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. Author manuscript; available in PMC: 2024 Aug 1.
Published in final edited form as: Alcohol Clin Exp Res (Hoboken). 2023 Jul 10;47(8):1467–1477. doi: 10.1111/acer.15138

Alcohol and vascular endothelial function; Biphasic effect highlights the importance of dose

Naresh K Rajendran *, Weimin Liu †, Paul A Cahill #, Eileen M Redmond §
PMCID: PMC10751391  NIHMSID: NIHMS1913237  PMID: 37369447

Abstract

Background.

Alcohol (ethanol/EtOH) consumption variously influences arterial disease, being protective or harmful depending on the amount and pattern of consumption. The mechanisms mediating these biphasic effects are unknown. Endothelial cells play a critical role in maintaining arterial health. The aim of our study was to compare the effect of moderate vs high alcohol concentrations on endothelial cell function.

Methods.

Human coronary artery endothelial cells (HCAEC) were treated with levels of ethanol associated with either low-risk/moderate drinking (i.e., 25 mM) or with high-risk/heavy drinking (i.e., 50 mM) before assessing endothelial function. The effect of ethanol’s primary metabolite acetaldehyde (10 μM and 25 μM) was also determined.

Results.

Moderate EtOH exposure (25 mM) improved HCAEC barrier integrity as determined by increased transendothelial electrical resistance (TEER), inhibited cell adhesion molecule (CAM) mRNA expression, decreased inflammatory cytokine (interferon-γ and interleukin 6) production, and inhibited monocyte chemotactic protein-1 (MCP-1) expression and monocyte adhesion, while stimulating homeostatic Notch signaling. In contrast, exposure to high level EtOH (50 mM) decreased TEER, increased CAM expression and inflammatory cytokine production, and stimulated MCP-1 and monocyte adhesion, with no effect on Notch signaling. Reactive oxygen species (ROS) generation and eNOS activity were increased by both alcohol treatments, and to a greater extent in the 50 mM EtOH group. Acetaldehyde-elicited responses were generally the same as those of the high level EtOH group.

Conclusions.

EtOH has biphasic effects on several endothelial functions such that moderate level alcohol maintains the endothelium in a non-activated state, whereas high level alcohol causes endothelial dysfunction, as does acetaldehyde. These data stress the importance of dose when considering alcohol’s effects in arterial endothelium, and may explain, in part, the J-shaped relationship between alcohol concentration and atherosclerosis reported in epidemiologic studies.

INTRODUCTION

Numerous studies over the last three decades indicate that alcohol consumption impacts atherosclerotic cardiovascular disease that is the leading cause of death worldwide (World Health Organization). While the relationship is complex and influenced by both the pattern of drinking and the amount consumed(Piano, 2017), in general, a J-shaped response is evident with both epidemiologic and animal studies pointing to a protective effect of frequent low-to-moderate consumption but a harmful effect of heavy drinking and chronic alcohol abuse on arterial disease and its associated morbidity and mortality(O’Keefe et al., 2014, Rehm and Roerecke, 2017, Ronksley et al., 2011) (Liu et al., 2011). These data suggest that alcohol is a double-edged sword when it comes to arterial disease, with the dose making it either an antidote or a toxin. Of note, recent analyses have highlighted methodological problems in some epidemiologic studies, (e.g. the confounding effects of sick quitters being classified as abstainers, and/or of coincident favorable lifestyle factors in low-risk drinkers) that challenge the purported cardioprotection of low level alcohol consumption (Stockwell and Zhao, 2017),(Biddinger et al., 2022) . In contrast, others report that the reduced risk of cardiovascular disease with moderate drinking is independent of such confounding variables(McEvoy et al., 2022). This controversy emphasizes the importance of gaining a complete basic science understanding of the effects of alcohol, at different levels, on arterial cells involved in atherogenesis.

The endothelium, a cell monolayer lining the blood vessel lumen, is an active interface between the circulating blood and the underlying vessel wall. The endothelium plays a critical role to preserve arterial health and function and to prevent atherogenesis(Cahill and Redmond, 2016). It acts not only as a semipermeable barrier, but by altering its release of vasoactive substances and/or its expression of surface proteins it can influence a variety of responses (e.g., platelet function, vascular tone, cell phenotype, monocyte adhesion, and inflammation) whose balance is influential to vessel homeostasis(Cahill and Redmond, 2016). Damage or ‘activation’ of the endothelium leading to its compromised function is considered a decisive initiating step in atherogenesis(Gimbrone and Garcia-Cardena, 2016).

Studies have previously investigated alcohol effects on endothelial function. Human studies looking at lifetime alcohol intake and using flow-mediated dilation (FMD) as a measure of endothelial-mediated vasodilation showed that consumption of up to 2 drinks/day was associated with increased FMD (Suzuki et al., 2009, Teragawa et al., 2002), while heavier consumption (> 3 drinks/day) was associated with reduced FMD(Tanaka et al., 2016); supportive of a biphasic endothelial response to alcohol. In vitro studies show that ethanol influences endothelial vasoactive activity both acutely and chronically(Hendrickson et al., 1999, Polikandriotis et al., 2005), alters angiogenesis(Morrow et al., 2008), and maintains endothelium in an non-activated state despite an atherogenic milieu(Rajendran et al., 2021). These studies, more often than not, have investigated a single dose of alcohol. Clinically, alcohol is known to have a hormetic influence on several diseases including atherosclerosis, whereby protective effects at moderate intake are reversed with higher exposures (Fernandez-Sola, 2015). The aim of our study, therefore, was to interrogate this concept by comparing the effect of ethanol at levels consistent with either low-risk/moderate drinking or increased risk/heavy drinking, as well as to determine the effect of alcohol’s primary metabolite, acetaldehyde, on various aspects of endothelial function relevant to vessel homeostasis and arterial disease.

MATERIALS AND METHODS

Cell Culture

Human coronary artery endothelial cells (HCAEC) were obtained from Lonza (Walkersville, MD, USA) and cultured in optimized endothelial cell medium (Cat# CC-3162, CloneticsR, Lonza, Walkersville, MD, USA) along with supplements (FBS, hydrocortisone, hFGF, VEGF, RE-IGF-1, ascorbic acid, hEGF, GA-1000 & heparin). HCAEC passage 5–14 were used for all experiments. EtOH (200 proof, ACS/USP Grade, Pharmco Products, Brookfield, CT, USA) was diluted in media to achieve the desired concentration before being added to cells for the time specified. Experimental plates were covered in parafilm to prevent evaporation. Under these conditions ethanol concentration (determined by diagnostic kit from Sigma) after 24 hrs of incubation +/− endothelial cells was ~92% of that at the start, indicating no significant metabolism of alcohol by these cells. Acetaldehyde was obtained from Sigma (St Louis, MO). A stock concentration of 100 mM ACT was made up in PBS and diluted in culture medium to the desired concentration.

Quantitative real-time reverse transcription PCR (qRT-PCR)

Total RNA was extracted using RNeasy Mini Kit (Cat# 74134, Hilden, Germany) and 1 μg RNA was reverse transcribed to cDNA using iScript cDNA Synthesis kit (Cat# 1708891, Biorad, Hercules, CA, USA). The mRNA expression was determined using SYBR Green master mix (Cat# 4309155, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol using a QuantStudio 3 (Applied Bosystems, Foster City, CA, USA). The RT-PCR cycling conditions were as follows; DNA polymerase activation at 95°C for 10 min, followed by 40 PCR cycles, each cycle consisting of 95°C for 15 s (denature) and 60°C for 1 min (anneal/extend).

Primer sequences were HRT 2 forward (5′-GTACCTGAGCTCCGTGGAAG-3′) and reverse (5′-AGTTGTGGAGAGGCGACAAG-3′); HRT 3 forward (5′-GGTGGGACAGGATTCTTTGA-3′) and reverse (5′-AGCTGTTGAGGTGGGAGAGA-3′); ICAM-1 forward (5′-AGGATGGCACTTTCCCACTG-3′ and reverse (5′-GGAGAGCACATTCACGGTCA-3′); VCAM-1 forward (5′-ACTGGCATGGTACGGAGATG-3′) and reverse (5′-CAATGTGACTAAAGGAGGCAGT-3′); MCP-1 forward (5′-AGCATGAAAGTCTCTGCCGC-3′) and reverse (5′-GGGTACCACGTCTGCTTGGA-3′); Caveolin-1 forward (5’-GCTGTCGGAGCGGTTAGTT-3’ and reverse (5′-TGTAGATGTTGCCCTGTTCCC-3’); GAPDH forward (5′-CGAGATCCCTCCAAAATCAA-3′) and reverse (5′-TTCACACCCATGGACGAACAT-3′). GAPDH was used as a housekeeping control. The data presented are the average (mean ± SEM). The results were analyzed with Applied Biosystems® qPCR analysis software (ThermoFisher Scientific).

Cell viability.

The cell metabolic activity/viability of HCAEC in 96-well plates following treatments (24 h) was assessed by MTT colorimetric assay (Abcam, Cambridge, MA) according to the manufacturer’s instructions. Absorbance readings of extracts were performed at λ 590 nm.

Trans-endothelial electrical resistance (TEER)

A fundamental role of the endothelium is to act as a semipermeable barrier controlling passage of macromolecules and cells between the lumen and the vessel wall (Chistiakov et al., 2015, Sluiter et al., 2021). Barrier function was assessed by TEER, measured according to Ghatak (Ghatak et al., 2016). In brief, HCAEC (P4 – P8) were grown in 12-well Transwells (0.4 μm polyester membrane, polystyrene plates) until ~90% confluency and treated with or without experimental agents as indicated for 1 hr at 37°C. Membrane resistance was then measured using chopstick electrodes attached to an Epithelial Voltohmmeter (EVOM2, World Precision Instruments, Inc., Sarasota, FL). The electrode was completely immersed in the media but not touching to the base of the wells. Readings were taken at three different positions (12, 4, 8 o’clock) of each Transwell insert.

ICAM-1 detection by Immunofluorescence

Cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) expressed on the surface of endothelial cells play a role in facilitating the adhesion and migration of inflammatory cells during atherogenesis (Fotis et al., 2012) (Huo and Ley, 2001, Jaipersad et al., 2014). HCAECs were seeded onto sterilized fibronectin coated cover slips in a 6-well plate. Following treatment, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, then permeabilized (0.1% Triton X-100, 15 min). Cells were blocked with 3% BSA (bovine serum albumin) for 30 min and probed with anti-ICAM-1 antibody (1:100), (Cat# MA5–13021, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4 °C. After washing, FITC-conjugated anti-rabbit IgG antibody (Cat# 62–6511, Thermo Fisher Scientific) was added and incubated for 2 h at 37°C. Cells were mounted with Sigma Fluoroshield with DAPI. Images were captured using a fluorescence microscope (Palmbeam LCM, Zeiss).

Cytokines: Enzyme-Linked Immunosorbant Assay (ELISA)

Increased expression of inflammatory cytokines by activated endothelium is instrumental to atherogenesis (Tedgui and Mallat, 2006). Supernatants of HCAEC cultures were collected following treatments as indicated and stored at −80 °C until analysis. Interferon-γ (IFN-γ, Cat# DIF50) and Interleukin-6 (IL-6, Cat# D6060) levels were determined using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Western blot analysis

Total cell lysate was obtained using ice-cold RIPA cell lysis buffer (cat#89900, ThermoFisher Scientific) and 1X Protease and Phosphatase Inhibitor cocktail mixture. The extracted protein was quantified using Bio-Rad protein assay kit II (cat#5000002; Bio-Rad, Hercules, CA). Proteins were separated by size using 4% to 20% Mini-Protean TGX Precast protein gels (cat#4561094; Bio-Rad) and then transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, MA, USA). The protein-blotted membrane was blocked with a 5% (w/v) skimmed milk solution in 0.1% (v/v) Tween-20 for 1hr at room temperature in a rocking platform. Primary antibodies to caveolin-1 (cat#3238), and beta-actin (cat#4970) (Cell Signaling Technology, Danvers, MA) were incubated overnight at 4oC. The secondary antibody-anti-rabbit IgG, HRP conjugated (cat#7074; Cell Signaling Technology) was incubated for 120 min at room temperature in rocking platform. Proteins were visualized with a SuperSignal west Pico Chemiluminescent Substrate (Cat # 34580; ThermoFisher Scientific) and ChemiDoc XR Imaging System (Bio-Rad, Hercules, CA).

THP-1 Monocyte Adhesion Assay

HCAEC were grown to 80% confluence in 96-well plates and treated with ethanol or acetaldehyde for 24 hr at 37°C. Human monocytic cell line (THP-1) cells were obtained from American Type Culture Collection (Cat# TIB-202, ATCC®, Manassas, VA, USA) and labelled with 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (10 μg/mL) (BCECF-AM; Invitrogen, Carlsbad, CA, USA) for 30 min in RPMI-1640 medium and washed twice with HBSS. THP-1 cells (1 ×103) were then added to HCAEC monolayers, and cultures were incubated at 37°C in a CO2 incubator for 3 hr. After incubation, medium was removed and HCAEC were washed twice with PBS to remove unbound THP-1 cells. The fluorescence intensity of remaining THP-1 cells was measured at λ 480nm Excitation/520nm Emission wavelength range using a SpectraMax 340PC spectrophotometer (Molecular Devices, CA, USA). The number of adherent cells was expressed as fluorescence intensity.

NOS activity

Endothelial nitric oxide synthase (eNOS) mediates the production of the vasodilator nitric oxide (NO). eNOS activity was measured in HCAEC lysates using a colorimetric Nitric Oxide Synthase Activity Assay Kit based on the Greiss reaction (Catalog # K205–100, BioVision Inc).

Reactive Oxygen Species Assay

Balancing of reactive oxygen species (ROS) and cell redox status is a pre-requisite for vessel health(Nowak et al., 2017). ROS was measured by incubating HCAEC with 10 μM 5-(and-6)-carboxy-21, 71-dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA, 30 min) (Cat# C400, Invitrogen, Carlsbad, CA, USA), a cell permeable indicator for ROS generation. Cells were then treated with experimental agents for 3 hr, before cells were counterstained with DAPI (1 μg/ml, 10 min) and ROS generation (red fluorescence) detected by microplate reader at λ 480nm excitation/520nm emission.

Data analysis

Data are expressed as mean ± SEM. Experimental points were performed in duplicate with a minimum of 3 independent experiments. An unpaired Student’s t-test was performed to determine differences between 2 groups, while multiple data sets were analysed by ordinary one-way ANOVA followed by a Tukey’s or Dunnett’s multiple comparisons test (GraphPad Prism software V 9.1.0). A probability (p) value of 0.05 was considered significant.

RESULTS

Endothelial inflammatory cytokine production; J-shaped response to EtOH

Human coronary artery endothelial cells (HCAEC) were treated with various doses of ethanol (EtOH 0–100 mM, 24 hr) before cytokine levels in supernatants were determined by specific ELISA. There was a j-shaped response between EtOH concentration and both interleukin-6 (IL-6) and interferon-γ (IFN-γ) cytokine production, with maximum inhibition by 25 mM EtOH, but significant stimulation at 50 and 100 mM EtOH (Fig 1). Based on this, we choose to use 25 mM (‘moderate’ dose) and 50 mM (‘high’ dose) for the remainder of our experiments. The effect of acetaldehyde (10 μM and 25 μM) was also determined. While there was no effect of acetaldehyde on IL-6, acetaldehyde (25 μM) significantly increased IFN-γ levels in the supernatant (Fig 1). HCAEC viability under the different treatments was assessed by MTT/ cell metabolic activity assay. While there was no effect on cell viability with either EtOH 25 mM or acetaldehyde 25 μM, there was a significant decrease (~20%) in HCAEC viability following both EtOH 50 mM and acetaldehyde 25 μM exposures (Fig 2a). z culture revealed no ethanol- or acetaldehyde-induced changes in cell morphology (Fig 2b).

Figure 1.

Figure 1.

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Biphasic effect of ethanol on endothelial inflammatory cytokine production. (a) Interleukin-6 (IL-6) and (b) interferon-γ (IFN-γ) levels in human coronary artery endothelial cells (HCAEC) supernatants following treatment (24 hr) with Ethanol (EtOH) or acetaldehyde as indicated. Ethanol dose-response (0–100 mM EtOH) curve is shown (top), together with bar graph data for EtOH 25 mM (E 25) and 50 mM (E 50), and for acetaldehyde 10 μM (A 10) and 25 μM (A 50) (bottom). Data are mean ± SEM, n=3. *P<0.05, **P<0.01 vs Control.

Figure 2.

Figure 2.

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HCAEC viability assessed by MTT/cell metabolic activity assay. (a) Effect of EtOH 25 mM (E 25), EtOH 50 mM (E 50), acetaldehyde 10 μM (A 10) and acetaldehyde 25 μM (A 25) treatment (24 hr). Data are mean ± SEM, n=3. *P<0.05 vs C. (b) Representative phase contrast images of HCAEC treated with either EtOH or acetaldehyde (Act) as indicated showing gross morphology in culture.

Biphasic effect of moderate and high EtOH on endothelial barrier integrity

Barrier function of HCAEC grown in Transwell plates was assessed by measuring trans-endothelial electrical resistance (TEER). Following treatment (1 hr), electrical resistance across the HCAEC monolayer was significantly increased compared to control in the moderate EtOH group (25 mM EtOH) (189±8.0 vs 160±7.3 Ω.cm2), but decreased in the high EtOH group (50 mM EtOH: 137±4.9 Ω.cm2), and in both acetaldehyde treatment groups (markedly so by 25 μM acetaldehyde) (Fig 3).

Figure 3.

Figure 3.

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Biphasic effect of ethanol on endothelial barrier integrity. Trans-endothelial electrical resistance (TEER) of HCAEC monolayers in Transwell inserts treated (2 hr) with/without EtOH 25 mM (E 25), EtOH 50 mM (E 50) or acetaldehyde 10 μM (A 10) and acetaldehyde 25 μM (A 25). Data are mean ± SEM, n=3. *P<0.05, **P<0.01 vs C.

Biphasic effect of moderate and high EtOH on cell adhesion molecule expression

Treatment with 25 mM EtOH significantly inhibited VCAM-1 and ICAM-1 mRNA expression compared to control cells, whereas 50 mM EtOH increased the mRNA of both adhesion molecules (Fig 4). Treatment with acetaldehyde also significantly increased VCAM-1 and ICAM-1 mRNA expression in these cells (Fig 4).

Figure 4.

Figure 4.

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Biphasic effect of ethanol on endothelial cell adhesion molecule expression. (a) VCAM-1 mRNA expression and (b) ICAM-1 expression in HCAEC (top) (representative immunofluoresence images shown, white scale bar = 50 μM), and ICAM-1 mRNA expression (bottom) in HCAEC determined by qRT-PCR following treatment (24 hr) as indicated: EtOH 25 mM or 50 mM (E 25, E 50), acetaldehyde 10 μM or 25 μM (A 10, A 25). Data are mean ± SEM, n=3. *P<0.05, **P<0.01 vs C.

Biphasic effect of moderate and high EtOH on MCP-1 expression and on monocyte adhesion

The CC chemokine monocyte chemotactic protein-1 (MCP-1), binding to its receptor CCR2, facilitates monocyte recruitment instrumental to the pathogenesis of atherosclerosis(Bianconi et al., 2018). Treatment of HCAEC with moderate level EtOH (25 mM) inhibited MCP-1 mRNA expression, whereas treatment with high level EtOH (50 mM) significantly increased MCP-1 expression (Fig 5a). Similarly to high level EtOH, exposure to acetaldehyde (25 μM, but not 10 μM) also stimulated MCP-1 mRNA levels (Fig 5a). THP-1, a human monocytic cell line, is extensively used to study monocyte activities(Chanput et al., 2014). We used THP-1 cells to interrogate monocyte adhesion to HCAEC under the different experimental conditions. Compared to the control group, the adhesion of THP-1 monocytes to HCAEC was significantly reduced following treatment with moderate EtOH (25 mM) (~65% decrease), but significantly increased following treatment with either high EtOH (50 mM) (~40% increase) or with acetaldehyde (25 μM) (~100% increase) (Fig 5b).

Figure 5.

Figure 5.

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Biphasic effect of ethanol on MCP-1 expression and monocyte adhesion. (a) Monocyte chemoattractant protein-1 (MCP-1) mRNA levels in HCAEC, and (b) THP-1 monocyte adhesion to HCAEC following treatment (24 hr) as indicated: EtOH 25 mM or 50 mM (E 25, E 50), acetaldehyde 10 μM or 25 μM (A 10, A 25). Data are mean ± SEM, n=3. *P<0.05, **P<0.01 vs C.

EtOH stimulates eNOS activity in HCAEC

Using a colorimetric assay, we measured eNOS activity in HCAEC lysates following treatment with or without EtOH. Ethanol, at both 25 mM and 50 mM doses, significantly increased eNOS activity (1.9 and 2.2 fold, respectively) compared to that in control untreated cells (Fig 6a).

Figure 6.

Figure 6.

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Ethanol stimulates eNOS activity and ROS generation. (a) eNOS activity in HCAEC lysates following treatment (2 hr) with moderate and high level EtOH (E 25 mM, E 50 mM). (b) ROS generation in HCAEC treated (3 hr) with EtOH (E 25 mM, E 50 mM) or acetaldehyde (A 10 μM, A 25 μM). Data are mean ± SEM, n=3. *P<0.05, **P<0.01 vs C, and # p<0.05 between experimental groups as indicated.

EtOH stimulates endothelial ROS generation

Intercellular ROS generation was measured using a cell-permeable fluorescent probe as described in Methods. Treatment with EtOH at both moderate (25 mM) and high (50 mM) doses elicited increased ROS generation in HCAEC over control non-treated cells, with a more pronounced response seen in the high EtOH group (i.e.,1.8 vs 1.3 fold increase) (Fig 6b). Acetaldehyde treatment also dose-dependently increased HCAEC ROS generation with a greater than 2 fold increase by 25 μM (Fig 6b).

Differential effect of moderate and high EtOH on caveolin-1 expression and Notch signalling

Notch signalling in vascular cells is key to maintaining vessel homeostasis(Aquila et al., 2019). In endothelium this pathway is negatively regulated by caveolin-1(Rajendran et al., 2023). We compared the effect of moderate and high levels of EtOH on caveolin-1 and Notch signalling in HCAEC. Treatment with moderate level EtOH (25 mM EtOH) inhibited caveolin-1 mRNA and protein levels and concomitantly stimulated Notch signalling in HCAEC as indicated by increased target gene (hrt2 and hrt3) expression (Fig 7). In contrast, both high level EtOH (50 mM EtOH) and acetaldehyde (25 μM) increased caveolin-1 mRNA and did not significantly affect Notch signalling (Fig 7).

Figure 7.

Figure 7.

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Moderate level EtOH, but not high level EtOH or acetaldehyde, stimulates Notch signalling in endothelial cells. (a) Caveolin-1 (Cav-1), (b) Hrt2, and (c) Hrt3 mRNA levels in HCAEC determined by qRT-PCR following treatment with EtOH (E 25 mM, 50 mM) or with acetaldehyde (A 10 μM, 25 μM). Data are mean ± SEM, n=3–4. *P<0.05, **P<0.01 vs Control (no treatment). Also shown in (a); a representative Western blot showing Caveolin-1 protein expression in HCAEC treated +/− EtOH as indicated. β-actin was used as a loading control.

DISCUSSION

Here we report that alcohol has striking biphasic effects on several endothelial functions such that at moderate levels it supports the endothelium in a non-activated state, whereas at high levels it causes endothelial dysfunction. These data highlight the importance of dose and emphasize the sharp ‘double-edged’ nature of alcohol with respect to endothelial homeostatic function and, thus potentially, to atherosclerotic vascular disease.

Drinking is a common modifiable behaviour worldwide. Approximately 63% of US adults use alcoholic beverages, and 25% of those report that they engaged in binge drinking or heavy alcohol use in the past month (NIAAA ‘Alcohol facts and statistics’ March 2022). Meta-analysis of epidemiologic data suggests that, compared to abstinence, light to moderate daily consumption (i.e., 1–4 drinks/day) is associated with the lowest risk of cardiovascular disease incidence and mortality, whereas episodic bingeing and chronic alcohol abuse are associated with a higher incidence of cardiovascular disease and increased mortality(O’Keefe et al., 2014, Rehm and Roerecke, 2017). In agreement with these data, a J-shaped relationship between alcohol intake and ischemic heart disease was observed in the Global Burden of Disease Study(Collaborators, 2018), and a differential effect of daily-moderate (protective) vs 2 day-binge alcohol (exacerbatory) on atherosclerotic plaque development was noted in a mouse model (Liu et al., 2011).

Here, based on J-shaped dose-response curves obtained in initial experiments as well as on our previous reports(Rajendran et al., 2023, Rajendran et al., 2021), we assigned 25 mM as ‘moderate level’ EtOH and 50 mM as ‘high level’ EtOH and compared the effects of the two doses. Keeping in mind that a person’s blood alcohol concentration (BAC) is affected by several factors including how much alcohol is consumed and how quickly, body weight, gender, pattern of drinking, and genetics, how do the concentrations we used in vitro relate to those found in alcohol consumers? Generally, for a male who weighs 150 lbs, 4 standard drinks is predicted to result in a BAC of 25 mM (i.e., 0.1 g%; ‘impaired’ range, consistent with social alcohol consumption), and 8 drinks in 2 hrs will result in a BAC of 50 mM (i.e., 0.2 g%; ‘intoxicated’ range, consistent with alcohol abuse/heavy drinking). Thus, by National Institute on Alcohol Abuse and Alcoholism (NIAAA) definitions(Alcohol Research: Current Reviews Editorial, 2018), 25 mM EtOH could be achieved by low-risk drinking, albeit at the upper limit (daily limit for low risk drinking for men is ‘no more than 4 drinks’), whereas 50 mM EtOH would be achieved following heavy binge drinking (5 or more drinks in ~2 hours) or high-intensity drinking (i.e, double or triple the standard binge drinking threshold)(Alcohol Research: Current Reviews Editorial, 2018).

The endothelium is multifaceted in its role as a gatekeeper of vessel health(Cahill and Redmond, 2016). It acts as a barrier to prevent vascular leakage and to control the passage of cells and lipoproteins from the lumen into the vessel wall, it regulates blood flow, it maintains a basal anti-inflammatory and anti-thrombotic state, and controls the balance of vascular cell proliferation and death, all of which impact vessel homeostasis (Cahill and Redmond, 2016) (Bianconi et al., 2018, Sluiter et al., 2021). Alterations in these endothelial homeostatic mechanisms contribute critically to the initiation and progression of atherosclerosis and its complications such as heart attack and stroke(Cahill and Redmond, 2016, Libby et al., 2006). Endothelial injury and dysfunction can be triggered by a variety of stimuli including hypoxia, turbulent blood flow, hypercholesterolemia, smoking, hypertension, and bacterial or viral infection(Abraham and Distler, 2007, Ross, 1999). These injurious stimuli all lead to inflammation and endothelial cell activation or dysfunction(Libby et al., 2006).

Our data indicate biphasic effects of ethanol (i.e., the type of alcohol found in alcoholic beverages) on several important aspects of endothelial biology including barrier function, cell adhesion molecule and MCP-1 expression, inflammatory cytokine production, and monocyte adhesion (Fig 8). Specifically, moderate level EtOH exposure generally boosted homeostatic endothelial function; it improved barrier integrity as determined by increased TEER, inhibited inflammatory cytokine release and cell adhesion molecule expression, and resulted in reduced MCP-1 expression and less monocyte adhesion, effects expected to protect against atherogenesis. On the other hand, high level EtOH exposure caused endothelial activation/dysfunction; it decreased TEER, stimulated inflammatory cytokine release and cell adhesion molecule expression, and resulted in greater MCP-1 expression and increased monocyte adhesion, effects expected to drive atherogenesis. These latter data strongly suggest that alcohol at levels associated with heavy drinking is injurious to the endothelium. We note that while TEER measurement is widely used to assess barrier integrity in endothelial monolayers in cell culture(Bednarek, 2022), TEER can be affected by changes in ionic absorption/secretion and therefore may not reflect changes in paracellular permeability(Bischoff et al., 2016). Whether the differential TEER changes we see here with moderate and high EtOH treatments correlate with changes in macromolecular flux and/or structural changes in tight junctions remains to be determined.

Figure 8.

Figure 8.

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While alcohol (at both moderate and high levels) stimulates ROS generation and eNOS activity, it has biphasic effects on endothelial barrier integrity, cell adhesion molecule and MCP-1 expression, inflammatory cytokine production, monocyte adhesion, and Notch signaling such that moderate level alcohol supports the endothelium in a non-activated state, whereas high level alcohol causes endothelial dysfunction. Acetaldehyde, alcohol’s primary metabolite, has similar activating effects as high level alcohol. Figure created using Biorender.

Unlike for the responses listed above, EtOH did not have a biphasic effect with respect to reactive oxygen species (ROS), but rather a dose-dependent effect; i.e., both EtOH exposures (moderate and high) elicited increased ROS generation from human arterial endothelial cells, but a more pronounced response was seen with high level EtOH. While certain levels of ROS are crucial for proper cell signalling under physiological conditions, sustained increased levels can lead to oxidative stress and be detrimental(Forstermann et al., 2017). Indeed, increased ROS levels are known contributors to endothelial dysfunction and activation of an inflammatory response(Nowak et al., 2017). Thus, it seems likely that the high level EtOH ROS response would be pro-atherogenic, whereas the low level EtOH ROS response may or may not be. Moreover, in a similar manner to ROS, both moderate and high levels of EtOH stimulated eNOS activity in HCAEC, data that are in general agreement with previous studies (Abou-Agag et al., 2005, Hendrickson et al., 1999, Rajendran et al., 2021). Given that nitric oxide (NO) produced by eNOS is an antioxidant(Hummel et al., 2006) as well as a vasodilator, and is believed to counterbalance intracellular ROS (Forstermann et al., 2017), increases in both eNOS activity (protective) and ROS (damaging) induced by EtOH might be expected to functionally cancel each other out with respect to effects on atherogenesis, a concept that warrants further investigation. Moreover, whether chronic alcohol exposure would elicit similar endothelial responses overall as the acute treatments (i.e., 1–24h) in this study remains to be determined.

Acetaldehyde, a cause of facial flushing and hangover symptoms, is the primary metabolite of ethanol(Abraham et al., 2011). It is metabolized to the less toxic acetate by aldehyde dehydrogenase (ALDH). Acetaldehyde can accumulate in the body due to a deficiency of/or lesser functioning ALDH (e.g., a variant present in Asians), or following excessive alcohol consumption. Acetaldehyde levels in blood and breath of alcoholics have been measured in the range 10–110 μM(Korsten et al., 1975, Lindros et al., 1980, Tsukamoto et al., 1989). Acetaldehyde can also be produced in the body from other sources, such as cigarette smoke and some foods. While acetaldehyde can cause DNA damage and is carcinogenic (Seitz and Stickel, 2010), its role in mediating cardiovascular effects in drinkers is less well known. Acetaldehyde reportedly stimulates arterial smooth muscle cell growth(Hatch et al., 2018). Here, we found that acetaldehyde, especially at the higher dose tested (25 μM), generally had similar pro-atherogenic effects as high level EtOH in endothelial cells, i.e., it stimulated inflammatory cytokines, reduced barrier integrity, increased cell adhesion molecule and MCP-1 expression, increased monocyte adhesion, and stimulated ROS generation. It is likely that following heavy bingeing and/or in a person with alcohol use disorder (AUD), high levels of both EtOH and acetaldehyde could be achieved and could act synergistically to cause endothelial dysfunction and promote atherogenesis.

Notch signaling is an important player in vessel homeostasis in the adult(Aquila et al., 2019). Notch activation results in the upregulation of genes that preserve endothelial function(Briot et al., 2016, Mack and Iruela-Arispe, 2018, Mack et al., 2017). Endothelial Notch signaling is negatively regulated by caveolin-1(Rajendran et al., 2023), and is mechanosensitive being affected by laminar shear stress(Mack et al., 2017). Our data demonstrate that whereas moderate EtOH inhibited caveolin-1 and thus stimulated Notch target genes in endothelial cells in agreement with our previous report(Rajendran et al., 2023), high level EtOH and acetaldehyde both increased caveolin-1 and did not stimulate Notch signaling. Given that Notch dysregulation is implicated in endothelial dysfunction and the pathophysiology of arterial disease, these differential effects of moderate and high EtOH on Notch signaling are likely relevant to their respective endothelial protective and endothelial damaging effects described herein.

Maintaining a healthy endothelium is paramount for vessel health and is a clinical goal for reducing the enormous health and financial burden associated with atherosclerotic cardiovascular disease(Khera et al., 2020, Virani et al., 2020). Overall, our data indicate that alcohol has biphasic effects on several critical endothelial functions, such that moderate levels of alcohol maintain endothelium in the non-activated state instrumental to vessel homeostasis, whereas high levels of alcohol and of acetaldehyde cause endothelial dysfunction (Fig. 8). These findings underscore the importance of dose when assessing alcohol’s effects on endothelium and arterial health, that may underpin the J-shaped effect of alcohol on arteriosclerotic vascular disease reported in epidemiologic and animal studies. Moreover, this study bolsters existing guidelines on alcohol consumption and re-emphasizes the likely harm to health of heavy drinking.

ACKNOWLEDGMENTS

The authors wish to thank Dr Heli Hamalainen-Laanaya for proof-reading. This work was supported by grant R01AA024082 (to EMR) from the National Institutes of Health.

Footnotes

CONFLICT OF INTEREST

None declared

Contributor Information

Naresh K. Rajendran, Department of Surgery University of Rochester Medical Center, Rochester, NY, USA

Weimin Liu, Department of Surgery University of Rochester Medical Center, Rochester, NY, USA.

Paul A. Cahill, Vascular Biology and Therapeutics Laboratory (PAC), School of Biotechnology, Dublin City University, Ireland

Eileen M. Redmond, Department of Surgery University of Rochester Medical Center, Rochester, NY, USA.

REFERENCES

  1. Abou-Agag LH, Khoo NK, Binsack R, White CR, Darley-Usmar V, Grenett HE, Booyse FM, Digerness SB, Zhou F, Parks DA (2005) Evidence of cardiovascular protection by moderate alcohol: role of nitric oxide. Free Radic Biol Med 39:540–548. [DOI] [PubMed] [Google Scholar]
  2. Abraham D, Distler O (2007) How does endothelial cell injury start? The role of endothelin in systemic sclerosis. Arthritis Res Ther 9 Suppl 2:S2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Abraham J, Balbo S, Crabb D, Brooks PJ (2011) Alcohol Metabolism in Human Cells Causes DNA Damage and Activates the Fanconi Anemia-Breast Cancer Susceptibility (FA-BRCA) DNA Damage Response Network. Alcoholism, clinical and experimental research. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alcohol Research: Current Reviews Editorial S (2018) Drinking Patterns and Their Definitions. Alcohol Res 39:17–18. [PMC free article] [PubMed] [Google Scholar]
  5. Aquila G, Kostina A, Vieceli Dalla Sega F, Shlyakhto E, Kostareva A, Marracino L, Ferrari R, Rizzo P, Malaschicheva A (2019) The Notch pathway: a novel therapeutic target for cardiovascular diseases? Expert Opin Ther Targets 23:695–710. [DOI] [PubMed] [Google Scholar]
  6. Bednarek R (2022) In Vitro Methods for Measuring the Permeability of Cell Monolayers. Methods Protoc 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bianconi V, Sahebkar A, Atkin SL, Pirro M (2018) The regulation and importance of monocyte chemoattractant protein-1. Current opinion in hematology 25:44–51. [DOI] [PubMed] [Google Scholar]
  8. Biddinger KJ, Emdin CA, Haas ME, Wang M, Hindy G, Ellinor PT, Kathiresan S, Khera AV, Aragam KG (2022) Association of Habitual Alcohol Intake With Risk of Cardiovascular Disease. JAMA Netw Open 5:e223849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bischoff I, Hornburger MC, Mayer BA, Beyerle A, Wegener J, Furst R (2016) Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay. Sci Rep 6:23671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Briot A, Bouloumie A, Iruela-Arispe ML (2016) Notch, lipids, and endothelial cells. Curr Opin Lipidol 27:513–520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cahill PA, Redmond EM (2016) Vascular endothelium - Gatekeeper of vessel health. Atherosclerosis 248:97–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chanput W, Mes JJ, Wichers HJ (2014) THP-1 cell line: an in vitro cell model for immune modulation approach. Int Immunopharmacol 23:37–45. [DOI] [PubMed] [Google Scholar]
  13. Chistiakov DA, Orekhov AN, Bobryshev YV (2015) Endothelial Barrier and Its Abnormalities in Cardiovascular Disease. Front Physiol 6:365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Collaborators GBDA (2018) Alcohol use and burden for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 392:1015–1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fernandez-Sola J (2015) Cardiovascular risks and benefits of moderate and heavy alcohol consumption. Nat Rev Cardiol 12:576–587. [DOI] [PubMed] [Google Scholar]
  16. Forstermann U, Xia N, Li H (2017) Roles of Vascular Oxidative Stress and Nitric Oxide in the Pathogenesis of Atherosclerosis. Circulation research 120:713–735. [DOI] [PubMed] [Google Scholar]
  17. Fotis L, Agrogiannis G, Vlachos IS, Pantopoulou A, Margoni A, Kostaki M, Verikokos C, Tzivras D, Mikhailidis DP, Perrea D (2012) Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 at the early stages of atherosclerosis in a rat model. In Vivo 26:243–250. [PubMed] [Google Scholar]
  18. Ghatak S, Reveiller M, Toia L, Ivanov AI, Zhou Z, Redmond EM, Godfrey TE, Peters JH (2016) Bile Salts at Low pH Cause Dilation of Intercellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt Signaling. J Gastrointest Surg 20:500–509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gimbrone MA Jr, Garcia-Cardena G (2016) Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis. Circulation research 118:620–636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hatch E, Morrow D, Liu W, Cahill PA, Redmond EM (2018) Differential effects of alcohol and its metabolite acetaldehyde on vascular smooth muscle cell Notch signaling and growth. American journal of physiology 314:H131–h137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hendrickson RJ, Cahill PA, Sitzmann JV, Redmond EM (1999) Ethanol enhances basal and flow-stimulated nitric oxide synthase activity in vitro by activating an inhibitory guanine nucleotide binding protein. The Journal of pharmacology and experimental therapeutics 289:1293–1300. [PubMed] [Google Scholar]
  22. Hummel SG, Fischer AJ, Martin SM, Schafer FQ, Buettner GR (2006) Nitric oxide as a cellular antioxidant: a little goes a long way. Free Radic Biol Med 40:501–506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huo Y, Ley K (2001) Adhesion molecules and atherogenesis. Acta Physiol Scand 173:35–43. [DOI] [PubMed] [Google Scholar]
  24. Jaipersad AS, Lip GY, Silverman S, Shantsila E (2014) The role of monocytes in angiogenesis and atherosclerosis. J Am Coll Cardiol 63:1–11. [DOI] [PubMed] [Google Scholar]
  25. Khera R, Valero-Elizondo J, Nasir K (2020) Financial Toxicity in Atherosclerotic Cardiovascular Disease in the United States: Current State and Future Directions. J Am Heart Assoc 9:e017793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Korsten MA, Matsuzaki S, Feinman L, Lieber CS (1975) High blood acetaldehyde levels after ethanol administration. Difference between alcoholic and nonalcoholic subjects. The New England journal of medicine 292:386–389. [DOI] [PubMed] [Google Scholar]
  27. Libby P, Aikawa M, Jain MK (2006) Vascular endothelium and atherosclerosis. Handb Exp Pharmacol:285–306. [DOI] [PubMed] [Google Scholar]
  28. Lindros KO, Stowell A, Pikkarainen P, Salaspuro M (1980) Elevated blood acetaldehyde in alcoholics with accelerated ethanol elimination. Pharmacol Biochem Behav 13 Suppl 1:119–124. [DOI] [PubMed] [Google Scholar]
  29. Liu W, Redmond EM, Morrow D, Cullen JP (2011) Differential effects of daily-moderate versus weekend binge alcohol consumption on atherosclerotic plaque development in mice. Atherosclerosis. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mack JJ, Iruela-Arispe ML (2018) NOTCH regulation of the endothelial cell phenotype. Current opinion in hematology 25:212–218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mack JJ, Mosqueiro TS, Archer BJ, Jones WM, Sunshine H, Faas GC, Briot A, Aragon RL, Su T, Romay MC, McDonald AI, Kuo CH, Lizama CO, Lane TF, Zovein AC, Fang Y, Tarling EJ, de Aguiar Vallim TQ, Navab M, Fogelman AM, Bouchard LS, Iruela-Arispe ML (2017) NOTCH1 is a mechanosensor in adult arteries. Nat Commun 8:1620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McEvoy LK, Bergstrom J, Tu X, Garduno AC, Cummins KM, Franz CE, Lyons MJ, Reynolds CA, Kremen WS, Panizzon MS, Laughlin GA (2022) Moderate Alcohol Use Is Associated with Reduced Cardiovascular Risk in Middle-Aged Men Independent of Health, Behavior, Psychosocial, and Earlier Life Factors. Nutrients 14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morrow D, Cullen JP, Cahill PA, Redmond EM (2008) Ethanol stimulates endothelial cell angiogenic activity via a Notch- and angiopoietin-1-dependent pathway. Cardiovascular research 79:313–321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nowak WN, Deng J, Ruan XZ, Xu Q (2017) Reactive Oxygen Species Generation and Atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology 37:e41–e52. [DOI] [PubMed] [Google Scholar]
  35. O’Keefe JH, Bhatti SK, Bajwa A, DiNicolantonio JJ, Lavie CJ (2014) Alcohol and cardiovascular health: the dose makes the poison…or the remedy. Mayo Clin Proc 89:382–393. [DOI] [PubMed] [Google Scholar]
  36. Piano MR (2017) Alcohol’s Effects on the Cardiovascular System. Alcohol Res 38:219–241. [PMC free article] [PubMed] [Google Scholar]
  37. Polikandriotis JA, Rupnow HL, Hart CM (2005) Chronic ethanol exposure stimulates endothelial cell nitric oxide production through PI-3 kinase-and hsp90-dependent mechanisms. Alcoholism, clinical and experimental research 29:1932–1938. [DOI] [PubMed] [Google Scholar]
  38. Rajendran NK, Liu W, Cahill PA, Redmond EM (2023) Caveolin-1 inhibition mediates the opposing effects of alcohol on gamma-secretase activity in arterial endothelial and smooth muscle cells. Physiol Rep 11:e15544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rajendran NK, Liu W, Chu CC, Cahill PA, Redmond EM (2021) Moderate dose alcohol protects against serum amyloid protein A1-induced endothelial dysfunction via both notch-dependent and notch-independent pathways. Alcoholism, clinical and experimental research 45:2217–2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rehm J, Roerecke M (2017) Cardiovascular effects of alcohol consumption. Trends Cardiovasc Med 27:534–538. [DOI] [PubMed] [Google Scholar]
  41. Ronksley PE, Brien SE, Turner BJ, Mukamal KJ, Ghali WA (2011) Association of alcohol consumption with selected cardiovascular disease outcomes: a systematic review and meta-analysis. BMJ (Clinical research ed 342:d671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ross R (1999) Atherosclerosis--an inflammatory disease. The New England journal of medicine 340:115–126. [DOI] [PubMed] [Google Scholar]
  43. Seitz HK, Stickel F (2010) Acetaldehyde as an underestimated risk factor for cancer development: role of genetics in ethanol metabolism. Genes Nutr 5:121–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sluiter TJ, van Buul JD, Huveneers S, Quax PHA, de Vries MR (2021) Endothelial Barrier Function and Leukocyte Transmigration in Atherosclerosis. Biomedicines 9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stockwell T, Zhao J (2017) Alcohol’s contribution to cancer is underestimated for exactly the same reason that its contribution to cardioprotection is overestimated. Addiction (Abingdon, England) 112:230–232. [DOI] [PubMed] [Google Scholar]
  46. Suzuki K, Elkind MS, Boden-Albala B, Jin Z, Berry G, Di Tullio MR, Sacco RL, Homma S (2009) Moderate alcohol consumption is associated with better endothelial function: a cross sectional study. BMC Cardiovasc Disord 9:8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tanaka A, Cui R, Kitamura A, Liu K, Imano H, Yamagishi K, Kiyama M, Okada T, Iso H, Investigators C (2016) Heavy Alcohol Consumption is Associated with Impaired Endothelial Function. J Atheroscler Thromb 23:1047–1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tedgui A, Mallat Z (2006) Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiological reviews 86:515–581. [DOI] [PubMed] [Google Scholar]
  49. Teragawa H, Fukuda Y, Matsuda K, Higashi Y, Yamagata T, Matsuura H, Chayama K (2002) Effect of alcohol consumption on endothelial function in men with coronary artery disease. Atherosclerosis 165:145–152. [DOI] [PubMed] [Google Scholar]
  50. Tsukamoto S, Muto T, Nagoya T, Shimamura M, Saito M, Tainaka H (1989) Determinations of ethanol, acetaldehyde and acetate in blood and urine during alcohol oxidation in man. Alcohol and alcoholism 24:101–108. [DOI] [PubMed] [Google Scholar]
  51. Virani SS, Alonso A, Benjamin EJ, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Delling FN, Djousse L, Elkind MSV, Ferguson JF, Fornage M, Khan SS, Kissela BM, Knutson KL, Kwan TW, Lackland DT, Lewis TT, Lichtman JH, Longenecker CT, Loop MS, Lutsey PL, Martin SS, Matsushita K, Moran AE, Mussolino ME, Perak AM, Rosamond WD, Roth GA, Sampson UKA, Satou GM, Schroeder EB, Shah SH, Shay CM, Spartano NL, Stokes A, Tirschwell DL, VanWagner LB, Tsao CW (2020) Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association. Circulation 141:e139–e596. [DOI] [PubMed] [Google Scholar]

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