Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice.

The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic ß-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.

Genetic variation is a key determinant of chromatin accessibility and drives differences in the regulatory landscape of C57BL/6J and 129S1/SvImJ mice.

Most common genetic variants associated with disease are located in non-coding regions of the genome. One mechanism by which they function is through altering transcription factor (TF) binding. In this study, we explore how genetic variation is connected to differences in the regulatory landscape of livers from C57BL/6J and 129S1/SvImJ mice fed either chow or a high-fat diet. To identify sites where regulatory variation affects TF binding and nearby gene expression, we employed an integrative analysis of H3K27ac ChIP-seq (active enhancers), ATAC-seq (chromatin accessibility) and RNA-seq (gene expression). We show that, across all these assays, the genetically driven (i.e. strain-specific) differences in the regulatory landscape are more pronounced than those modified by diet. Most notably, our analysis revealed that differentially accessible regions (DARs, N = 29635, FDR < 0.01 and fold change > 50%) are almost always strain-specific and enriched with genetic variation. Moreover, proximal DARs are highly correlated with differentially expressed genes. We also show that TF binding is affected by genetic variation, which we validate experimentally using ChIP-seq for TCF7L2 and CTCF. This study provides detailed insights into how non-coding genetic variation alters the gene regulatory landscape, and demonstrates how this can be used to study the regulatory variation influencing TF binding.

Western Diet Decreases Hepatic Drug Metabolism in Male LDLr-/-ApoB100/100 Mice.

Consumption of a Western diet is an important risk factor for several chronic diseases including nonalcoholic fatty liver disease (NAFLD), but its effect on the xenobiotic metabolizing enzyme activities in the liver has been studied incompletely. In this study, male LDLr-/-ApoB100/100 mice were fed with Western diet (WD) or a standard diet for five months to reveal the effects on drug metabolism such as cytochrome P450 (CYP) oxidation and conjugation activities in the liver. Hepatic steatosis, lobular inflammation, and early fibrosis were observed in WD fed mice, but not in chow diet control mice. When compared to the controls, the WD-fed mice had significantly decreased protein-normalized CYP probe activities of 7-ethoxyresorufinO-deethylation (52%), coumarin 7-hydroxylation (26%), 7-hydroxylation of 3-(3-fluoro-4-hydroxyphenyl)-6-methoxycoumarin (70%), 7-hydroxylation of 3-(4-trifluoromethoxyphenyl)-6-methoxycoumarin (78%), 7-hydroxylation of 3-(3-methoxyphenyl)coumarin (81%), and pentoxyresorufin O-depentylation (66%). Increased activity was seen significantly in sulfonation of 3-(4-methylphenyl)-7-hydroxycoumarin (289%) and cytosol catechol O-methyltranferase (COMT, 148%) in the WD group when compared to the controls. In conclusion, the WD-induced steatosis in male LDLr-/-ApoB100/100 mice was associated with decreased CYP oxidation reactions but had no clear effects on conjugation reactions of glucuronidation, sulfonation, and cytosolic catechol O-methylation. Consequently, the WD may decrease the metabolic elimination of drugs compared to healthier low-fat diets.

Deletion of the murine ortholog of human 9p21.3 locus promotes atherosclerosis by increasing macrophage proinflammatory activity.

Background: Several genome-wide association studies have reported a risk locus for coronary artery disease (CAD) in the 9p21. 3 chromosomal region. This region encodes a lncRNA in the INK4 locus (ANRIL) and its genetic variance has a strong association with CAD, but its mechanisms in atherogenesis remain unclear. Objectives: This study aimed to investigate the role of the murine ortholog of human 9p21.3 locus in atherogenesis in hypercholesterolemic mice. Methods: Murine 9p21.3 ortholog knockout mice (Chr4Δ70kb/Δ70kb ) were crossbred with Ldlr -/- ApoB 100/100 mice, and atherosclerotic plaque size and morphology were analyzed on a standard or a high-fat diet (HFD). The hematopoietic cell-specific effect of Chr4Δ70kb/Δ70kb on atherosclerotic plaque development was studied via bone marrow (BM) transplantation, where Chr4Δ70kb/Δ70kb or wild-type BM was transplanted into Ldlr -/- ApoB 100/100 mice. The role of Chr4Δ70kb/Δ70kb in macrophage M1/M2 polarization was studied. In addition, single-cell sequencing data from human and mouse atheroma were analyzed to show the expression profiles of ANRIL and its murine equivalent, Ak148321, in the plaques. Results: Both systemic and hematopoietic Chr4Δ70kb/Δ70kb increased atherosclerosis in Ldlr -/- ApoB 100/100 mice after 12 weeks of HFD. The systemic Chr4Δ70kb/Δ70kb also elevated the number of circulating leukocytes. Chr4Δ70kb/Δ70kb BMDMs showed enhanced M1 polarization in vitro. Single-cell sequencing data from human and mouse atheroma revealed that ANRIL and Ak148321 were mainly expressed in the immune cells. Conclusion: These data demonstrate that both systemic and BM-specific deletion of the murine 9p21.3 risk locus ortholog promotes atherosclerosis and regulates macrophage pro-inflammatory activity, suggesting the inflammation-driven mechanisms of the risk locus on atherogenesis.

RNA interference-based therapies for the control of atherosclerosis risk factors.

PURPOSE OF REVIEW: Atherosclerosis, characterized by lipid accumulation and chronic inflammation in the arterial wall, is the leading causes of death worldwide. The purpose of this article is to review the status of RNA interference (RNAi) based therapies in clinical trials for the treatment and prevention of atherosclerosis risk factors. RECENT FINDINGS: There is a growing interest on using RNAi technology for the control of atherosclerosis risk factors. Current clinical trials utilizing RNAi for atherosclerosis are targeting lipid metabolism regulating genes including proprotein convertase subtilisin/kexin 9, apolipoprotein C-III, lipoprotein (a) and angiopoietin-like protein 3. Currently, three RNAi-based drugs have been approved by U.S. Food and Drug Administration, but there are several therapies in clinical trials at the moment, and potentially entering the market in near future. In addition, recent preclinical studies on regulating vascular inflammation have shown promising results. SUMMARY: In recent years, RNAi based technologies and therapies have been intensively developed for the treatment of atherosclerosis risk factors, such as hyperlipidemia and vascular inflammation. Multiple potential therapeutic targets have emerged, and many of the reported clinical trials have already been successful in plasma lipid lowering. The scope of RNAi therapies is well recognized and recent approvals are encouraging for the treatment of cardiovascular and metabolic disorders.

SUR1-E1506K mutation impairs glucose tolerance and promotes vulnerable atherosclerotic plaque phenotype in hypercholesterolemic mice.

BACKGROUND AND AIMS: Diabetes is a major risk factor of atherosclerosis and its complications. The loss-of-function mutation E1506K in the sulfonylurea receptor 1 (SUR1-E1506K) induces hyperinsulinemia in infancy, leading to impaired glucose tolerance and increased risk of type 2 diabetes. In this study, we investigate the effect of SUR1-E1506K mutation on atherogenesis in hypercholesterolemic LDLR-/- mice. METHODS: SUR1-E1506K mutated mice were cross-bred with LDLR-/- mice (SUR1Δ/LDLR-/-), 6 months old mice were fed a western-diet (WD) for 6 months to induce advanced atherosclerotic plaques. At the age of 12 months, atherosclerosis and plaque morphology were analyzed and mRNA gene expression were measured from aortic sections and macrophages. Glucose metabolism was characterized before and after WD. Results were compared to age-matched LDLR-/- mice. RESULTS: Advanced atherosclerotic plaques did not differ in size between the two strains. However, in SUR1Δ/LDLR-/- mice, plaque necrotic area was increased and smooth muscle cell number was reduced, resulting in higher plaque vulnerability index in SUR1Δ/LDLR-/- mice compared to LDLR-/- mice. SUR1Δ/LDLR-/- mice exhibited impaired glucose tolerance and elevated fasting glucose after WD. The positive staining area of IL-1ß and NLRP3 inflammasome were increased in aortic sections in SUR1Δ/LDLR-/- mice compared to LDLR-/- mice, and IL-18 plasma level was elevated in SUR1Δ/LDLR-/- mice. Finally, the mRNA expression of IL-1ß and IL-18 were increased in SUR1Δ/LDLR-/- bone marrow derived macrophages in comparison to LDLR-/- macrophages in response to LPS. CONCLUSIONS: SUR1-E1506K mutation impairs glucose tolerance and increases arterial inflammation, which promotes a vulnerable atherosclerotic plaque phenotype in LDLR-/- mice.

Novel RNAi-Based Therapies for Atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis, defined by inflammation and accumulation of cholesterol, extracellular matrix, and cell debris into the arteries is a common factor behind cardiovascular diseases (CVD), such as coronary artery disease, peripheral artery disease, and stroke. In this review, we discuss and describe novel RNA interference (RNAi)-based therapies in clinical trials and on the market. RECENT FINDINGS: The first RNAi-based therapies have entered clinical use for the control of atherosclerosis risk factors, i.e., blood cholesterol levels. The most advanced treatment is silencing of proprotein convertase subtilisin/kexin type 9 (PCSK9) with a drug called inclisiran, which has been approved for the treatment of hypercholesterolemia in late 2020, and results in a robust decrease in plasma cholesterol levels. As the new RNAi therapies for atherosclerosis are now entering markets, the usefulness of these therapies will be further evaluated in larger patient cohorts. Thus, it remains to be seen how fast, effectively and eminently these new drugs consolidate their niche within the cardiovascular disease drug palette.

Menaquinone 4 increases plasma lipid levels in hypercholesterolemic mice.

In calcific aortic valve disease (CAVD) progressive valvular calcification causes aortic valve dysfunction. CAVD has several risk factors such as age and dyslipidemia. Vitamin K was shown to inhibit vascular calcification in mice and valvular calcification in patients with CAVD. We studied the effect of menaquinone 4 (MK4/vitamin K2) on valvular calcification in the hypercholesterolemic mouse model of CAVD. LDLr-/-ApoB100/100 male mice were fed with a Western diet for 5 months, with (n = 10) or without (n = 10) added 0.2 mg/g MK4. Body weight gain was followed weekly. Morphology of aortic valves and liver was assessed with immunohistochemistry. Plasma cholesterol levels and cytokines from hepatic tissue were assessed in the end of the study. Hepatic gene expression of lipid metabolism regulating genes were assessed after 18 h diet. MK4 exacerbated the lipoprotein lipid profile without affecting aortic valve morphology in hypercholesterolemic LDLr-/- ApoB100/100 mice. The MK4-containing WD diet increased plasma levels of LDL and triglycerides, hepatic steatosis, and mRNA expression of genes required for triglyceride and cholesterol synthesis. MK4 diminished levels of several cytokines and chemokines in liver, including IL-6, TNFα and MCP1, as measured by hepatic cytokine array. Consequently, MK4 may exert non-beneficial effects on circulating lipid levels, especially in hypercholesterolemic individuals.

LDL Receptor Regulates the Reverse Transport of Macrophage-Derived Unesterified Cholesterol via Concerted Action of the HDL-LDL Axis: Insight From Mouse Models.

RATIONALE: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. OBJECTIVE: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice. METHODS AND RESULTS: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. CONCLUSIONS: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.

Nucleic Acid-Based Therapies for Atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is characterized by accumulation of lipids and chronic inflammation in medium size to large arteries. Recently, RNA-based antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are being developed, along with small molecule-based drugs and monoclonal antibodies, for the treatment of risk factors associated with atherosclerosis.. The purpose of this review is to describe nucleic acid-based therapeutics and introduce novel RNAs that might become future tools for treatment of atherosclerosis. RECENT FINDINGS: RNA-based inhibitors for PCSK9, Lp(a), ApoCIII, and ANGPTL3 have been successfully tested in phase II-III clinical trials. Moreover, multiple microRNA and long non-coding RNAs have been found to reduce atherogenesis in preclinical animal models. Clinical trials especially with ASOs and siRNAs directed to liver, targeting cholesterol and lipoprotein metabolism, have shown promising results. Additional research in larger patient cohorts is needed to fully evaluate the therapeutic potential of these new drugs.

Characterizing valve dynamics in mice by high-resolution cine-MRI.

AIMS: In calcific aortic valve disease (CAVD), progressive valvular sclerosis and calcification cause narrowing of the orifice and an impairment of the valve's function. We applied high-resolution cine-MRI to perform quantitative analysis of the dynamics of the aortic valve in a mice model of CAVD. METHODS AND RESULTS: LDLr-/- ApoB100/100 mice were fed a Western diet (WD) or a standard diet (control) for 22 weeks. The mice were imaged in a 7 T horizontal MRI scanner, and aortic valve dynamics was examined by imaging the cross-section of the aorta at valve level using cine sequences. From these images, the area of the aortic valve orifice was determined during the heart cycle. MRI results were compared with echocardiographic and histopathologic results. The data revealed evidence of clear aortic valve dysfunction in WD mice as compared with control mice (interaction P < 0.001). MRI showed narrowing (14%, P < 0.05) of the orifice area, and this was also seen in histology (34%, P < 0.05), indicating more severe aortic stenosis after WD than in controls. Additionally, MRI revealed a reduction in the ejection fraction (EF) (-11%, P < 0.01), a result confirmed with echocardiography (-27%, P < 0.001) in mice fed with WD. EF detected by MRI and echocardiography also correlated strongly with the degree of stenosis assessed by histology. CONCLUSIONS: Cine-MRI can be used for quantitative analysis of the aortic valve orifice over the cardiac cycle in mice. MRI showed the cusps clearly, and we were able to detect aortic valve dysfunction over time through the cardiac cycle.

Nuclear factor E2-related factor 2 deficiency impairs atherosclerotic lesion development but promotes features of plaque instability in hypercholesterolaemic mice.

Aims: Oxidative stress and inflammation play an important role in the progression of atherosclerosis. Transcription factor NF-E2-related factor 2 (Nrf2) has antioxidant and anti-inflammatory effects in the vessel wall, but paradoxically, global loss of Nrf2 in apoE deficient mice alleviates atherosclerosis. In this study, we investigated the effect of global Nrf2 deficiency on early and advanced atherogenesis in alternative models of atherosclerosis, LDL receptor deficient mice (LDLR-/-), and LDLR-/- mice expressing apoB-100 only (LDLR-/- ApoB100/100) having a humanized lipoprotein profile. Methods and results: LDLR-/- mice were fed a high-fat diet (HFD) for 6 or 12 weeks and LDLR-/-ApoB100/100 mice a regular chow diet for 6 or 12 months. Nrf2 deficiency significantly reduced early and more advanced atherosclerosis assessed by lesion size and coverage in the aorta in both models. Nrf2 deficiency in LDLR-/- mice reduced total plasma cholesterol after 6 weeks of HFD and triglycerides in LDLR-/-ApoB100/100 mice on a chow diet. Nrf2 deficiency aggravated aortic plaque maturation in aged LDLR-/-ApoB100/100 mice as it increased plaque calcification. Moreover, ∼36% of Nrf2-/-LDLR-/-ApoB100/100 females developed spontaneous myocardial infarction (MI) or sudden death at 5 to 12 months of age. Interestingly, Nrf2 deficiency increased plaque instability index, enhanced plaque inflammation and calcification, and reduced fibrous cap thickness in brachiocephalic arteries of LDLR-/-ApoB100/100 female mice at age of 12 months. Conclusions: Absence of Nrf2 reduced atherosclerotic lesion size in both atherosclerosis models, likely via systemic effects on lipid metabolism. However, Nrf2 deficiency in aged LDLR-/-ApoB100/100 mice led to an enhanced atherosclerotic plaque instability likely via increased plaque inflammation and oxidative stress, which possibly predisposed to MI and sudden death.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Nitro-Oleic Acid Regulates Endothelin Signaling in Human Endothelial Cells.

Nitro-fatty acids are reactive signaling mediators that are formed when unsaturated fatty acids react with nitric oxide or nitric oxide-derived species. Nitro-fatty acids can modify specific signaling pathways via post-translational modifications of Cys residues in key regulatory proteins. One of the signaling cascades activated by nitro-fatty acids is the Keap1-Nrf2 pathway. We have previously studied the effects of nitro-oleic acid (OA-NO2) on the human endothelial cell transcriptome. We observed that endothelin receptor B [ET-B (gene name EDNRB)], the receptor mediating the vasodilatory effects of endothelin-1 (ET-1) is induced by OA-NO2 Inasmuch as ET-1 is one of the key regulators of vascular tone, we chose to examine in more detail the effect of OA-NO2 on endothelin signaling in human endothelial cells. Nrf2 was found to regulate the OA-NO2-induced transcription of ET-B in human and mouse endothelial cells. Furthermore, chromatin immunoprecipitation analysis revealed that OA-NO2 increased the binding of Nrf2 to an antioxidant response element in the enhancer region of the EDNRB gene. In addition, we show that the overexpression of both OA-NO2 and Nrf2 substantially decreased and that Nrf2 silencing increased the ET-1 concentration in the culture media of endothelial cells. The change in the extracellular ET-1 concentration was dependent on ET-B receptor expression. These data suggest that OA-NO2 modulates endothelin signaling by increasing Nrf2-dependent expression of the ET-B receptor in endothelial cells, which in turn mediates the decrease in extracellular ET-1 concentration. Based on these results, we propose that OA-NO2 and Nrf2 may alleviate the vasoconstrictive effects of ET-1 by removing it from the circulation.

Western-type diet modulates inflammatory responses and impairs functional outcome following permanent middle cerebral artery occlusion in aged mice expressing the human apolipoprotein E4 allele.

BACKGROUND: Numerous clinical trials in stroke have failed, most probably partially due to preclinical studies using young, healthy male rodents with little relevance to the heterogenic conditions of human stroke. Co-morbid conditions such as atherosclerosis and infections coupled with advanced age are known to contribute to increased risk of cerebrovascular diseases. Clinical and preclinical studies have shown that the E4 allele of human apolipoprotein (ApoE4) is linked to poorer outcome in various conditions of brain injury and neurodegeneration, including cerebral ischemia. Since ApoE is a known regulator of lipid homeostasis, we studied the impact of a high-cholesterol diet in aged mice in the context of relevant human ApoE isoforms on the outcome of focal brain ischemia. METHODS: Aged mice expressing human E3 and E4 isoforms of ApoE in C57BL/6J background and C57BL/6J mice fed on either a high-fat diet or a normal diet underwent permanent middle cerebral artery occlusion. The impact of a high-cholesterol diet was assessed by measuring the serum cholesterol level and the infarction volume was determined by magnetic resonance imaging. Sensorimotor deficits were assessed using an adhesive removal test and the findings were correlated with inflammatory markers. RESULTS: We show that expression of human ApoE4 renders aged mice fed with a western-type diet more susceptible to sensorimotor deficits upon stroke. These deficits are not associated with atherosclerosis but are accompanied with altered astroglial activation, neurogenesis, cyclooxygenase-2 immunoreactivity and increased plasma IL-6. CONCLUSIONS: Our results support the hypothesis that ApoE alleles modify the inflammatory responses in the brain and the periphery, thus contributing to altered functional outcome following stroke.

The absence of macrophage Nrf2 promotes early atherogenesis.

AIMS: The loss of nuclear factor E2-related factor 2 (Nrf2) has been shown to protect against atherogenesis in apoE-deficient mice. The mechanism by which Nrf2 deficiency affords atheroprotection in this model is currently unknown, but combined systemic and local vascular effects on lesion macrophages have been proposed. We investigated the effect of bone marrow-specific loss of Nrf2 on early atherogenesis in low-density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice, and assessed the effect of Nrf2 on cellular accumulation of modified LDLs and the expression of inflammatory markers in macrophages. METHODS AND RESULTS: The effect of bone marrow-specific loss of Nrf2 on atherogenesis was studied using bone marrow transplantation of wild-type (WT) or Nrf2(-/-) bone marrow to LDLR(-/-) mice. Mice transplanted with Nrf2(-/-) bone marrow and fed a high-fat diet for 6 weeks exhibited significantly larger atherosclerotic lesions than WT bone marrow transplanted mice. Moreover, in thioglycollate-elicited Nrf2(-/-) macrophages, the uptake of acetylated and malondialdehyde-modified LDLs was increased in comparison with WT controls, with the concomitant increase in the expression of scavenger receptor A and toll-like receptor 4. In addition, the expression of pro-inflammatory monocyte chemoattractant protein-1 and interleukin-6 were increased in Nrf2(-/-) vs. WT macrophages. CONCLUSION: Nrf2 deficiency specific to bone marrow-derived cells aggravates atherosclerosis in LDLR(-/-) mice. Furthermore, the loss of Nrf2 in macrophages enhances foam cell formation and promotes the pro-inflammatory phenotype.

Oxidative stress-regulated lentiviral TK/GCV gene therapy for lung cancer treatment.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.