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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. Cancer Res; 72(23); 6227–35. ©2012 AACR.

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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. Keywords: Aging, Autophagy, Degeneration, Oxidative stress, Protein aggregation, Proteasome

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. Summary 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.

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. Summary 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.

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.

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

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.

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.

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. Cancer Res; 72(23); 6227–35. ©2012 AACR.

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.

OBJECTIVE Transcription factor NF-E2-related factor 2 (Nrf2) regulates antioxidant and detoxifying enzymes affording cytoprotection in the cardiovascular system. We have previously reported that Nrf2 deficiency specific to bone marrow derived cells aggravates atherosclerosis in LDL receptor deficient (LDLR-/-) mice. Furthermore, Nrf2 deficiency in macrophages enhances foam cell formation and promotes the proinflammatory phenotype. In contrast, the total loss of Nrf2 has been shown to protect against atherogenesis in apoE-deficient mice. The mechanism by which Nrf2 deficiency affords atheroprotection in apoE-model is currently unknown, but combined systemic and local vascular effects have been proposed. Given these contrasting results, we aimed at assessing the effect of total loss of Nrf2 on atherogenesis in hypercholesterolemic LDLR-/- mice. METHODS AND RESULTS Nrf2-/- mice were cross-bred to LDLR-/- mice and fed a high fat diet (HFD) for 6 or 12 weeks. The degree of atherosclerosis was assessed from the cross-sections of proximal aorta. Nrf2 deficiency decreased atherosclerosis in females from 17.2±8.7 % (mean ±SD, n=11-25) to 12.3±4.6 % (p=0.1) and in males from 11.2±3.9 % to 7.6±4.6 % (p=0.025) after 6 weeks on HFD. After 12 weeks on HFD the effects were more pronounced, as Nrf2 deficiency decreased atherosclerosis from 36.5±5.5 % to 30.3±3.5 % in females (p=0.001) and from 30.6±5.7 % to 21.3±6.9 % in males (p=0.005). Nrf2 deficiency also increased the macrophage content relative to lesion area. Supporting systemic effects, Nrf2 deficiency reduced plasma total cholesterol from 24.0±8.4 mmol/l to 10.6±6,7 mmol/l (p=0.006) and triglyceride levels from 3.6±1.4 mmol/l to 1.7±1.2 mmol/l (p=0.008) in males after 6 weeks on HFD. CONCLUSIONS In contrast to bone marrow specific Nrf2 deficiency, total loss of Nrf2 aggravates atherosclerosis in LDLR-/- mice likely via systemic effects on lipid metabolism.

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.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.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.