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 micereconstituted 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.

We have previously demonstrated that shear stress increases transcription of the endothelial nitric-oxide synthase (eNOS) by a pathway involving activation of the tyrosine kinase c-Src and extracellular signal-related kinase 1/2 (ERK1/2). In the present study sought to determine the events downstream of this pathway. Shear stress activated a human eNOS promoter chloramphenicol acetyl-CoA transferase chimeric construct in a time-dependent fashion, and this could be prevented by inhibition of the c-Src and MEK1/2. Studies using electromobility shift assays, promoter deletions, and promoter mutations revealed that shear activation of the eNOS promoter was due to binding of nuclear factor B subunits p50 and p65 to a GAGACC sequence ؊990 to ؊984 base pairs upstream of the eNOS transcription start site. Shear induced nuclear translocation of p50 and p65, and activation of the eNOS promoter by shear could be prevented by co-transfection with a dominant negative I kappa B␣. Exposure of endothelial cells to shear resulted in I kinase phosphorylation, and this was blocked by the MEK1/2 inhibitor PD98059 and the cSrc inhibitor PP1, suggesting these signaling molecules are upstream of NF B activation. These experiments indicate that shear stress increases eNOS transcription by NF B activation and p50/p65 binding to a GAGACC sequence present of the human eNOS promoter. While NF B activation is generally viewed as a proinflammatory stimulus, the current data indicate that its transient activation by shear may increase expression of eNOS, which via production of nitric oxide could convey anti-inflammatory and anti-atherosclerotic properties.Unidirectional laminar shear stress, the frictional force of blood over the surface of the endothelium, exerts atheroprotective effects by preventing adhesion molecule expression, reducing platelet aggregation, and inhibiting both smooth muscle cell proliferation and endothelial cell apoptosis (1). In contrast, areas of the vasculature exposed to low levels of shear stress are prone to atherosclerotic lesion formation (2, 3). At least a portion of the beneficial effects of laminar shear stress is due to modulation of nitric oxide (NO ⅐ ) production via two mechanisms. Immediately after the onset of shear, there is an acute activation of the endothelial NO synthase (eNOS) leading to NO ⅐ release within seconds thereafter (4). Over several hours, shear stress stimulates an increase in eNOS mRNA and protein expression (5). Recent work from our laboratory has shown that this latter effect occurs by two distinct pathways regulating eNOS transcription and mRNA stability, with transcription peaking at 1 h and returning to baseline levels shortly thereafter. Both of these pathways share a need for activation of c-Src; however, eNOS transcription, as measured by nuclear run-on analysis, involves c-Src activation of the mitogen-activated protein kinases Raf, MEK1/2, 1 and extracellular-signal related kinase 1/2 (ERK1/2) (6).The precise nuclear events that lead to an increase in eNOS transcription in ...

Abstract-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. Key Words: basic helix-loop-helix proteins Ⅲ differentiation Ⅲ gene regulation Ⅲ adiponectin Ⅲ adipocytes A diponectin is produced by adipocytes and is secreted into the circulation. 1,2 Adiponectin levels are reduced in obese, insulin-resistant, and diabetic rodents, 3 monkeys, 4 and humans. 5 Administration of adiponectin in rodent models increases insulin sensitivity and lowers plasma glucose. 6,7 Low adiponectin levels are associated with the development of coronary artery disease 8,9 and ApoE Ϫ/Ϫ mice overexpressing adiponectin demonstrate significantly less atherosclerosis in response to high-fat feeding. 10,11 Although these studies implicate adiponectin as an important modulator of insulin sensitivity and the development of atherosclerosis, the transcriptional mechanisms that regulate adiponectin expression are incompletely understood.Both the human and mouse adiponectin promoters contain binding sites for transcription factors including sterol regulatory elements (SREs), peroxisome proliferator-activated receptor (PPAR)-response elements, C/EBP sites, and E-boxes. 12 Accordingly, many factors influence adiponectin expression either directly or indirectly, including sterol regulatory element binding protein (SREBP)-1c, 12 PPAR␥, 12,13 and C/EBP. 12,14 To date, no study has addressed the role of the 3 putative E-boxes present in the adiponectin promoter. 12 SREBP-1c is a member of the basic helix-loop-helix (bHLH)-leucine zipper (bHLH-LZ) family of proteins that binds to SREs. 15 Originally implicated in cholesterolregulated gene expression, 16 SREBP-1c is also a major regulator of adipogenic genes, including fatty acid synthase and adiponectin. 12,17 Also known as adipocyte determinationand differentiation-dependent factor-1 (ADD1), SREBP-1c is expressed preferentially in adipose tissue and is upregulated as adipocyte differentiation progresses. 12,15 The inhibitor of differentiation (Id) family of proteins contains 4 members, Id1 to -4, which have both redundant and unique functions. 18 Ids are HLH proteins that function as dominant-negative transcription factors that are incapable of binding to DNA. Instead, the Ids bind to a subset of bHLH factors known as E-proteins, including E12, E47, ITF2, and HEB, thereby preventing their dimerization and DNA binding. Although the Ids bind preferentially to E-proteins, 1 report has suggested that Id3 binds to and inhibits SREBP-1c directly. 19 Previous studies have proposed a role for Id proteins in adipocytes. The mRNAs for Ids 1 to 3 are expressed in 3T3-L1 preadipocytes and decrease to...

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 high fat diet (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 WT 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 WT controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction (SVF) and Id3−/− mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to WT. 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. Conclusions 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.