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The article examines the role of the orphan nuclear receptor NOR1 in the development of atherosclerosis. According to the authors, NOR1 positively regulates monocyte recruitment to the vascular wall, thereby performing an atherogenic role. The authors were also able to identify a pathway underlying monocyte adhesion which explains how the nuclear receptor affects the process of atherosclerosis development.

The article presents a discussion concerning the inhibition of vascular smooth muscle cell (VSMC) proliferation on peroxisome proliferation-activated receptor (PPAR)α/p16INK4a pathway by repressing cell cycle-dependent telomerase activation. It also demonstrates that PPARα's antiproliferative effects in VSMCs depend on the suppression of tolemerase activity by targeting the p16/RB/E2F transcriptional cascade.

Objective: Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive., Methods and Results: In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA-mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G(1) phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip). Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury., Conclusions: These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.

Objective: Telomerase serves as a critical regulator of tissue renewal. Although telomerase activity is inducible in response to various environmental cues, it remains unknown whether telomerase is activated during the inflammatory remodeling underlying atherosclerosis formation. To address this question, we investigated in the present study the regulation of telomerase in macrophages and during atherosclerosis development in low-density lipoprotein receptor-deficient mice., Methods and Results: We demonstrate that inflammatory stimuli activate telomerase in macrophages by inducing the expression of the catalytic subunit telomerase reverse transcriptase (TERT). Reporter and chromatin immunoprecipitation assays identified a previously unrecognized nuclear factor-κB (NF-κB) response element in the TERT promoter, to which NF-κB is recruited during inflammation. Inhibition of NF-κB signaling completely abolished the induction of TERT expression, characterizing TERT as a bona fide NF-κB target gene. Furthermore, functional experiments revealed that TERT deficiency results in a senescent cell phenotype. Finally, we demonstrate high levels of TERT expression in macrophages of human atherosclerotic lesions and establish that telomerase is activated during atherosclerosis development in low-density lipoprotein receptor-deficient mice., Conclusions: These results characterize TERT as a previously unrecognized NF-κB target gene in macrophages and demonstrate that telomerase is activated during atherosclerosis. This induction of TERT expression prevents macrophage senescence and may have important implications for the development of atherosclerosis.

Objective: Abdominal aortic aneurysms (AAA) are an age-related vascular disease and an important cause of morbidity and mortality. In this study, we sought to determine whether the catalytic component of telomerase, telomerase reverse transcriptase (TERT), modulates angiotensin (Ang) II-induced AAA formation., Methods and Results: Low-density lipoprotein receptor-deficient (LDLr-/-) mice were lethally irradiated and reconstituted with bone marrow-derived cells from TERT-deficient (TERT-/-) mice or littermate wild-type mice. Mice were placed on a diet enriched in cholesterol, and AAA formation was quantified after 4 weeks of Ang II infusion. Repopulation of LDLr-/- mice with TERT-/- bone marrow-derived cells attenuated Ang II-induced AAA formation. TERT-deficient recipient mice revealed modest telomere attrition in circulating leukocytes at the study end point without any overt effect of the donor genotype on white blood cell counts. In mice repopulated with TERT-/- bone marrow, aortic matrix metalloproteinase-2 (MMP-2) activity was reduced, and TERT-/- macrophages exhibited decreased expression and activity of MMP-2 in response to stimulation with Ang II. Finally, we demonstrated in transient transfection studies that TERT overexpression activates the MMP-2 promoter in macrophages., Conclusions: TERT deficiency in bone marrow-derived macrophages attenuates Ang II-induced AAA formation in LDLr-/- mice and decreases MMP-2 expression. These results point to a previously unrecognized role of TERT in the pathogenesis of AAA.

Rationale: The orphan nuclear receptor NOR1 is a member of the evolutionary highly conserved and ligand-independent NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily have been characterized as early response genes regulating essential biological processes including inflammation and proliferation; however, the role of NOR1 in atherosclerosis remains unknown., Objective: The goal of the present study was to determine the causal contribution of NOR1 to atherosclerosis development and to identify the mechanism by which this nuclear receptor participates in the disease process., Methods and Results: In the present study, we demonstrate expression of NOR1 in endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through a nuclear factor kappaB-dependent transactivation of the NOR1 promoter. Overexpression of NOR1 in human endothelial cells increased the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule-1, whereas NOR1 deficiency altered adhesion molecule expression in response to inflammatory stimuli. Transient transfection experiments and chromatin immunoprecipitation assays revealed that NOR1 induces VCAM-1 promoter activity by binding to a canonical response element for NR4A receptors in the VCAM-1 promoter. Further functional studies confirmed that NOR1 mediates monocyte adhesion by inducing VCAM-1 and intercellular adhesion molecule-1 expression in endothelial cells. Finally, we demonstrate that NOR1 deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE(-/-) mice by decreasing the macrophage content of the lesion., Conclusions: In concert, these studies identify a novel pathway underlying monocyte adhesion and establish that NOR1 serves a previously unrecognized atherogenic role in mice by positively regulating monocyte recruitment to the vascular wall.

Background: The neuron-derived orphan receptor-1 (NOR1) belongs to the evolutionary highly conserved and most ancient NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily function as early-response genes regulating key cellular processes, including proliferation, differentiation, and survival. Although NOR1 has previously been demonstrated to be required for smooth muscle cell proliferation in vitro, the role of this nuclear receptor for the proliferative response underlying neointima formation and target genes trans-activated by NOR1 remain to be defined., Methods and Results: Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in NOR1(-/-) mice compared with wild-type mice. In vitro, NOR1-deficient smooth muscle cells exhibit decreased proliferation as a result of a G(1)-->S phase arrest of the cell cycle and increased apoptosis in response to serum deprivation. NOR1 deficiency alters phosphorylation of the retinoblastoma protein by preventing mitogen-induced cyclin D1 and D2 expression. Conversely, overexpression of NOR1 induces cyclin D1 expression and the transcriptional activity of the cyclin D1 promoter in transient reporter assays. Gel shift and chromatin immunoprecipitation assays identified a putative response element for NR4A receptors in the cyclin D1 promoter, to which NOR1 is recruited in response to mitogenic stimulation. Finally, we provide evidence that these observations are applicable in vivo by demonstrating decreased cyclin D1 expression during neointima formation in NOR1-deficient mice., Conclusions: These experiments characterize cyclin D1 as an NOR1-regulated target gene in smooth muscle cells and demonstrate that NOR1 deficiency decreases neointima formation in response to vascular injury.

Peroxisome proliferator-activated receptor (PPAR)alpha, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARalpha activation suppresses G(1)-->S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16(INK4a) (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARalpha is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARalpha activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1-dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect that was dependent on p16. The inhibition of cell proliferation by PPARalpha activation was lost in VSMCs following TERT overexpression or knockdown, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARalpha. Finally, we demonstrate that PPARalpha agonists suppress telomerase activation during the proliferative response following vascular injury, indicating that these findings are applicable in vivo. In concert, these results demonstrate that the antiproliferative effects of PPARalpha in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade.

Activation of the peroxisome proliferator-activated receptor (PPAR) gamma, the molecular target for insulin sensitizing thiazolidinediones used in patients with type 2 diabetes, inhibits vascular smooth muscle cell (VSMC) proliferation and prevents atherosclerosis and neointima formation. Emerging evidence indicates that telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation. In the present study, we demonstrate that ligand-induced and constitutive PPARgamma activation inhibits telomerase activity in VSMCs. Telomerase reverse transcriptase (TERT) confers the catalytic activity of telomerase, and PPARgamma ligands inhibit TERT expression through a receptor-dependent suppression of the TERT promoter. 5'-deletion analysis, site-directed mutagenesis, and transactivation studies using overexpression of Ets-1 revealed that suppression of TERT transcription by PPARgamma is mediated through negative cross-talk with Ets-1-dependent transactivation of the TERT promoter. Chromatin immunoprecipitation assays further demonstrated that PPARgamma ligands inhibit Ets-1 binding to the TERT promoter, which is mediated at least in part through an inhibition of Ets-1 expression by PPARgamma ligands. In VSMCs overexpressing TERT, the efficacy of PPARgamma ligands to inhibit cell proliferation is lost, indicating that TERT constitutes an important molecular target for the antiproliferative effects of PPARgamma ligands. Finally, we demonstrate that telomerase activation during the proliferative response after vascular injury is effectively inhibited by PPARgamma ligands. These findings provide a previously unrecognized mechanism for the antiproliferative effects of PPARgamma ligands and support the concept that PPARgamma ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases.