Amanda C. Doran

Role of Smooth Muscle Cells in the Initiation and Early Progression of Atherosclerosis

The initiation of atherosclerosis results from complex interactions of circulating factors and various cell types in the vessel wall, including endothelial cells, lymphocytes, monocytes, and smooth muscle cells (SMCs). Recent reviews highlight the role of activated endothelium and inflammatory cell recruitment in the initiation of and progression of early atherosclerosis. Yet, human autopsy studies, in vitro mechanistic studies, and in vivo correlative data suggest an important role for SMCs in the initiation of atherosclerosis. SMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli can modify the type of matrix proteins produced. In turn, the type of matrix present can affect the lipid content of the developing plaque and the proliferative index of the cells that are adherent to it. SMCs are also capable of functions typically attributed to other cell types. Like macrophages, SMCs can express a variety of receptors for lipid uptake and can form foam-like cells, thereby participating in the early accumulation of plaque lipid. Like endothelial cells, SMCs can also express a variety of adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to which monocytes and lymphocytes can adhere and migrate into the vessel wall. In addition, through these adhesion molecules, SMCs can also stabilize these cells against apoptosis, thus contributing to the early cellularity of the lesion. Like many cells within the developing plaque, SMCs also produce many cytokines such as PDGF, transforming growth factor-beta, IFNgamma, and MCP-1, all of which contribute to the initiation and propagation of the inflammatory response to lipid. Recent advances in SMC-specific gene modulation have enhanced our ability to determine the role of SMCs in early atherogenesis.

Identification of a Novel Macrophage Phenotype That Develops in Response to Atherogenic Phospholipids via Nrf2

Rationale: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known. Objective: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. Methods and Results: We show that oxidized phospholipid-treated murine macrophages develop into a novel phenotype (Mox) that is strikingly different from the conventional M1 and M2 macrophage phenotypes. Compared to M1 and M2, Mox macrophages show a different gene expression pattern, as well as decreased phagocytotic and chemotactic capacity. Treatment with oxidized phospholipids induces both M1 and M2 macrophages to switch to the Mox phenotype. Whole-genome expression array analysis and subsequent gene ontology clustering revealed that the Mox phenotype was characterized by abundant overrepresentation of Nrf2-mediated expression of redox-regulatory genes. In macrophages isolated from Nrf2−/− mice, oxidized phospholipid-induced gene expression and regulation of redox status were compromised. Moreover, we found that Mox macrophages comprise 30% of all macrophages in advanced atherosclerotic lesions of low-density lipoprotein receptor knockout (LDLR−/−) mice. Conclusions: Together, we identify Nrf2 as a key regulator in the formation of a novel macrophage phenotype (Mox) that develops in response to oxidative tissue damage. The unique biological properties of Mox macrophages suggest this phenotype may play an important role in atherosclerotic lesion development as well as in other settings of chronic inflammation.

Lymphocytes and the Adventitial Immune Response in Atherosclerosis

Although much of the research on atherosclerosis has focused on the intimal accumulation of lipids and inflammatory cells, there is an increasing amount of interest in the role of the adventitia in coordinating the immune response in atherosclerosis. In this review of the contributions of the adventitia and adventitial lymphocytes to the development of atherosclerosis, we discuss recent research on the formation and structural nature of adventitial immune aggregates, potential mechanisms of crosstalk between the intima, media, and adventitia, specific contributions of B lymphocytes and T lymphocytes, and the role of the vasa vasorum and surrounding perivascular adipose tissue. Furthermore, we highlight techniques for the imaging of lymphocytes in the vasculature.

B-Cell Aortic Homing and Atheroprotection Depend on Id3

Rationale: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. Objective: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. Methods and Results: Loss of Id3 in Apoe−/− mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3−/− Apoe−/− mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3−/− Apoe−/− mice into B-cell–deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3+/+ Apoe−/− mice, but aortic reconstitution and B-cell–mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. Conclusions: Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell–mediated protection from early atherosclerosis.

The Helix–Loop–Helix Factors Id3 and E47 Are Novel Regulators of Adiponectin

Adiponectin is an adipocyte-derived cytokine with beneficial effects on insulin sensitivity and the development of atherosclerosis. Id3 is a helix-loop-helix factor that binds to E-proteins such as E47 and inhibits their binding to DNA. Although the helix-loop-helix factor sterol regulatory element binding protein (SREBP)-1c is a known activator of adiponectin transcription, this study provides the first evidence of a role for Id3 and E47 in adiponectin expression. Decreased Id3 in differentiating adipocytes correlates with increased adiponectin expression and forced expression of Id3 inhibits adiponectin expression. Moreover, Id3-null mice have increased adiponectin expression in visceral fat tissue and in serum. We demonstrate that E47 potentiates SREBP-1c-mediated adiponectin promoter activation and that Id3 can dose-dependently inhibit this action via interaction with E47. Mutation of a consensus E47 binding site results in nearly complete loss of promoter activation. Furthermore, we demonstrate E47 binding to the endogenous adiponectin promoter both in vitro and in vivo by chromatin immunoprecipitation analysis. Binding is not detected in undifferentiated cells which express Id3 but peaks during differentiation in parallel with Id3 decline. This promoter binding can be completely abolished by the overexpression of Id3 and is enhanced in adipose tissue null for Id3. These data establish Id3 and E47 as novel regulators of SREBP-1c-mediated adiponectin expression in differentiating adipocytes and provide evidence that Id3 regulates adiponectin expression in vivo.

Id3 Is a Novel Atheroprotective Factor Containing a Functionally Significant Single-Nucleotide Polymorphism Associated With Intima–Media Thickness in Humans

Rationale: The gene encoding the helix–loop–helix transcription factor Id3 (inhibitor of differentiation-3) is located within atherosclerosis susceptibility loci of both mice and humans, yet its influence on atherosclerosis is not known. Objective: The present study sought to determine whether polymorphisms in the ID3 gene were associated with indices of atherosclerosis in humans and if loss of Id3 function modulated atherogenesis in mice. Methods and Results: Six tagging single-nucleotide polymorphisms (SNPs) (tagSNPs) in the human ID3 gene were assessed in participants of the Diabetes Heart Study. One tagSNP, rs11574, was independently associated with carotid intima–media thickness (IMT). The human ID3 variant at rs11574 results in an alanine to threonine substitution in the C terminus. To determine the effect of this polymorphism on the basic function of Id3, site-directed mutagenesis of the human ID3 gene at rs11574 was performed. Results demonstrated a significant reduction in coimmunoprecipitation of the known E-protein partner, E12, with Id3 when it contains the sequence encoded by the risk allele (Id3105T). Further, Id3105T had an attenuated ability to modulate E12-mediated transcriptional activation compared to Id3 containing the ancestral allele (Id3105A). Microarray analysis of vascular smooth muscle cells from WT and Id3−/− mice revealed significant modulation of multiple gene pathways implicated in atherogenesis. Moreover, Id3−/−ApoE−/− mice developed significantly more atherosclerosis in response to 32 weeks of Chow or Western diet feeding than Id3+/+ApoE−/− mice. Conclusions: Taken together, results provide novel evidence that Id3 is an atheroprotective factor and link a common SNP in the human ID3 gene to loss of Id3 function and increased IMT.

Inhibitor of Differentiation-3 Mediates High Fat Diet-Induced Visceral Fat Expansion

Objective—Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. Methods and Results—Id3−/− mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3−/− mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. Conclusion—Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Loss of Id3 Increases VCAM-1 Expression, Macrophage Accumulation, and Atherogenesis in Ldlr–/– Mice

Objective—Inhibitor of differention-3 (Id3) promotes B cells homing to the aorta and atheroprotection in Apoe−/− mice. We sought to determine the impact of loss of Id3 in the Ldlr−/− mouse model of diet-induced atherosclerosis and identify novel Id3 targets in the vessel wall. Methods and Results—Ex vivo optical imaging confirmed that Id3−/− Ldlr−/− mice have significantly fewer aortic B cells than Id3+/+ Ldlr−/− mice. After 8 and 16 weeks of Western diet, Id3−/− Ldlr−/− mice developed significantly more atherosclerosis than Id3+/+ Ldlr−/− mice, with Id3+/− Ldlr−/− mice demonstrating an intermediate phenotype. There were no differences in serum lipid levels between genotypes. Immunostaining demonstrated that aortas from Id3−/− Ldlr−/− mice had greater intimal macrophage density and C-C chemokine ligand 20 and vascular cell adhesion molecule 1 (VCAM-1) expression compared with Id3+/+ Ldlr−/− mice. Real-time polymerase chain reaction demonstrated increased VCAM-1 mRNA levels in the aortas of Id3−/− Ldlr−/− mice. Primary vascular smooth muscle cells from Id3−/− mice expressed greater amounts of VCAM-1 protein compared with control. Gain and loss of function studies in primary vascular smooth muscle cells identified a role for Id3 in repressing VCAM-1 promoter activation. Chromatin immunoprecipitation demonstrated interaction of E12 with the VCAM-1 promoter, which is inhibited by Id3. Conclusion—Id3 is an atheroprotective transcription regulator with targets in both B cells and vessel wall cells leading to reduced macrophage accumulation and reduced atherosclerosis formation.

Deficiency of a Transcriptional Regulator, Inhibitor of Differentiation 3, Induces Glomerulonephritis in Apolipoprotein E-Deficient Mice A Model Linking Hyperlipidemia and Renal Disease

The clinical association between hyperlipidemia and renal disease is well established, yet hyperlipidemia as a cause for renal disease is rare. Apolipoprotein E-deficient (ApoE(-/-)) mice develop hyperlipidemia and are a model for atherosclerosis. Introducing deficiency of inhibitor of differentiation 3 (Id3) in ApoE(-/-) mice further exacerbates atherosclerosis. ID3 is a transcription regulator expressed in multiple cell types. Id3(-/-) mice develop antibodies to self-antigens and salivary gland autoimmunity. This study was undertaken to investigate a link between hyperlipidemia, autoimmunity, and renal disease. ApoE(-/-), Id3(-/-), and ApoE(-/-)Id3(-/-) double-knockout (DKO) mice were studied at different ages for renal pathological features and function. Serum samples were analyzed for the presence of autoantibodies. At 16 weeks, DKO mice developed mesangioproliferative glomerulonephritis (GN), leading to severe proteinuria. GN was associated with glomerular deposition of lipids and immune complexes and with macrophage infiltration. DKO mice had high levels of circulating autoantibodies. Although ApoE(-/-) mice had glomerular lipid deposits and Id3(-/-) mice had circulating autoantibodies, neither group of age-matched single-knockout mice developed GN. These data provide support for the hypothesis that induction of renal disease in hyperlipidemia is dictated by additional factors. Our study shows that some of these factors are regulated by ID3. Thus, ID3 is a novel risk factor linking cardiovascular and renal disease.

Comment on Conventional B2 B Cell Depletion Ameliorates whereas Its Adoptive Transfer Aggravates Atherosclerosis

We read with interest the paper by Kyaw and colleagues ([1][1]), which used adoptive transfer of B cells into μMT Apoe−/− mice to determine if these cells were atherogenic in a model selectively deficient in B lymphocytes. When compared with PBS control, injection of 5 × 106 C57BL/6 splenic B