Fen Yin

Heme Oxygenase-1, Oxidation, Inflammation, and Atherosclerosis

Atherosclerosis is an inflammatory process of the vascular wall characterized by the infiltration of lipids and inflammatory cells. Oxidative modifications of infiltrating low-density lipoproteins and induction of oxidative stress play a major role in lipid retention in the vascular wall, uptake by macrophages and generation of foam cells, a hallmark of this disorder. The vasculature has a plethora of protective resources against oxidation and inflammation, many of them regulated by the Nrf2 transcription factor. Heme oxygenase-1 (HO-1) is a Nrf2-regulated gene that plays a critical role in the prevention of vascular inflammation. It is the inducible isoform of HO, responsible for the oxidative cleavage of heme groups leading to the generation of biliverdin, carbon monoxide, and release of ferrous iron. HO-1 has important antioxidant, antiinflammatory, antiapoptotic, antiproliferative, and immunomodulatory effects in vascular cells, most of which play a significant role in the protection against atherogenesis. HO-1 may also be an important feature in macrophage differentiation and polarization to certain subtypes. The biological effects of HO-1 are largely attributable to its enzymatic activity, which can be conceived as a system with three arms of action, corresponding to its three enzymatic byproducts. HO-1 mediated vascular protection may be due to a combination of systemic and vascular local effects. It is usually expressed at low levels but can be highly upregulated in the presence of several proatherogenic stimuli. The HO-1 system is amenable for use in the development of new therapies, some of them currently under experimental and clinical trials. Interestingly, in contrast to the HO-1 antiatherogenic actions, the expression of its transcriptional regulator Nrf2 leads to proatherogenic effects instead. This suggests that a potential intervention on HO-1 or its byproducts may need to take into account any potential alteration in the status of Nrf2 activation. This article reviews the available evidence that supports the antiatherogenic role of HO-1 as well as the potential pathways and mechanisms mediating vascular protection.

NF-E2–Related Factor 2 Promotes Atherosclerosis by Effects on Plasma Lipoproteins and Cholesterol Transport That Overshadow Antioxidant Protection

Objective—To test the hypothesis that NF-E2–related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties. Methods and Results—Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E–null background by sequential breeding, resulting in Nrf2−/−, apoE−/− (KO), Nrf2−/+, apoE−/− (HET) and Nrf2+/+, and apoE−/− wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36. Conclusion—Nrf2 expression unexpectedly promotes atherosclerotic lesion formation in a sex-dependent manner, most likely by a combination of systemic metabolic and local vascular effects.

Diesel Exhaust Induces Systemic Lipid Peroxidation and Development of Dysfunctional Pro-Oxidant and Pro-Inflammatory High-Density Lipoprotein

Objective—To evaluate whether exposure to air pollutants induces oxidative modifications of plasma lipoproteins, resulting in alteration of the protective capacities of high-density lipoproteins (HDLs). Approach and Results—We exposed apolipoprotein E–deficient mice to diesel exhaust (DE) at ≈250 µg/m3 for 2 weeks, filtered air (FA) for 2 weeks, or DE for 2 weeks, followed by FA for 1 week (DE+FA). DE led to enhanced lipid peroxidation in the brochoalveolar lavage fluid that was accompanied by effects on HDL functionality. HDL antioxidant capacity was assessed by an assay that evaluated the ability of HDL to inhibit low-density lipoprotein oxidation estimated by 2′,7′-dichlorofluorescein fluorescence. HDL from DE-exposed mice exhibited 23 053±2844 relative fluorescence units, higher than FA-exposed mice (10 282±1135 relative fluorescence units, P<0.001) but similar to the HDL from DE+FA-exposed mice (22 448±3115 relative fluorescence units). DE effects on HDL antioxidant capacity were negatively correlated with paraoxonase enzymatic activity, but positively correlated with levels of plasma 8-isoprostanes, 12-hydroxyeicosatetraenoic acid, 13-hydroxyoctadecadienoic acid, liver malondialdehyde, and accompanied by perturbed HDL anti-inflammatory capacity and activation of the 5-lipoxygenase pathway in the liver. Conclusions—DE emissions induced systemic pro-oxidant effects that led to the development of dysfunctional HDL. This may be one of the mechanisms by which air pollution contributes to enhanced atherosclerosis.

Network for Activation of Human Endothelial Cells by Oxidized Phospholipids: A Critical Role of Heme Oxygenase 1

Rationale: Oxidized palmitoyl arachidonyl phosphatidylcholine (Ox-PAPC) accumulates in atherosclerotic lesions, is proatherogenic, and influences the expression of more than 1000 genes in endothelial cells. Objective: To elucidate the major pathways involved in Ox-PAPC action, we conducted a systems analysis of endothelial cell gene expression after exposure to Ox-PAPC. Methods and Results: We used the variable responses of primary endothelial cells from 149 individuals exposed to Ox-PAPC to construct a network that consisted of 11 groups of genes, or modules. Modules were enriched for a broad range of Gene Ontology pathways, some of which have not been identified previously as major Ox-PAPC targets. Further validating our method of network construction, modules were consistent with relationships established by cell biology studies of Ox-PAPC effects on endothelial cells. This network provides novel hypotheses about molecular interactions, as well as candidate molecular regulators of inflammation and atherosclerosis. We validated several hypotheses based on network connections and genomic association. Our network analysis predicted that the hub gene CHAC1 (cation transport regulator homolog 1) was regulated by the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway, and here we showed that ATF4 directly activates an element in the CHAC1 promoter. We showed that variation in basal levels of heme oxygenase 1 (HMOX1) contribute to the response to Ox-PAPC, consistent with its position as a hub in our network. We also identified G-protein–coupled receptor 39 (GPR39) as a regulator of HMOX1 levels and showed that it modulates the promoter activity of HMOX1. We further showed that OKL38/OSGN1 (oxidative stress–induced growth inhibitor), the hub gene in the blue module, is a key regulator of both inflammatory and antiinflammatory molecules. Conclusions: Our systems genetics approach has provided a broad view of the pathways involved in the response of endothelial cells to Ox-PAPC and also identified novel regulatory mechanisms.

HDL anti-oxidant function associates with LDL level in young adults

OBJECTIVES The primary objective was to evaluate predictors of HDL anti-oxidant function in young adults. BACKGROUND High-density lipoprotein (HDL) cholesterol is considered a protective factor for cardiovascular disease (CVD). However, increased levels are not always associated with decreased cardiovascular risk. A better understanding of the importance of HDL functionality and how it affects CVD risk is needed. METHODS Fifty non-Hispanic white subjects from the Testing Responses on Youth (TROY) study were randomly selected to investigate whether differences in HDL anti-oxidant function are associated with traditional cardiovascular risk factors, including carotid intima media thickness (CIMT), arterial stiffness and other inflammatory/metabolic parameters. HDL anti-oxidant capacity was evaluated by assessing its ability to inhibit low-density lipoprotein (LDL) cholesterol oxidation by air using a DCF-based fluorescent assay and expressed as a HDL oxidant index (HOI). The associations between HOI and other variables were assessed using both linear and logistic regression. RESULTS Eleven subjects (25%) had an HOI ≥ 1, indicating a pro-oxidant HDL. Age, LDL, high sensitivity C-reactive protein (hsCRP), and paraoxonase activity (PON1), but not HDL, were all associated with HOI level in univariate linear regression models. In multivariate models that mutually adjusted for these variables, LDL remained the strongest predictor of HOI (0.13 increase in HOI per 1 SD increase in LDL, 95% CI 0.04, 0.22). Atherogenic index of plasma, pulse pressure, homocysteine, glucose, insulin, CIMT and measurements of arterial stiffness were not associated with HOI in this population. CONCLUSIONS These results suggest LDL, hsCRP and DBP might predict HDL anti-oxidant function at an early age.

Increased Cardiac Sympathetic Activity and Oxidative Stress in Habitual Electronic Cigarette Users: Implications for Cardiovascular Risk

Importance Electronic cigarettes (e-cigarettes) have gained unprecedented popularity, but virtually nothing is known about their cardiovascular risks. Objective To test the hypothesis that an imbalance of cardiac autonomic tone and increased systemic oxidative stress and inflammation are detectable in otherwise healthy humans who habitually use e-cigarettes. Design, Setting, and Participants Cross-sectional case-control study of habitual e-cigarette users and nonuser control individuals from 2015 to 2016 at the University of California, Los Angeles. Otherwise healthy habitual e-cigarette users between the ages of 21 and 45 years meeting study criteria, including no current tobacco cigarette smoking and no known health problems or prescription medications, were eligible for enrollment. Healthy volunteers meeting these inclusion criteria who were not e-cigarette users were eligible to be enrolled as control individuals. A total of 42 participants meeting these criteria were enrolled in the study including 23 self-identified habitual e-cigarette users and 19 self-identified non–tobacco cigarette, non–e-cigarette user control participants. Main Outcomes and Measures Heart rate variability components were analyzed for the high-frequency component (0.15-0.4 Hz), an indicator of vagal activity, the low-frequency component (0.04-0.15 Hz), a mixture of both vagal and sympathetic activity, and the ratio of the low frequency to high frequency, reflecting the cardiac sympathovagal balance. Three parameters of oxidative stress were measured in plasma: (1) low-density lipoprotein oxidizability, (2) high-density lipoprotein antioxidant/anti-inflammatory capacity, and (3) paraoxonase-1 activity. Results Of the 42 participants, 35% were women, 35% were white, and the mean age was 27.6 years. The high-frequency component was significantly decreased in the e-cigarette users compared with nonuser control participants (mean [SEM], 46.5 [3.7] nu vs 57.8 [3.6] nu; P = .04). The low-frequency component (mean [SEM], 52.7 [4.0] nu vs 39.9 [3.8] nu; P = .03) and the low frequency to high frequency ratio (mean [SEM], 1.37 [0.19] vs 0.85 [0.18]; P = .05) were significantly increased in the e-cigarette users compared with nonuser control participants, consistent with sympathetic predominance. Low-density lipoprotein oxidizability, indicative of the susceptibility of apolipoprotein B–containing lipoproteins to oxidation, was significantly increased in e-cigarette users compared with nonuser control individuals (mean [SEM], 3801.0 [415.7] U vs 2413.3 [325.0] U; P = .01) consistent with increased oxidative stress, but differences in high-density antioxidant/anti-inflammatory capacity and paraoxonase-1 activity were not significant. Conclusions and Relevance In this study, habitual e-cigarette use was associated with a shift in cardiac autonomic balance toward sympathetic predominance and increased oxidative stress, both associated with increased cardiovascular risk.

Prooxidative Effects of Ambient Pollutant Chemicals Are Inhibited by HDL

Exposure to air pollution leads to adverse pulmonary and systemic vascular effects. High levels of plasma high‐density lipoproteins (HDL) cholesterol reduce cardiovascular risk. We explored whether HDL could protect against the prooxidative effects of an organic extract of diesel exhaust particles (DEP) in vascular cells. We used a cell‐free fluorescent assay to evaluate DEP oxidation by air, estimated by the degree of dichlorofluorescein (DCF) fluorescence and tested the ability of HDL to inhibit it. We also evaluated DEP prooxidative effects in bovine aortic endothelial cells and RAW264.7 macrophages by DCF fluorescence. DEP oxidation and prooxidative cellular effects occurred in concentration‐ and time‐dependent manners. Normal HDL inhibited DEP oxidation and prooxidative cellular effects, whereas dysfunctional HDL failed to inhibit DEP oxidation and instead, it promoted further oxidation. In conclusion, DEP prooxidative effects in endothelial cells and macrophages are inhibited by normal HDL. Therefore, HDL may protect against air pollution mediated adverse vascular effects.

Cigarette smoking is associated with dose-dependent adverse effects on paraoxonase activity and fibrinogen in young women.

Abstract Context: Smoking is associated with increased fibrinogen and decreased paraoxonase (PON) activity, markers of inflammation and oxidative stress, in patients with coronary artery disease. Objective: We tested the hypothesis that the adverse effect of smoking on these biomarkers of inflammation and oxidative stress would be detectable in otherwise healthy young female habitual smokers. Materials and methods: Thirty-eight young women participated in the study (n = 20 habitual smokers, n = 18 non-smokers). Fibrinogen, PON-1 activity and HDL oxidant index (HOI) were measured. Results: Mean values of fibrinogen, PON-1 activity and log HOI were not different between the groups. Importantly, however, decreased PON-1 activity (rs = −0.51, p = 0.03) and increased fibrinogen (rs = 0.49, p = 0.04) were significantly correlated with increasing number of cigarettes smoked per day in habitual smokers. Discussion and conclusion: Cigarette smoking is associated with a dose-dependent adverse effect on PON-1 activity and fibrinogen in young women, which may have implications for future cardiovascular risk.

No effect of acute exposure to coarse particulate matter air pollution in a rural location on high-density lipoprotein function

Abstract Context: High-density lipoprotein (HDL) particles perform numerous vascular-protective functions. Animal studies demonstrate that exposure to fine or ultrafine particulate matter (PM) can promote HDL dysfunction. However, the impact of PM on humans remains unknown. Objective: We aimed to determine the effect of exposure to coarse concentrated ambient particles (CAP) on several metrics of HDL function in healthy humans. Methods: Thirty-two adults (25.9 ± 6.6 years) were exposed to coarse CAP [76.2 ± 51.5 µg·m−3] in a rural location and filtered air (FA) for 2 h in a randomized double-blind crossover study. Venous blood collected 2- and 20-h post-exposures was measured for HDL-mediated efflux of [3H]-cholesterol from cells and 20-h exposures for HDL anti-oxidant capacity by a fluorescent assay and paraoxonase activity. The changes [median (first, third quartiles)] between exposures among 29 subjects with available results were compared by matched Wilcoxon tests. Results: HDL-mediated cholesterol efflux capacity did not differ between exposures at either time point [16.60% (15.17, 19.19) 2-h post-CAP versus 17.56% (13.43, 20.98) post-FA, p  =  0.768 and 14.90% (12.47, 19.15) 20-h post-CAP versus 17.75% (13.22, 23.95) post-FA, p  =  0.216]. HOI [0.26 (0.24, 0.35) versus 0.28 (0.25, 0.40), p = 0.198] and paraoxonase activity [0.54 (0.39, 0.82) versus 0.60 μmol·min−1 ml plasma−1 (0.40, 0.85), p = 0.137] did not differ 20-h post-CAP versus FA, respectively. Conclusions: Brief inhalation of coarse PM from a rural location did not acutely impair several facets of HDL functionality. Whether coarse PM derived from urban sites, fine particles or longer term PM exposures can promote HDL dysfunction warrant future investigations.

Sympathomimetic Effects of Acute E‐Cigarette Use: Role of Nicotine and Non‐Nicotine Constituents

Background Chronic electronic (e) cigarette users have increased resting cardiac sympathetic nerve activity and increased susceptibility to oxidative stress. The purpose of the present study is to determine the role of nicotine versus non‐nicotine constituents in e‐cigarette emissions in causing these pathologies in otherwise healthy humans. Methods and Results Thirty‐three healthy volunteers who were not current e‐cigarette or tobacco cigarette smokers were studied. On different days, each participant used an e‐cigarette with nicotine, an e‐cigarette without nicotine, or a sham control. Cardiac sympathetic nerve activity was determined by heart rate variability, and susceptibility to oxidative stress was determined by plasma paraoxonase activity. Following exposure to the e‐cigarette with nicotine, but not to the e‐cigarette without nicotine or the sham control, there was a significant and marked shift in cardiac sympathovagal balance towards sympathetic predominance. The decrease in high‐frequency component and the increases in the low‐frequency component and the low‐frequency to high‐frequency ratio were significantly greater following exposure to the e‐cigarette with nicotine compared with exposure to the e‐cigarette without nicotine or to sham control. Oxidative stress, as estimated by plasma paraoxonase, did not increase following any of the 3 exposures. Conclusions The acute sympathomimetic effect of e‐cigarettes is attributable to the inhaled nicotine, not to non‐nicotine constituents in e‐cigarette aerosol, recapitulating the same heart rate variability pattern associated with increased cardiac risk in multiple populations with and without known cardiac disease. Evidence of oxidative stress, as estimated by plasma paraoxonase activity, was not uncovered following acute e‐cigarette exposure.