Vav guanine nucleotide exchange factors regulate atherosclerotic lesion development in mice

Abstract

Objective

Atherosclerosis requires migration of monocytes to the arterial intima with subsequent differentiation into foam cells. We showed previously that the scavenger receptor CD36 contributes to activation of Vav family guanine nucleotide exchange factors (Vavs) in aortae from hyperlipidemic apoe null mice, and that oxidatively modified LDL (oxLDL) induced CD36-dependent activation of macrophage Vavs in vitro. We also discovered that CD36-dependent uptake of oxLDL and foam cell formation were reduced in Vav-deficient macrophages. We now tested the hypothesis that Vavs play a role in atherosclerotic lesion development.

Approach and Results

We showed that apoe/vav1 double null mice fed a western diet had significant reduction in total aortic lesion area (by en face analysis) compared with apoe null mice with no significant differences in body weight or plasma lipid profiles. Histologic analysis of aortic sinus lesions showed fewer macrophages and foam cells in double null mice compared to apoe null, indicating impaired foam cell generation and/or homing of macrophages to atherosclerotic lesions. An intravital video microscopy-based adhesion assay with fluorescent (Qtracker655)-labeled monocytes showed reduced adhesion of vav1 null monocytes to hyperlipidemic carotid arteries compared to WT monocytes. Furthermore, fewer fluorescently labeled vav1 null monocytes accumulated in aortic sinus lesions in hyperlipidemic apoe null mice. We also found that activation of RhoGTPase Rac and MAP kinase c-Jun N-terminal kinase-2 (JNK2) by CD36-specific oxidized phospholipids was dependent on Vavs.

Conclusions

These results for the first time link Vavs to atherosclerotic lesion development and suggest that Vavs act as critical molecular links coupling hyperlipidemia with pro-atherogenic monocyte/macrophage responses.

Introduction

Atherosclerosis is a complex inflammatory process driven in part by macrophage internalization of oxidized low density lipoproteins (oxLDL) by scavenger receptors including CD36 and scavenger receptor A with subsequent formation of lipid-laden “foam cells,” the primary component of the atherosclerotic lesions.^1-3 Cumulative studies from our group and others demonstrated that CD36 accounts for a large proportion of foam cell formation in vitro and in vivo and that interruption of CD36 expression or downstream signaling blocks oxLDL uptake and limits experimental atherosclerosis in mice.^3-10 The degree of athero-protection in hyperlipidemic cd36 null mice differs in different genetic and diet models, but all showed either smaller lesions or less complex lesions which may be related to the level of systemic inflammation and oxidant stress.^7,10,11-13 CD36 is a multifunctional, multiligand transmembrane receptor expressed in a diverse array of cells including monocytes/macrophages, platelets, adipocytes, and myocytes.¹⁴ Numerous studies have revealed that CD36 acts as a signaling receptor, transmitting signals via Src family kinases Lyn and Fyn, and MAP kinases JNK and p38, in response to multiple endogenous and exogenous ligands, including microbial pathogens, oxLDL, apoptotic cells, cell-derived microparticles, thrombospondin-related proteins, advanced glycation end products and amyloid peptides.^14-16 The precise molecular details, however, by which ligation of CD36 leads to recruitment and activation of a signaling complex are not fully understood. We showed previously that the CD36 contributes to activation of Vav family guanine nucleotide exchange factors (Vavs) in aortae from hyperlipidemic apoe null mice, and that oxLDL induced CD36-dependent activation of macrophage Vavs in vitro.¹⁷ We also discovered that CD36-dependent internalization of oxLDL and foam cell formation were reduced in Vav-deficient macrophages.¹⁷ Our subsequent studies revealed that Vavs regulate CD36-mediated macrophage foam cell formation via calcium and dynamin-dependent processes.¹⁸ Here, we showed that apoe/vav1 double null mice fed a western diet had >60% reduction in total aortic lesion area (by en face analysis) compared with apoe null mice, suggesting Vavs as a critical regulator of atherosclerosis.

The three members of the Vav family are multidomain signaling proteins that act as guanine nucleotide exchange factors for RhoGTPases and also function as adaptor proteins, interacting directly with signaling molecules including dynamin, phospholipase C-(X003B3), ZAP70, and Lyn.^19-24 In these capacities Vavs may regulate multiple processes, including NADPH oxidase-mediated generation of reactive oxygen species and fission of endocytic vesicles from the plasma membrane.^19-24 Among the three members, Vav1 is expressed exclusively in hematopoietic cells, whereas Vav2 and Vav3 are ubiquitously expressed. Vavs have both unique and overlapping functions and can be activated by multiple pathways, including ligation of antigen receptors, integrins, growth factor receptors, and chemokine receptors.^19-24 Since Vav signaling impacts processes relevant to atherogenesis and involves pathways common to CD36 signaling, we hypothesized that Vavs could mechanistically link oxLDL-mediated CD36 signaling in macrophages to proatherogenic responses, and therefore, tested the hypothesis that Vavs play a role in atherosclerotic lesion development. In this manuscript, we present results for the first time linking Vavs to atherosclerotic lesion development and suggest that Vavs act as critical molecular links coupling hyperlipidemia with pro-atherogenic monocyte/macrophage responses. Mechanistically we demonstrate that Vavs regulate atherosclerotic lesion development by controlling foam cell generation and/or homing of macrophages to atherosclerotic lesions. We also found that activation of Rac and JNK2 by CD36-specific oxidized phospholipids was regulated by Vavs.

Materials and Methods

Vav1 null mice were created by Dr. V. Tybulewicz and were provided by Dr. J. Rivera (National Institutes of Health, Bethesda, MD). Vav1/vav3 double null mice were provided by Dr. W. Swat (Washington University). Vav1 null mice were crossed with apoe null mice to obtain apoe/vav1 double null mice. Mice were backcrossed 10 times to C57B1/6 and background matched WT control lines were used in all studies. Details of the Materials and Methods can be found in the online only Supplement section.

Results and Discussion

Atherosclerotic lesion development in apoe null mice is ameliorated by deletion of vav1

To test the hypothesis that Vavs play a role in atherosclerosis we generated apoe/vav1 double null mice and examined atherosclerotic plaque development after 12 weeks of high fat “western” diet. We found by en face analysis of aortic trees that lesion surface area was ∼ 60% less (p<0.02) in both male and female apoe/vav1 null mice compared to apoe null mice (Figures 1A and B). Cross sectional analysis of lesion development at the level of the aortic sinus was also assessed and revealed significantly smaller lesions in apoe/vav1 null compared to apoe null mice (Figures 1C). Mice maintained on chow diet for 12 weeks showed much smaller lesions than high fat diet fed animals, but no differences between the groups tested (data not shown), consistent with our published studies in which we found that Vavs were phosphorylated in aorta from high fat diet fed mice but not aorta from chow fed mice. This is likely to be related to western diet-dependent generation of CD36-specific atherogenic lipids in apoE-/- mice.^6,17. There were no significant difference in body weight gain, plasma lipoprotein profiles, plasma total cholesterol (1128 ± 204 vs 1098 ± 179 mg/dL) or triglyceride (124 ± 12 vs 114 ± 11 mg/dL) levels comparing apoe/vav1 null to apoe null mice (Supplemental Figures IA and B), suggesting that the observed athero-protective effect may be linked to absence of Vav-dependent functions in this model.

Figure 1. Disruption of vav1 decreases atherosclerotic lesion development in aortic tree and sinuses in apoe null mice.

(A and B) Lesion area in the aortic tree was assessed by en face oil red-o positive staining. Total oil red-o positive area was determined using Adobe Photoshop. Each symbol represents data from a single mouse. Mann-Whitney test was used for statistical analysis. (C). Lesion area in the aortic sinuses was assessed by oil red-o positive staining. Total oil red-o positive area was determined using Adobe Photoshop. Each symbol represents data from a single mouse. Mann-Whitney test was used for statistical analysis. (D). The aortic sinus cross-sectional area containing macrophages was assessed by anti-CD68 positivity measured using NIH ImageJ software. Mann-Whitney test was used for statistical analysis (n = 5). Representative images are shown. Control sections were stained with IgG.

Since Vav1 is exclusively expressed in hematopoietic cells our data suggest that the atheroprotective phenotype is most likely related to loss of monocyte/macrophage Vav1. We thus examined cross sections of lesions obtained at the level of the aortic sinus histologically and found that the lesions in the apoe/vav1 double null mice had markedly fewer macrophages than those in the apoe nulls (Figure 1D), as determined by immunostaining with anti-CD68 IgG. Animals maintained on normal chow diet showed no differences in lesion size or macrophage content (data not shown). In view of the functional redundancy of Vavs and our earlier data showing that all 3 Vavs were activated in vivo in western diet-fed apoe null we speculate that the degree of athero-protection would be even greater if Vav 2 and/or Vav3 were also absent.

Disruption of vav1 causes reduced adhesion of monocytes to carotid arteries and decreased accumulation of macrophages in atherosclerotic lesions in hyperlipidemic apoe null mice

Macrophage accumulation in atherosclerotic plaque is a complex process requiring adhesion of circulating monocytes to “injured” arterial endothelium and transmigration of the adherent cells into the intima where they differentiate into macrophages, internalize lipid to form foam cells, and then become trapped within the neointima. Our previous studies showed a role for CD36 signaling in foam cell formation and entrapment and other studies showed a role for Vav proteins in monocyte migration.^{4,5,7,8,25,26-29} We thus developed an in vivo fluorescence tracking method to assess monocyte adhesion to atherosclerosis-prone arterial surfaces and accumulation in plaque. The fate of Qtracker655-labeled vav1 null or control C57Bl/6 (WT) monocytes injected into hyperlipidemic apoe null mice was monitored by intravital video-microscopy. Figure 2A shows significantly reduced adhesion of vav1 null monocytes to the endothelium of atherosclerosis-prone carotid arteries compared to that of WT monocytes (p = 0.04).

Figure 2. Adhesion and homing of monocytes to carotid arteries in hyperlipidemic apoe null mice is dependent on Vav1.

(A) Images from intravital video microscopy showing in vivo adhesion of Qtracker655-labeled monocytes injected into carotid artery of apoe null mice. Saline injected mice were used as control (data not shown). Images are recorded for 3 min along a region of interest (2000um in length), which permits counting of the number of labeled monocyte firmly adhered to aorta. Mann-Whitney test was used for statistical analysis (n = 6). Representative images are shown. (B) Microscopy images showing homing of Qtracker655-labeled injected monocytes in aortic sinus lesion in apoe null mice. DAPI stain showing total number of cells. Mann-Whitney test was used for statistical analysis (n = 6) and the representative images are shown.

We then examined aortic sinus lesions 4 hours after injection of Qtracker655-labeled vav1 null or WT monocytes and found fewer fluorescently tagged cells in the plaques after infusion of vav1 null cells compared to WT cells (Figure 2B). Together, these results showed that in hyperlipidemic conditions Vavs play a critical role in regulating homing of monocytic cells to atherosclerotic lesions.

Activation of Rac and JNK2 by CD36-specific oxidized phospholipids was dependent on Vavs

Rho family GTPases are downstream effector of Vavs and are important regulators of cytoskeletal remodeling associated with cell migration.^26-29 In light of our observation that macrophage homing to plaque in apoe null mice was reduced in the absence of Vav1 (Figure 2B), we hypothesized that Vav-dependent activation of RhoGTPases regulates monocyte migration in the hyperlipidemic pro-atherogenic milieu. To support this hypothesis we measured activation of Rac and RhoA in WT, vav1 null and vav1/vav3 double null monocytes exposed to NO₂LDL, a form of oxidized LDL that is a highly specific ligand for CD36.^4,5 As shown in Supplemental Figure IIA, Rac1 was rapidly activated by NO₂LDL in WT cells and activation was reduced by more than 50% in cells deficient in Vav1 or Vav1/Vav3, suggesting a critical role for Vav1 proteins in oxLDL-induced activation of RhoGTPases. As shown previously, GTP-bound RhoA was not changed by NO₂LDL in WT and Vav null monocytes (Supplemental Figure IIB). The lack of change of GTP-RhoA may be related to the presence of Vav2 in the vav1 null or vav1/vav3 double null cells. Given that Rac is implicated in JNK activation ^30,31, we also hypothesize that the Vav-RhoGTPase signaling axis may be involved in activation of JNK2. In support of this hypothesis we found that activation of JNK2 in vav1 null and vav1/vav3 double null monocytes exposed to specific oxidized phospholipid CD36 ligands - POV-PC was 2-3 fold reduced compared to WT cells (Supplemental Figure IIC).

Adhesion of monocytic cells to specific atherosclerosis-prone regions of arterial endothelium is a critical early event in the atherosclerotic process. Adherent monocytes subsequently enter the vessel wall where they become trapped and eventually differentiate into foam cells as a result of dysregulated internalization of modified lipoproteins. We now found that in the apoe null model monocyte adhesion to aortic endothelium and their accumulation at lesion-prone areas was partially dependent on Vav1, suggesting an important role for Vav1 in homing or migration of monocytes under hyperlipidemic conditions. Macrophage foam cells are present in all stages of atherosclerosis and participate in many pathologic processes including inflammatory responses, tissue remodeling within the arterial intima, and rupture of plaques. Various pro-inflammatory cytokines and growth factors secreted by foam cells induce local inflammatory responses, increase ROS in the lesion, and promote the migration of hematopoietic cells to the intima. Our recent data revealed that CD36-dependent endocytosis of oxLDL and foam cell formation was significantly reduced in vav null macrophages ¹⁷, supporting the notion that Vav-dependent processes are involved in foam cell formation. Thus we conclude that Vavs act as a critical molecular link that couples hyperlipidemia with multiple pro-atherogenic monocyte/macrophage responses and, consequently, plays an important role in atherogenesis.

In summary, we utilized a multipronged approach involving apoe/vav1 double null mice, monocytes/macrophages from vav1 and vav1/3 null mice, highly specific oxidized phospholipid CD36 ligands, and in vitro and in vivo assays to identify mechanisms that link CD36 to atherosclerotic lesion development via Vav proteins. We reported previously that CD36-dependent activation of Vavs and JNK2 play essential role in macrophage oxLDL internalization and foam cell formation.^17,18 We now show that these pathways are important for atherosclerotic lesion development in vivo and that Vavs are involved in oxLDLmediated Rac and JNK activation in monocyte/macrophages. Based on current data and our previously published reports we propose that oxLDL binding to CD36 results in Src family kinase-dependent activation of Vav proteins, which subsequently induce activation of RhoGTPase Rac which in turn leads to activation of JNK2 and promotion of monocyte homing to the endothelium of pro-atherogenic arteries and migration into plaque. These studies suggest new targets for therapeutic intervention in hyperlipidemia-induced vascular diseases.

Significance.

This work combines use of a well-established mouse atherosclerosis model system (apoe null), in vitro mechanistic approaches, and new in vivo mechanistic studies to identify a role for a specific guanine nucleotide exchange factor family (Vav) in the pathogenesis of atherosclerosis and links the phenotype directly to Rac and Jnk activation and to altered monocyte trafficking to the atherogenic arterial wall. To our knowledge this is the first direct report of a specific Rac/Rho GEF playing a pathogenic role in murine atherosclerosis, thus identifying potential new targets for therapeutic intervention.