorphine via IP injection following the procedure and recovered for 7 days or 28 days without complication.
Rosuvastatin administration
When stated, rosuvastatin (10 mg/kg/day; AstraZeneca, London, UK) was administrated once per day via oral gavage. Rosuvastatin treatment commenced 7 days prior to carotid artery injury and continued through tissue harvest at 28 days post-injury. PBS was given in an equivalent volume as a control.
Morphometric analysis
Van Gieson-stained arterial cross sections 400, 800 and 1200 μm proximal to the ligation were analyzed for neointima formation using Image J (National Institute of Health, Bethesda, MD, USA). Lumen area, area inside the internal elastic lamina (IEL) and area inside the external elastic lamina (EEL) were measured for each cross section. To account for potential thrombus formation, arteries containing significant thrombus (>50% lumen occlusion) at 400 μm proximal to the aorta were excluded from analysis. Intima area was calculated by subtracting the lumen area from the IEL area, and the media area was calculated by subtracting the IEL area from the EEL area. I/M ratio was calculated as intima area divided by media area. Percentage of lumen stenosis was calculated as intima area divided by IEL area and reported as a percentage value. Finally, due to variability in arterial remodeling over the length of the injured artery, photomicrographs in each of the figures demonstrate representative arterial cross sections between 800 and 1200 μm proximal to the carotid bifurcation.
Histopathology and immunohistochemistry
Twenty-eight days after ligation, whole ligated and contralateral uninjured carotid arteries were harvested from mice as previously described (14,15). Briefly, mice were anesthetized with an isoflurane (2%)/oxygen (98%) mixture and were perfusion fixed at constant pressure (100 mmHg) with 10 ml of 125 mm adenosine plus 0.0125 mm sodium nitroprusside for 5 min followed by Z-fix solution (Anatech, Battle Creek, MI, USA). Under a dissecting scope, the injured and uninjured common carotid arteries were excised, then fixed overnight at 4°C in Z-fix solution and paraffin embedded. Serial 7 μm arterial cross sections were made at 200 μm intervals across the length of the carotid artery. Van Gieson staining was performed according to standard methods (EMS, Hatfield, PA, USA).
For immunohistochemistry, sections were blocked for endogenous peroxidase activity with 3% hydrogen peroxide in methanol, following antigen retrieval in Antigen Unmasking Solution (Vector Laboratories, Berlingame, CA, USA) at 95°C. Sections were blocked with Protein Block (Dako, Denmark) for 1 h and were incubated with anti-Mac3 (1:50; BD Biosciences, San Jose, CA, USA), anti-NIMP-R14 (1:50; Abcam, Cambridge, MA, USA) or anti-Ki67 (1:25; Dako) primary antibodies. Sections were incubated with appropriate biotinylated secondary antibody (Vector Laboratories) and visualized by 3,3′-diaminobenidine (Vector Laboratories) and counterstained with hematoxylin. Sections were examined, and images of sections were collected using a Zeiss Axioskop microscope (Carl Zeiss, Chester, VA, USA) with a 20× or 40 × CP-ACHROMAT/0.12NA objective. Images were acquired using a SPOT RT color camera (Diagnostic Instruments, Sterling Heights, MI, USA).
Polychromatic flow cytometry
Blood samples were obtained via tail vein at days 0, 7 and 28, following injury and collected into EDTA tubes (BD Biosciences, San Jose, CA, USA). Peripheral blood was collected separately for white blood cell count using the Hemavet 950 (Drew Scientific, Dallas, TX, USA). Red blood cell lysis was performed (Qiagen, Germantown, MD, USA), and cells were washed in 2% fetal bovine serum (FBS) in PBS. Following centrifugation, each sample was resuspended in 2% FBS in PBS and incubated with murine Fc Blocking Reagent (Miltenyi Biotec, Cologne, Germany) for 10 min on ice. Cell aliquots were incubated with either CD 115-PE (eBioscience, San Diego, CA, USA), CD11c-PERCPCy5.5 (BD Biosciences), CD11b-APC-Cy7 (BD Biosciences), Ly6C-PE-Cy7 (BD Biosciences), F4/80-APC (Serotech, Oxford, UK), F7/4-FITC (Serotech), TER119-Pac Blue (eBioscience) and LIVE/DEAD violet-fixable dead cell stain (Invitrogen, Grand Island, NY, USA) or separately CD34-FITC (BD Biosciences), CD117-APC (BD Biosciences), FceR1a-PE (eBioscience), CD19-PECy7 (BD Biosciences), CD3-APCCy7 (BD Biosciences), TER-119-Pac Blue (eBioscience) and LIVE/DEAD violet-fixable dead cell stain (Invitrogen) for 30 min and resuspended in 2% FBS in PBS. Antibodies were titered for optimal staining, and ‘fluorescence-minus-one’ gating controls were performed to determine true positive and negative events. Stained samples were acquired on a BD LSRII flow cytometer equipped with a 405-nm violet laser, 488-nm blue laser and 633-nm red laser. At least 100 000 events were collected for samples. Data were collected uncompensated and analyzed using FlowJo software version 8.7.3 (Tree Star). After excluding red blood cells, apoptotic and dead cells, total Ly6Chi monocytes were calculated by multiplying the percentage of Ly6Chi cells per total live cells by the peripheral white blood cell count for each individual sample.
Isolation of bone marrow-derived macrophages and characterization
Bone marrow-derived macrophage isolation was performed as described (15). Macrophage proliferation, migration and adhesion assays were performed as described (15). Where indicated, cells were pretreated with the indicated concentration of rosuvastatin or vehicle for 1 h.
VSMC isolation and proliferation
VSMC isolation and subsequent proliferation assays were performed as described (12). Where indicated, cells were pretreated with the indicated concentration of rosuvastatin or vehicle for 1 h.
Statistical analysis
All values are presented as mean ± SEM. Circulating monocyte frequency was assessed by one-way ANOVA. Intima area and I/M ratio analysis was assessed by one-way ANOVA with a Tukey's post hoc test using GraphPad Prism version 5.0d (GraphPad Software, San Diego, CA, USA). P < 0.05 were considered significant.
Conflict of Interest statement. None declared.
FUNDING
This work was supported by the National Institutes of Health (P50 NS052606 to D.A.I. and S.J.C.). Brian Stansfield is a Fellow of the Pediatric Scientist Development Program and was supported by (K12HD000850 to B.K.S.) from the Eunice Kennedy Shriver National Institute of Child Health & Human Development.
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