LXRα Regulates oxLDL-Induced Trained Immunity in Macrophages.

Reprogramming of metabolic pathways in monocytes and macrophages can induce a proatherosclerotic inflammatory memory called trained innate immunity. Here, we have analyzed the role of the Liver X receptor (LXR), a crucial regulator of metabolism and inflammation, in oxidized low-density lipoprotein (oxLDL)-induced trained innate immunity. Human monocytes were incubated with LXR agonists, antagonists, and oxLDL for 24 h. After five days of resting time, cells were restimulated with the TLR-2 agonist Pam3cys. OxLDL priming induced the expression of LXRα but not LXRß. Pharmacologic LXR activation was enhanced, while LXR inhibition prevented the oxLDL-induced inflammatory response. Furthermore, LXR inhibition blocked the metabolic changes necessary for epigenetic reprogramming associated with trained immunity. In fact, enrichment of activating histone marks at the IL-6 and TNFα promotor was reduced following LXR inhibition. Based on the differential expression of the LXR isoforms, we inhibited LXRα and LXRß genes using siRNA in THP1 cells. As expected, siRNA-mediated knock-down of LXRα blocked the oxLDL-induced inflammatory response, while knock-down of LXRß had no effect. We demonstrate a specific and novel role of the LXRα isoform in the regulation of oxLDL-induced trained immunity. Our data reveal important aspects of LXR signaling in innate immunity with relevance to atherosclerosis formation.

OxLDL-mediated immunologic memory in endothelial cells.

Trained innate immunity describes the metabolic reprogramming and long-term proinflammatory activation of innate immune cells in response to different pathogen or damage associated molecular patterns, such as oxidized low-density lipoprotein (oxLDL). Here, we have investigated whether the regulatory networks of trained innate immunity also control endothelial cell activation following oxLDL treatment. Human aortic endothelial cells (HAECs) were primed with oxLDL for 24 h. After a resting time of 4 days, cells were restimulated with the TLR2-agonist PAM3cys4. OxLDL priming induced a proinflammatory memory with increased production of inflammatory cytokines such as IL-6, IL-8 and MCP-1 in response to PAM3cys4 restimulation. This memory formation was dependent on TLR2 activation. Furthermore, oxLDL priming of HAECs caused characteristic metabolic and epigenetic reprogramming, including activation of mTOR-HIF1α-signaling with increases in glucose consumption and lactate production, as well as epigenetic modifications in inflammatory gene promoters. Inhibition of mTOR-HIF1α-signaling or histone methyltransferases blocked the observed phenotype. Furthermore, primed HAECs showed epigenetic activation of ICAM-1 and increased ICAM-1 expression in a HIF1α-dependent manner. Accordingly, live cell imaging revealed increased monocyte adhesion and transmigration following oxLDL priming. In summary, we demonstrate that oxLDL-mediated endothelial cell activation represents an immunologic event, which triggers metabolic and epigenetic reprogramming. Molecular mechanisms regulating trained innate immunity in innate immune cells also regulate this sustained proinflammatory phenotype in HAECs with enhanced atheroprone cell functions. Further research is necessary to elucidate the detailed metabolic regulation and the functional relevance for atherosclerosis formation in vivo.

Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

OBJECTIVE: Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. APPROACH AND RESULTS: We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. CONCLUSIONS: These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.

Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors.

Whole-Body MR Imaging Including Angiography: Predicting Recurrent Events in Diabetics.

OBJECTIVES: Whether whole-body MRI can predict occurrence of recurrent events in patients with diabetes mellitus. METHODS: Whole-body MRI was prospectively applied to 61 diabetics and assessed for arteriosclerosis and ischemic cerebral/myocardial changes. Occurrence of cardiocerebral events and diabetic comorbidites was determined. Patients were stratified whether no, a single or recurrent events arose. As a secondary endpoint, events were stratified into organ system-specific groups. RESULTS: During a median follow-up of 70 months, 26 diabetics developed a total of 39 events; 18 (30%) developed one, 8 (13%) recurrent events. Between diabetics with no, a single and recurrent events, a stepwise higher burden was observed for presence of left ventricular (LV) hypo-/akinesia (3/28/75%, p < 0.0001), myocardial delayed-contrast-enhancement (17/33/63%, p = 0.001), carotid artery stenosis (11/17/63%, p = 0.005), peripheral artery stenosis (26/56/88%, p = 0.0006) and vessel score (1.00/1.30/1.76, p < 0.0001). After adjusting for clinical characteristics, LV hypo-/akinesia (hazard rate ratio = 6.57, p < 0.0001) and vessel score (hazard rate ratio = 12.29, p < 0.0001) remained independently associated. Assessing organ system risk, cardiac and cerebral MR findings predicted more strongly events in their respective organ system. Vessel-score predicted both cardiac and cerebral, but not non-cardiocerebral, events. CONCLUSION: Whole-body MR findings predict occurrence of recurrent events in diabetics independent of clinical characteristics, and may concurrently provide organ system-specific risk. KEY POINTS: • Patients with long-standing diabetes mellitus are at high risk for recurrent events. • Whole-body MRI predicts occurrence of recurrent events independently of clinical characteristics. • The vessel score derived from whole-body angiography is a good general risk-marker. • Whole-body MRI may also provide organ-specific risk assessment. • Current findings may indicate benefits of whole-body MRI for risk stratification.

PDE4 inhibition reduces neointima formation and inhibits VCAM-1 expression and histone methylation in an Epac-dependent manner.

Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation following vascular injury. In this study we have investigated the effects of specific PDE4 inhibition with roflumilast on SMC proliferation and inflammatory activation in vitro and neointima formation following guide wire-induced injury of the femoral artery in mice in vivo. In vitro, roflumilast did not affect SMC proliferation, but diminished TNF-α induced expression of the vascular cell adhesion molecule 1 (VCAM-1). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued following inhibition of Epac. TNF-α induced NFκB p65 translocation and VCAM-1 promoter activity were not altered by roflumilast in SMCs. However, roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, while PKA activation showed no effect. Furthermore, HDAC inhibition blocked the inhibitory effect of roflumilast on VCAM-1 expression. Both, roflumilast and Epac activation reduced monocyte adhesion to SMCs in vitro. Finally, roflumilast treatment attenuated femoral artery intima-media ratio by more than 50% after 4weeks. In summary, PDE4 inhibition regulates VCAM-1 through a novel Epac-dependent mechanism, which involves regulatory epigenetic components and reduces neointima formation following vascular injury. PDE4 inhibition and Epac activation might represent novel approaches for the treatment of vascular diseases, including atherosclerosis and in-stent restenosis.

Protocol for the induction of innate immune memory in human smooth muscle cells and endothelial cells in vitro.

Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2.

Diabetes mellitus: long-term prognostic value of whole-body MR imaging for the occurrence of cardiac and cerebrovascular events.

PURPOSE: To study the predictive value of whole-body magnetic resonance (MR) imaging for the occurrence of cardiac and cerebrovascular events in a cohort of patients with diabetes mellitus (DM). MATERIALS AND METHODS: This HIPAA-compliant study was approved by the institutional review board. Informed consent was obtained from all patients before enrollment into the study. The authors followed up 65 patients with DM (types 1 and 2) who underwent a comprehensive, contrast material-enhanced whole-body MR imaging protocol, including brain, cardiac, and vascular sequences at baseline. Follow-up was performed by phone interview. The primary endpoint was a major adverse cardiac and cerebrovascular event (MACCE), which was defined as composite cardiac-cerebrovascular death, myocardial infarction, cerebrovascular event, or revascularization. MR images were assessed for the presence of systemic atherosclerotic vessel changes, white matter lesions, and myocardial changes. Kaplan-Meier survival and Cox regression analyses were performed to determine associations. RESULTS: Follow-up was completed in 61 patients (94%; median age, 67.5 years; 30 women [49%]; median follow-up, 70 months); 14 of the 61 patients (23%) experienced MACCE. Although normal whole-body MR imaging excluded MACCE during the follow-up period (0%; 95% confidence interval [CI]: 0%, 17%), any detectable ischemic and/or atherosclerotic changes at whole-body MR imaging (prevalence, 66%) conferred a cumulative event rate of 20% at 3 years and 35% at 6 years. Whole-body MR imaging summary estimate of disease was strongly predictive for MACCE (one increment of vessel score and each territory with atherosclerotic changes: hazard ratio, 13.2 [95% CI: 4.5, 40.1] and 3.9 [95% CI: 2.2, 7.5], respectively), also beyond clinical characteristics as well as individual cardiac or cerebrovascular MR findings. CONCLUSION: These initial data indicate that disease burden as assessed with whole-body MR imaging confers strong prognostic information in patients with DM. Online supplemental material is available for this article.

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

Transcriptional regulation of S phase kinase-associated protein 2 by NR4A orphan nuclear receptor NOR1 in vascular smooth muscle cells.

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27(KIP1) through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation.

Deficiency of the NR4A orphan nuclear receptor NOR1 decreases monocyte adhesion and atherosclerosis.

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.

Telomerase activation in atherosclerosis and induction of telomerase reverse transcriptase expression by inflammatory stimuli in macrophages.

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.

Epigenetic regulation of vascular smooth muscle cell proliferation and neointima formation by histone deacetylase inhibition.

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.

Telomerase deficiency in bone marrow-derived cells attenuates angiotensin II-induced abdominal aortic aneurysm formation.

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.

Deficiency of the NR4A neuron-derived orphan receptor-1 attenuates neointima formation after vascular injury.

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.

Metabolic syndrome predicts vascular changes in whole body magnetic resonance imaging in patients with long standing diabetes mellitus.

BACKGROUND: Although diabetic patients have an increased rate of cardio-vascular events, there is considerable heterogeneity with respect to cardiovascular risk, requiring new approaches to individual cardiovascular risk factor assessment. In this study we used whole body-MR-angiography (WB-MRA) to assess the degree of atherosclerosis in patients with long-standing diabetes and to determine the association between metabolic syndrome (MetS) and atherosclerotic burden. METHODS: Long standing (> or = 10 years) type 1 and type 2 diabetic patients (n = 59; 31 males; 63.3 +/- 1.7 years) were examined by WB-MRA. Based on the findings in each vessel, we developed an overall score representing the patient's vascular atherosclerotic burden (MRI-score). The score's association with components of the MetS was assessed. RESULTS: The median MRI-score was 1.18 [range: 1.00-2.41] and MetS was present in 58% of the cohort (type 2 diabetics: 73%; type 1 diabetics: 26%). Age (p = 0.0002), HDL-cholesterol (p = 0.016), hypertension (p = 0.0008), nephropathy (p = 0.0093), CHD (p = 0.001) and MetS (p = 0.0011) were significantly associated with the score. Adjusted for age and sex, the score was significantly (p = 0.02) higher in diabetics with MetS (1.450 [1.328-1.572]) compared to those without MetS (1.108 [0.966-1.50]). The number of MetS components was associated with a linear increase in the MRI-score (increase in score: 0.09/MetS component; r2 = 0.24, p = 0.038). Finally, using an established risk algorithm, we found a significant association between MRI-score and 10-year risk for CHD, fatal CHD and stroke. CONCLUSION: In this high-risk diabetic population, WB-MRA revealed large heterogeneity in the degree of systemic atherosclerosis. Presence and number of traits of the MetS are associated with the extent of atherosclerotic burden. These results support the perspective that diabetic patients are a heterogeneous population with increased but varying prevalence of atherosclerosis and risk.

The PPARalpha/p16INK4a pathway inhibits vascular smooth muscle cell proliferation by repressing cell cycle-dependent telomerase activation.

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.

LXR Activation Induces a Proinflammatory Trained Innate Immunity-Phenotype in Human Monocytes.

Objectives: The concept of trained innate immunity describes a long-term proinflammatory memory in innate immune cells. Trained innate immunity is regulated through reprogramming of cellular metabolic pathways including cholesterol and fatty acid synthesis. Here, we have analyzed the role of Liver X Receptor (LXR), a key regulator of cholesterol and fatty acid homeostasis, in trained innate immunity. Methods and Results: Human monocytes were isolated and incubated with different stimuli for 24 h, including LXR agonists, antagonists and Bacillus Calmette-Guerin (BCG) vaccine. After 5 days resting time, cells were restimulated with the TLR2-agonist Pam3cys. LXR activation did not only increase BCG trained immunity, but also induced a long-term inflammatory activation by itself. This inflammatory activation by LXR agonists was accompanied by characteristic features of trained innate immunity, such as activating histone marks on inflammatory gene promoters and metabolic reprogramming with increased lactate production and decreased oxygen consumption rate. Mechanistically, LXR priming increased cellular acetyl-CoA levels and was dependent on the activation of the mevalonate pathway and IL-1ß signaling. In contrast to mevalonate pathway inhibition, blocking fatty acid synthesis further increased proinflammatory priming by LXR. Conclusion: We demonstrate that LXR activation induces a proinflammatory trained immunity phenotype in human monocytes through epigenetic and metabolic reprogramming. Our data reveal important novel aspects of LXR signaling in innate immunity.

Mechanisms of Trained Innate Immunity in oxLDL Primed Human Coronary Smooth Muscle Cells.

Objective: Damage and pathogen associated molecular patterns such as oxidized low-density lipoprotein (oxLDL) or bacillus Calmette-Guerin (BCG) vaccine can induce long term pro-inflammatory priming in monocytes and macrophages due to metabolic and epigenetic reprogramming-an emerging new concept called trained innate immunity. Vascular smooth muscle cells express pattern recognition receptors involved in trained innate immunity in monocytes. Here we investigated whether the mechanisms of trained innate immunity also control a proinflammatory phenotype in human coronary smooth muscle cells. Methods: Human coronary smooth muscle cells were primed with oxLDL or BCG for 24 h. After a resting time of 4 to 7 days, the cells were restimulated with either PAM3cys4, LPS or TNFα and cytokine production or mRNA expression were measured. Then, mechanisms of monocyte trained innate immunity were analyzed in smooth muscle cells, including receptors, intracellular pathways as well as metabolic and epigenetic reprogramming. Results: Priming with oxLDL or BCG lead to a significantly increased production of IL6, IL8 and MCP-1 following restimulation. OxLDL priming had little effect on the expression of macrophage or SMC marker genes. Proinflammatory priming of smooth muscle cells induced mTOR-HIF1α-signaling and could be blocked by mTOR-, TLR2-, and TLR4-inhibition. Finally, metabolic and epigenetic mechanisms of trained innate immunity in monocytes could be replicated in smooth muscle cells, including increased glucose consumption, lactate production, responsiveness to 6-fluoromevalonate and mevalonate treatment and inhibition of priming by the histone methyltransferase inhibitor methylthioadenosine (MTA). Conclusion: We demonstrate for the first time that mechanisms of the so called trained innate immunity control a proinflammatory phenotype in non-immune cells of the vascular wall. Our findings warrant further research into the specificity of trained innate immunity as an immune cell response as well as the mechanisms of vascular smooth muscle cells inflammation.