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ORIGINAL ARTICLES

ALDH2 mutation results in excessive basal nitric oxide production and a delayed response to nitroglycerin

Zhu, Hongming1; Hu, Jingjing2; Dong, Zhen2; Liu, Yang1; Sun, Xiaolei2; Sun, Aijun2,3,4,∗

Author Information
doi: 10.1097/CP9.0000000000000011

Abstract

Introduction

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes superoxides and aldehydes[1]. Approximately 8% of the world's population carry a genetic variant of the ALDH2 gene, ALDH2+/−, and have much lower ALDH2 enzymatic activity. This variant is particularly prevalent in people of East Asian descent.

Coronary artery spasm contributes to the etiology ischemic heart diseases[2]. Patients carrying a mutant ALDH2 gene tend to have reduced response to nitroglycerin since nitroglycerin must be metabolized by ALDH2 to nitric oxide (NO) to produce vasodilatory action[3,4]. In comparison to people carrying wildtype (WT) ALDH2 gene, the vasodilatory response to nitroglycerin is reduced by 40% in those carrying the ALDH2+/− gene[5].

A reliable disease model that faithfully recapitulates the phenotype of individuals with the ALDH2+/− variant is helpful in the investigation of underlying mechanisms of nitroglycerin tolerance and development of novel treatment in patients carrying the ALDH2 mutation. Previous studies have largely relied on human samples or immortalized cell lines. Human induced pluripotent stem cells (iPSCs) provide a powerful and versatile tool to screen drug targets and study disease-specific mechanisms. In the current study, we characterized the phenotypic features of ALDH2 genotype-specific iPSC-derived endothelial cells (iPSC-ECs). We also examined coronary artery response to nitroglycerin in a group of human subjects and analyzed the relationship between nitroglycerin response to ALDH2 genotype.

Materials and Methods

Human study

Study population

A total of 151 adult human subjects suspected of having coronary artery disease (CAD) but angiographically verified as not having CAD (<40% stenosis of the coronary arteries) were recruited. Exclusion criteria are presented as Supplemental Material 1, https://links.lww.com/CARDIOPLUS/A5. This study was approved by the ethics committee of Zhongshan Hospital, Fudan University (approval number: B2020-391R; date: January 14, 2021), and was conducted in compliance to the ethical guidelines of the Declaration of Helsinki (as revised in 2013). All participants provided written informed consent.

Genotyping

Genomic DNA was extracted from peripheral blood using a standard DNA isolation kit (GenTLE, Takara Bio, Otsu, Japan). ALDH2 genotypes at codon 487 were determined by PCR and restriction fragment length polymorphism analysis. The PCR primer sequences were 5′-TTGGTGGCTACAAGATGTCG-3′ (forward) and 5′-AAACACTGATGGCCT-CAAGC-3′ (reverse). The 324-bp PCR products were digested with TspRI (New England BioLabs, Ipswich, MA) at 65°C for 20 minutes. The products were separated on a 3% agarose gel and visualized with ethidium bromide. Genotype was determined by the visualization of 255-bp and/or 323-bp fragments: digestion of the wildtype ALDH gene (ALDH21) yielded three fragments (68, 255, and 1 bp) whereas the mutant ALDH gene (ALDH22) yielded only two fragments (323 and 1 bp).

Nitroglycerin challenge test

Participants were asked to refrain from drinking alcohol and smoking cigarettes the night before testing. Diagnostic coronary angiography was performed using the percutaneous brachial approach. A 6-Fr guiding catheter was inserted into the left main coronary artery. A 0.014-inch Doppler flow guidewire (FloWire, Cardio-metrics, Mountain View, CA) was advanced through the guiding catheter into the proximal segment of the left anterior descending (LAD) coronary artery. The wire tip was positioned in a straight segment without visible atherosclerotic lesions and minimal luminal irregularities. Coronary angiography was performed under prior to as well as immediately after intracoronary injection of 200-μg nitroglycerin. Arterial pressure, heart rate, and electrocardiography were continuously monitored and recorded using a multichannel recorder (Polygraph 1600, Nihon Electric, Tokyo, Japan).

Luminal diameter of the LAD coronary artery was measured 1 min after nitroglycerin administration by an experienced investigator. Flow measurements were taken at the same vessel region as the luminal diameter (2–3 mm distal to the wire tip), where no visible atherosclerotic lesions or minimal luminal irregularities were present. Luminal diameters were measured in the end-diastolic cine frame using a computer-assisted coronary angiographic analysis system (CAAS II/QUANTCOR, Siemens AG, Berlin and Munich, Germany). For each subject, luminal diameter was measured three times, and the average was used for further analysis. Vasodilatory response to 200-μg NTG intracoronary injection was calculated using the equation ([Dmax − D0]/D0 × 100%), where D0 is the baseline diameter and Dmax is the maximum diameter after nitroglycerin treatment[6].

Experiments in cultured cells

Preparation of iPSC-ECs

iPSCs (Stanford Stem Cell Bank) were differentiated as previously described[7]. Briefly, the cells were cultured in EGM-2 endothelial cell growth media (Lonza) supplemented with vascular endothelial growth factor (VEGF, 50 ng/mL, Peprotech), bone morphogenetic protein 4 (BMP4, 20 ng/mL, Peprotech), and fibroblast growth factor 2 (FGF2, 20 ng/mL, Peprotech). By day 12, TrypLE and 4% paraformaldehyde (PFA) were used to dissociate and fix the cells. Harvested cells were resuspended in phosphate buffered saline (PBS) and stained with a phycoerythrin (PE)-conjugated antibody against human CD144 (1:400, BD Biosciences) for 30 min on ice. iPSC-ECs were isolated using a FACSAria II cell sorter (BD Biosciences) and passaged upon 80–90% confluence. Experiments were conducted using cells at passage 2–5.

Oxidized low-density lipoprotein (oxLDL) uptake

iPSC-ECs were incubated with DiI-conjugated oxLDL (L34358, Invitrogen) overnight. After washing with PBS, cells were visualized under a fluorescence microscope.

Proliferation assay

Cell proliferation assay was conducted using Cell Counting Kit-8 from Dojindo Laboratories (Japan). Cells were seeded in Millicell EZ slide (millipore C86024) at 5 × 103 cells per well. The cells floating in the medium were collected, and the adherent cells were filled with 100 μL of 50 μM EdU medium (Reagent A, diluted with a cell complete medium at a ratio of 1000:1) to each well for 2 h. Stained cells were fixed with 4% paraformaldehyde (50 μL/well) and then neutralized with glycine (2 mg/mL). 0.5% Triton X-100 in PBS were added to enhance cell membrane permeability. Finally, cells were stained with apollo staining and DAPI. Stained cells were photographed using a fluorescent inverted microscope in five random high power fields. Finally, the proliferated cells were counted with Image J.

Migration assay

Cell migration was assessed using a wound healing assay[8]. Briefly, iPSC-ECs were seeded into 24-well plates and allowed to reach full confluence. The cell layer was scratched with a pipette tip; detached cells were washed away with PBS, and pictures of the initial wounds were taken. After 6 h of culture, pictures were taken again. The initial and final areas covered by the cells were measured using ImageJ software. The difference between these two areas was used to determine cell migration.

Tube formation assay

Tube formation assay was conducted according to a previously published protocol[9]. Briefly, iPSC-ECs were dissociated using TyrpLE, resuspended in EGM2 medium and seeded into 24-well plates (104 cells/well) precoated with 250-μL growth factor reduced Matrigel (BD Biosciences). After 6 h of incubation, the tube branches were imaged and quantified.

Reactive oxygen species (ROS) and cell death detection assays

ROS and cell death detection assays were performed using the ROS dihydroethidium (DHE) probe and LIVE/DEAD Viability/Cytotoxicity Kit (L3224, Invitrogen) after 6 h of treatment with H2O2, as previously described[10]. The samples were imaged and positive-staining signals were quantified and analyzed using ImageJ software.

NO detection assay

A colorimetric Griess reaction kit (Nanjing Jiancheng, China) was used to determine the total amount of nitrite converted from nitrate in supernatant. The absorbance was measured at 540 nm.

Statistical analysis

SPSS 13.0 (SPSS Science, Chicago, IL) for Windows was used for statistical analysis.

The Kolmogorov-Smirnov test was used to evaluate distribution normality of continuous variables. Continuous variables were presented as the mean (±SD), and analyzed using one-way analysis of variance (ANOVA). Categorical variables were presented as percentages, and analyzed using the χ2 test. Changes from the baseline were analyzed using Student's t-test for paired data. P < 0.05 was considered statistically significant.

Results

Experiments in cultured cells

Differentiation of iPSC-ECs

After differentiation, the WT and ALDH2+/− iPSC-ECs displayed similar endothelial cell morphologies as well as similar level of vascular endothelial (VE)-cadherin (CD144) expression (Figure 1A and B).

F1
Figure 1:
IPSC-EC differentiation and characterization.

Impaired endothelial functions of the ALDH2+/− iPSC-ECs

Lipid uptake capacity did not differ between WT and ALDH2+/− iPSC-ECs (Figure 2A and B). EdU and CCK-8 assays revealed decreased cell proliferation in the ALDH2+/− iPSC-ECs compared with the WT control (Figure 2A and B). The percentage of EdU-positive cells was 65.67% in the ALDH2+/− iPSC-ECs vs 86.67% in the WT control (P < 0.01). In the CCK-8 assay, the optical density (OD) was significantly lower in the ALDH2+/− iPSC-ECs (0.276 ± 0.05 vs 0.523 ± 0.02 in the WT control, P < 0.01). The ALDH2+/− iPSC-ECs had lower migration capability (750.0 ± 51.00 in ALDH2+/− vs 1,236.67 ± 247.0 in the WT control, P = 0.035; Figure 3A and B) and tube formation (Figure 3C). The iPSC-ECs also had higher ROS level upon H2O2 exposure (26.67 ± 5.51 vs 16.67 ± 4.04 in WT, P < 0.05) and higher level of cell death (red signal, 34 ± 6.56 vs. 13 ± 3.61 in WT) (Figure 4).

F2
Figure 2:
ALDH2 mutant inhibited cell proliferation but not LDL uptake.
F3
Figure 3:
ALDH2 mutant depressed migration and tube formation, Migration and tube formation of IPSC-ECs.
F4
Figure 4:
ALDH2 mutant enhanced ROS production and cell death ROS and cell death of IPSC-ECs.

Excessive baseline NO production but delayed nitroglycerin response in the ALDH2+/− iPSC-ECs

NO production in the absence of nitroglycerin was significantly higher in ALDH2+/− iPSC-ECs than the WT control (2.15-fold after 2 h, P < 0.01; 1.67-fold after 4 h, P < 0.05), However, the response to nitroglycerin was delayed in the ALDH2+/− iPSC-ECs vs the WT control (Figure 5). Exposure of WT iPSC-ECs to 10-μg/mL nitroglycerin for 2 h increased NO production by 2.83-fold. In contrast, 4-h exposure to nitroglycerin of the same concentration was required to induce significant NO release from the ALDH2+/− iPSC-ECs.

F5
Figure 5:
ALDH2 mutant regulated NO production.

Vasodilatory effects of nitroglycerin vs ALDH2 genotypes

The genotype frequency in the 151 human subjects was 74% (111/151) for ALDH2WT; 31% (32/151) for ALDH2+/−; and 5% (8/151) for ALDH2−/−. Subjects with the 3 genotypes were generally comparable in age, sex, smoking status, hypertension, and the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (Table 1).

Table 1 - Demographic and clinical characteristics of the study participants
Total (n = 151) +/+ (n = 111) +/− (n = 32) −/− (n = 8) P
Male (%) 92 (61) 68 (61) 19 (59) 5 (63) 0.95
Age (years) 61.8 ± 9.2 61.9 ± 9.2 60.8 ± 8.8 65.3 ± 11.2 0.47
Smoker, n (%) 24 (16) 17 (15) 5 (16) 2 (25) 0.77
Body Mass Index (kg/m2) 25.0 ± 3.3 25.0 ± 3.2 25.2 ± 3.4 24.2 ± 4.6 0.74
Hypertension, n (%) 90 (60) 70 (63) 15 (47) 5 (63) 0.24
Diabetes mellitus, n (%) 20 (13) 14 (13) 5 (16) 1 (13) 0.91
ARB, n (%) 29 (19) 22 (20) 5 (16) 2 (25) 0.75
ACE inhibitors, n (%) 8 (5) 6 (5) 1 (3) 1 (1) 0.49
Fasting glucose (mmol/L) 7.2 ± 4.7 7.6 ± 5.3 6.4 ± 2.5 5.7 ± 1.7 0.27
Triglyceride (mmol) 1.8 ± 1.2 1.8 ± 1.2 1.7 ± 0.9 1.8 ± 1.6 0.98
Total cholesterol (mmol) 4.0 ± 1.0 4.0 ± 1.1 4.0 ± 0.9 4.2 ± 0.7 0.87
LDL (mmol) 2.0 ± 0.9 2.0 ± 0.9 1.9 ± 0.7 2.2 ± 0.6 0.65
HDL (mmol/L) 1.3 ± 0.4 1.3 ± 0.4 1.3 ± 0.4 1.3 ± 0.5 0.97
LVEF (%) 65 ± 3.9 65 ± 3.8 65 ± 4.5 67 ± 3.0 0.43
ACE: angiotensin-converting enzyme; ARB: angiotensin receptor blocker; HDL: high-density lipoprotein; LDL: low-density lipoprotein; LVEF: left ventricular ejection fraction.

The three genotype groups did not differ significantly in the changes in pulse rate and blood pressure upon nitroglycerin treatment (Table 2). Prior to nitroglycerin challenge, the LAD coronary artery diameter in the ALDH2 mutant group (ALDH2MUT, including both ALDH2+/− and ALDH2−/− individuals) were larger than those in the WT group (ALDH2WT) under baseline conditions (3.6 vs 3.4 P = 0.0975). Vasodilatory response was significantly lower in the ALDH2MUT group vs the WT control (7.1 ± 0.6% vs 10.1 ± 0.8%, P = 0.024).

Table 2 - Hemodynamic change and coronary artery response to intracoronary injection of 200-μg nitroglycerin in the human subjects with 3 different ALDH2 genotypes
Before injection (n = 151) After injection (n = 151)


+/+ +/− −/− P +/+ +/− −/− P
Heart rate (beats/min) 75 ± 11 76 ± 12 81 ± 8 0.38 81 ± 12 82 ± 12 86 ± 15 0.50
Systolic aortic pressure (mm Hg) 125 ± 20 132 ± 17 118 ± 25 0.09 107 ± 21 112 ± 19 102 ± 24 0.41
Diastolic aortic pressure (mm Hg) 77 ± 11 81 ± 12 79 ± 11 0.15 72 ± 12 77 ± 16 75 ± 13 0.16
Diameter of LAD (mm) 3.4 ± 0.7 3.5 ± 0.8 3.8 ± 0.5 0.17 3.7 ± 0.7 3.8 ± 0.8 4.1 ± 0.5 0.35
LAD: left anterior descending.

Discussion

The results from the current study demonstrated impaired endothelial functions in the ALDH2+/− iPSC-ECs. Specifically, the ALDH2+/− iPSC-ECs exhibited an aberrant increase in endothelial NO production under baseline conditions and a substantially delayed response to nitroglycerin treatment. Consistently, the human subjects carrying the ALDH2 mutation tended to have larger LAD coronary artery diameter at the baseline, but smaller vasodilatory response upon nitroglycerin treatment.

Endothelial cells play a vital role in the regulation of vascular homeostasis and function[11–13] and therefore are one of the primary targets for treating coronary artery spasm[14]. Primary human endothelial cells are difficult to expand in vitro. The current study demonstrated the feasibility of using endothelial cells differentiated from WT and ALDH2+/− iPSCs as a tool to examine the impact of ALDH2 mutation on endothelial functions. NO produced by endothelial cells is a critical component of the mechanisms that regulate the contraction of smooth muscles cells in coronary artery and therefore the coronary blood flow[15]. The current study demonstrated higher NO production by ALDH2+/− iPSC-ECs under baseline conditions but delayed response to nitroglycerin. Specifically, 2-h exposure to 10-μg/mL nitroglycerin increased NO production by 3 folds in WT iPSC-ECs, but failed to increase NO production in ALDH2+/− iPSC-ECs. Prolonged exposure to nitroglycerin (4 h) was needed to increase NO production in ALDH2+/− iPSC-ECs. These in vitro findings were verified by the study in human subjects. Specifically, the human subjects carrying the ALDH2 mutation tended to have larger LAD coronary artery diameter at the baseline, but smaller vasodilatory response upon nitroglycerin treatment.

The efficacy of nitroglycerin is impaired in patients carrying ALDH2 mutation. Nonetheless, it remains an important drug to treat coronary artery spasms. A previous study showed that continuous use of nitroglycerin can worsen vasospasm, particularly in individuals carrying an ALDH2 mutation[16]. ALDH2 mutation has been previously associated with the incidence of coronary artery spasm[17]. The findings from the current study are particularly relevant to patients with East Asian heritage since approximately 40% of this population carry an ALDH2 mutation[18]. In a recent study by Gu et al.[19], overexpressing ALDH2 via lentiviral transduction enhanced the migration and angiogenic capacity of ALDH2+−- iPSC-ECs, providing a novel strategy to alleviate ALDH2 mutant-induced endothelial cell impairment. We further speculate that using an ALDH2 activator (eg, lipoic acid) could improve the coronary vasodilation response to nitroglycerin in patients carrying an ALDH2 mutation. Future studies are needed to test such a hypothesis.

In summary, ALDH2+/− iPSC-ECs had higher level of NO production in basal conditions but poor response to nitroglycerin. Similarly, coronary artery in human subjects carrying ALDH2 mutation had impaired vasodilatory response to nitroglycerin. These findings highlight the importance of individualized approach in the management of ischemic heart disease in general, and more specifically coronary artery spasm and nitroglycerin intolerance.

Funding

This study was supported by grants to Aijun Sun from the National Science Fund for Distinguished Young Scholars (81725002).

Author contributions

AS conceived and designed the study. HZ and JH wrote the manuscript. HZ, JH, ZD, YL, and XS performed most of the experiments and analyzed the data. AS guided and revised the manuscript.

Conflict of interest statement

Aijun Sun is an Editorial Board member of Cardiology Plus. The article was subject to the journal's standard procedures, with peer review handled independently of this Editorial Board member and their research groups.

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Keywords:

Aldehyde dehydrogenase 2; Coronary artery disease; iPSC-ECs; Nitric oxide; Nitroglycerin

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