MicroRNAa-155 Deficiency Leads to Decreased Atherosclerosis

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Nov 17, 2016 - atherosclerosis; 2) ApoE-/-/miR-155-/- (DKO) mice show HF .... differentially in aorta, liver and white adipose tissue (WAT). ...... SDS] containing 0.4mg/mL proteinase K (EMD. Millipore, MA) at 55°C ..... vessel and pinned open on a black silicone tray for imaging. ... nitrocellulose membrane (GE). Membranes ...
JBC Papers in Press. Published on November 17, 2016 as Manuscript M116.739839 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.739839 MicroRNA-155 downregulation leads to obesity paradox

MicroRNA-155 deficiency leads to decreased atherosclerosis, increased white adipose tissue obesity and nonalcoholic fatty liver disease–a novel mouse model of obesity paradox Anthony Virtue1, Candice Johnson1*, Jahaira Lopez-Pastraña1*, Ying Shao1#, Hangfei Fu1#, Xinyuan Li1#, Ya-Feng Li1#, Ying Yin1#, Jietang Mai1#, Victor Rizzo1, Michael Tordoff2, Zsolt Bagi3, Huimin Shan1, Xiaohua Jiang1, Hong Wang1, Xiao-Feng Yang1 Centers for Metabolic Disease Research, Cardiovascular Research, & Thrombosis Research, Department of Pharmacology, Temple University Lewis Katz School of Medicine1, Philadelphia, PA, 19140, USA; Monell Chemical Senses Center2, Philadelphia, PA 19104, USA; Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA3

*Authors contributed equally to this work # Authors contributed equally to this work Running title: MicroRNA-155 downregulation leads to obesity paradox *Corresponding author: Xiao-Feng Yang, MD, PhD, FAHA, Centers for Metabolic Disease Research and Cardiovascular Research, Temple University Lewis Katz School of Medicine, 3500 North Broad Street, MERB-1059, Philadelphia, PA 19140, USA; Email: [email protected] Keywords: microRNA-155, inflammation, atherosclerosis, diet-induced obesity, nonalcoholic fatty liver disease

ABSTRACT Obesity paradox (OP) describes a widely observed clinical finding of improved cardiovascular fitness and survival in some overweight or obese patients. The molecular mechanisms underlying OP remain enigmatic partly due to a lack of animal models mirroring OP in patients. Using apolipoprotein E knockout (ApoE-/-) mice on high-fat (HF) diet as an atherosclerotic obesity model, we demonstrated: 1) microRNA-155 (miRNA-155, miR-155) is significantly upregulated in aortas of ApoE-/- mice; and miR-155 deficiency in ApoE-/- mice inhibits atherosclerosis; 2) ApoE-/-/miR-155-/- (DKO) mice show HF diet-induced obesity, adipocyte hypertrophy and present with nonalcoholic fatty liver disease (NAFLD); 3) DKO mice demonstrate HF diet-induced elevations of plasma leptin, resistin, fed-state and fasting insulin, increased expression of adipogenic transcription factors, but

lack glucose intolerance and insulin resistance. Our results are the first to present an OP model using DKO mice with features of decreased atherosclerosis, increased obesity and NAFLD. Our findings suggest the mechanistic role of reduced miR-155 expression in OP and present a new OP working model based on a single miRNA deficiency in diet-induced obese atherogenic mice. Furthermore, our results serve as a breakthrough in understanding the potential mechanism underlying OP and provide a new biomarker and novel therapeutic target for OP-related metabolic diseases. INTRODUCTION Metabolic syndrome (MetS) is a recently coined clinical term that refers to the presence of at least three of the following five conditions: central obesity, elevated triglycerides, diminished high density lipoprotein cholesterol, hypertension,

1 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MicroRNA-155 downregulation leads to obesity paradox and insulin resistance. Individuals diagnosed with MetS have a two-fold increased risk of suffering from stroke, myocardial infarction, atherosclerosis, or succumbing to cardiovascular disease (CVD)-associated mortality, and are five times more likely to develop type II diabetes1. In addition, non-alcoholic fatty liver disease (NAFLD) is a clinical condition hallmarked by the abnormal accumulation of triglycerides within the liver, and is considered a hepatic manifestation of MetS2. In contrast to the low mortality risk found in the normal body mass index (BMI) range of 20– 24.9 kg/m, BMI values in the overweight and obese categories usually have adverse effects on cardiac structure and function, with prevalence of CVD markedly increasing with the degree of obesity3. As an exception to this widely-accepted concept, recent clinical data has shown that in some patients with chronic diseases, including chronic heart failure or coronary artery disease, the best survival outcome is observed in overweight or obese patients. This phenomenon is termed “obesity paradox”4-7. However, the molecular and regulatory mechanisms underlying the obesity paradox remain unknown, which results partially from the fact that animal models mirroring obesity paradox have not been established. Chronic inflammation is a common mechanism of both obesity8 and atherosclerosis9, the latter of which can give rise to complications, such as myocardial infarction, stroke and peripheral artery disease, which are the leading cause of morbidity and mortality in the US10. We and others previously reported that hyperlipidemia and other risk factors promote vascular inflammation in various aspects, such as endothelial cell (EC) activation and injury11, 12, increasing monocyte recruitment and differentiation13-15, and decreasing regulatory T cell inflammation suppression16, 17. However, the interplay between obesity and atherosclerosis in the pathology of obesity paradox remains unknown. As we recently reviewed, the discovery of non-coding RNAs, including microRNAs (miRs), has revolutionized the way that we examine the genome, RNA products, and the regulation of transcription and translation18, 19. By facilitating mRNA degradation and translation repression, miRs regulate inflammatory responses20, EC

activation21, atherosclerosis22, obesity23, and NAFLD24, etc. Due to the well-characterized proinflammatory nature of miR-155, its role in chronic vascular inflammatory diseases has been critically investigated. However, the reported roles of miR-155 in vascular pathology have been controversial. MiR-155 promotes EC dysfunction by targeting the 3’-untranslated region of endothelial nitric oxide synthase (eNOS). In fact, inhibition of miR-155 increases eNOS expression and improves EC function25. However, other reports found that miR-155 reduces the expression of vascular cell adhesion molecule-1 (VCAM-1), leukocyte-EC interaction and EC migration26 via inhibition of pro-inflammatory extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in ECs27 and upregulation of anti-inflammatory heme oxygenase-128. Along the same line, the role of miR-155 in atherosclerosis also remains controversial. MiR-155 expression can be induced by tumor necrosis factor-α (TNF)-α29 in atherogenic apolipoprotein E-deficient (ApoE-/-) mouse aorta and in plasma samples from patients with coronary artery disease. In addition, it has been shown that hematopoietic deficiency of miR155 leads to more advanced atherosclerotic lesions in low density lipoprotein receptor knockout (LDLR-/-) mice25 while intravenous delivery of miR-155 “agomiRs” to overexpress miR-155 reduces atherosclerotic lesion in ApoE-/- mice30. In contrast to these findings, however, it has been reported that miR-155 promotes type 1 CD4+ T helper cell (Th1) differentiation31, 32 and that leukocyte-specific miR-155 deficiency reduces plaque size in ApoE-/- mice via directly targeting an NF-kB negative regulator, Bcl-633. Thus, the controversial roles of miR-155 in EC activation and atherogenesis need to be further elucidated. In addition, miR-155 appears to play an important role in adipose tissue. It is enriched in mouse brown adipose tissue, and miR-155deficient mice exhibit increased brown adipose tissue function and browning of white fat tissue34. MiR-155 is also expressed in human omental and subcutaneous adipose tissue where miR-155 expression is negatively correlated with adipocyte volume35. However, an important question remains whether miR-155 regulates the fat deposition differentially in aorta, liver and white adipose tissue (WAT).

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MicroRNA-155 downregulation leads to obesity paradox In this study, we examined the role of miR-155 in atherosclerosis and obesity using our newly generated ApoE-/-/miR-155-/- double knockout (DKO) mice. By using PCR array-, histological-, flow cytometry-, nuclear magnetic resonance- (NMR) and metabolic analyses, we found that miR-155 promotes aortic EC activation and atherosclerosis; and consequently that ApoE-//miR-155-/- DKO mice have less atherosclerotic plaques. Paradoxically, we showed that miR-155 inhibits WAT adipogenesis and NAFLD; and consequently that DKO mice have significantly increased body weight and augmented WAT and NAFLD. These results suggest that miR-155 promotes aortic EC activation and atherogenesis but inhibits WAT development and the pathogenesis of NAFLD, which make miR-155 deficiency in ApoE-/- background a novel model for studying the pathogenesis of obesity paradox. The establishment of this novel mouse model for the investigation of obesity paradox would have significant impact on the determination of the molecular mechanisms underlying obesity paradox and the identification of novel therapeutics for related diseases. RESULTS MiR-155 is significantly upregulated in the aortas of ApoE-/- mice during early atherosclerosis. In order to determine the roles of miRNAs in atherosclerosis development as well as measure their relevancies against other miRNAs, we utilized a PCR array to screen 84 of the most abundant and well characterized miRNAs in miRBase. We compared the aortic miRNA expression profiles of ApoE-/- mice fed normal chow diet versus ApoE-/- mice fed high fat diet for 12 weeks. Analysis of the array data revealed several miRNAs that were differentially regulated (Fig. 1A). We found that 16 miRNAs were upregulated by at least four-fold (miR-880, miR295, miR-155, miR-21a, miR-302d, miR-291a, and miR-744) or downregulated by at least fourfold (miR-99a, miR-199a, miR-335, miR-142-3p, miR-142-5p, miR-19b, miR-101a, miR-19a, and miR-22) (Fig. 1B). The atherosclerosis-induced downregulations of miR-19b and miR-22 were predicted in our bioinformatics analysis18. Of note, atherosclerosis-induced upregulations of five

miRs, including miR-880, miR-295, miR-302d, miR-291a and miR-744, in mouse aorta have not been reported38, 39. In addition, atherosclerosisinduced downregulations of eight miRs have not been reported in the atherosclerotic aorta, the exception being miR-19a38, 39. MiR-155 was one of three miRNAs with a 10-fold or greater change in expression and its well-characterized proinflammatory role suggested that it might be involved in the development of atherosclerosis. Of note, the unavailability of miR-880- and miR-295deficient mice prevented us from performing further atherosclerotic studies on those miRs right away. Next, we confirmed our miR-155 screening results with qRT-PCR (Fig. 1C). The statistical significance for miR-155 upregulation was observed after only 3 weeks of HF diet. Moreover, the fold-change and significance continued to increase with duration of feeding (Fig. 1C). Our data on the increased plasma levels of cholesterol and triglycerides in ApoE-/- mice fed with high fat diet for 0 weeks, 3 weeks, 6 weeks and 12 weeks (Figs. 1D and 1E) suggest that upregulation of miR-155 in aortas is correlated well with elevated levels of plasma cholesterol and triglycerides. This dramatic shift in miR-155 expression during atherosclerosis indicated that it might play a critical role in atherosclerosis and that it could be one of the most pertinent contributing miRNAs. In addition, since lipopolysaccharide (LPS) has been found to contribute to atherosclerosis40, we also examined the expression of miR-155 and other miRs in LPS-stimulated wild-type mouse aortas using PCR array. The results showed that LPS significantly upregulated miR-155 and miR22 in WT mouse aortas (Figs. 1F and 1G). Quantitative PCR confirmed that LPS induced miR-155 upregulation by more than 10-fold in WT mouse aortas (Fig. 1H). Collectively, these findings demonstrate that aortic miR-155 expression is upregulated during atherogenesis, indicating a pertinent role for miR-155 in atherosclerosis. MiR-155 promotes atherosclerosis endothelial cell activation in ApoE-/- mice.

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Thus far, there have been contradicting reports on the contributions of miR-155 in atherosclerosis development. A previous paper published by Nazari-Jahantigh et al. detailed a

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MicroRNA-155 downregulation leads to obesity paradox pro-atherogenic role for miR-155 through its promotion of a pro-inflammatory macrophage response33. In contrast, Donners et al. have reported that hematopoietic miR-155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice25. Due to the conflicting reports within the scientific community, we felt that atherosclerosis assessment within our model would provide useful and significant insight into the topic. As part of this assessment, we organized a table identifying direct targets of miR-155 in atherosclerosis from previous studies (Suppl. Table 1). Furthermore, our new results, demonstrating that four-hour LPS stimulation significantly upregulates miR-155 expression in WT aortas, uniquely highlight the potential contributions of vascular cell miR-155 expression to atherogenesis in the absence of significant macrophage recruitment into the aorta under these conditions41. We performed several experiments to determine the role of miR-155 in vascular ECs and atherosclerosis. Investigation of atherosclerosis within our models yielded novel results. First, we found aortic intercellular adhesion molecule-1 (ICAM-1), a prototypic EC adhesion molecule, to be significantly diminished in miR-155-/- mice in comparison to that of WT mice in non-stimulated and LPS-stimulated mice (Figs. 2A and 2C). This suggests that miR-155 promotes ICAM-1 expression under both physiological conditions and during times of inflammation where it facilitates aortic EC activation. The constitutive expression of ICAM-1 and insignificant upregulation of ICAM-1 in WT aortas in response to four-hour LPS stimulation may be due to a high constitutive expression of ICAM-1 or possibly a delayed ICAM-1 upregulation in response to LPS stimulation. Second, we found no difference in the expression of aortic VCAM-1, another prototypical EC adhesion molecule, between miR-155-/- and WT mice (Figs. 2A and 2B). This suggests that miR155 selectively impacts ICAM-1 expression. In order to further extend this finding, we examined miR-155 expression in human aortic ECs (HAECs) stimulated by LPS, pro-atherogenic inflammatory cytokines, TNF-α and IL-1β, as well as oxidized low-density lipoprotein (oxLDL)derived pro-atherogenic lipids, lysophosphatidic acid (lysoPA) and lysophosphatidylcholine (lysoPC). Although the results of TNF-α-induced

upregulation of miR-155 in human umbilical vein ECs have been reported29, we are the first to report that stimulation by LPS, TNF-α, IL-1β, lysoPA and lysoPC induces miR-155 upregulation in atherosclerosis-related human aortic ECs (Fig. 2D). Similar to our data, other studies show that TNF-α, IL-1β and IL-6 serve as pro-inflammatory mediators of miR-155’s pro-atherosclerotic function33, 43. Our findings of miR-155 upregulation in aortic ECs in response to proatherogenic stimuli and of miR-155 promotion of ICAM-1 expression in mouse aortas strongly suggest an important role for miR-155 in promoting aortic EC activation. To directly examine this issue, we used a static EC adhesion assay as a functional read-out for aortic EC activation. HAECs were transfected with various doses of miR-155 mimic (6.25 ng, 12.5 ng, 25 ng and 50 ng) and then permitted to interact with nontreated human peripheral blood mononuclear cells as we have previously reported12. Our results showed that enhanced miR-155 expression in aortic ECs significantly increased monocyte adhesion to miR-155 mimic-transfected HAECs in comparison to HAECs transfected with mimic controls, indicating that miR-155 plays a functional role in EC activation and leukocyte adhesion (Fig. 2E). Pro-inflammatory immune responses play a critical role in the pathogenesis of MetS, obesity, and atherosclerosis42, and miR-155 has been indicated as a mediator of several cellular processes related to these pathologies33. In addition, our results on miR-155 promotion of aortic EC activation implicate miR-155 as a player in atherogenesis. We used ApoE-/-/miR-155-/DKO mice to determine whether miR-155 promotes early atherosclerosis, which was not examined previously33, 43. At 8 weeks of age, several groups of male mice were fed with high fat diet for 0 weeks, 3 weeks, 6 weeks, or 12 weeks, after which aortas were excised for en face analysis (Figs. 3A and 3B). As expected, we observed virtually no plaque area in the 0 and 3 week high fat-fed mice, as well as no discernible differences between ApoE-/- and DKO mice. However, after 6 weeks of high fat diet, a noticeable phenotype began to develop despite being statistically insignificant, as DKO mice had 32% less aortic lesions. This indicates that miR-

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MicroRNA-155 downregulation leads to obesity paradox 155 in vascular cells, including aortic ECs, contributes to early atherosclerosis (i.e., the stage prior to intermediate lesion formation in ApoE-/mouse aortas44) as we reported in a similar study11. Observation at 12 weeks of high fat diet resulted in statistically significant findings, translating to a 45% decrease in lesion area in DKO mice in comparison to their ApoE-/- equivalents (p