Abstract
Aims: Oxidative stress is a central factor in the pathogenesis of atherosclerosis and potentially exhibits sexual dimorphism. The induction of heme oxygenase-1 (HO-1) serves as a crucial mechanism against reactive oxygen species toxicity in the vascular wall, and this induction is regulated by the promoter (GT)n repeat length. We aim to investigate whether or notHO-1 gene (GT)n polymorphism is associated with the occurrence of large-artery atherosclerotic (LAA) stroke.
Methods: We consecutively recruited stroke patients, with a control group comprising age- and sex-matched non-stroke individuals.HO-1 (GT)n genotypes were determined using DNA extracted from the peripheral leukocytes.HO-1 (GT)n polymorphism was classified as short [S, ≤ 24 (GT)n], medium [M, 25 ≤ (GT)n ＜31], or long [L, 31 ≤ (GT)n]. Clinical data were collected, and stroke patients were categorized into LAA and non-LAA groups according to the TOAST classification. A multivariable logistic regression analysis was conducted to evaluate the association betweenHO-1 (GT)n variants and LAA occurrence stratified by sex.
Results: There was no significant difference in the distribution ofHO-1 (GT)n genotypes between the stroke and non-stroke populations. However, the proportion of S/S genotype was significantly lower in the LAA stroke patients than in the non-LAA stroke patients (7.08% vs. 21.78%,p＜0.001). A multivariable logistic regression analysis indicated that non-SS genotypes were associated with a significantly increased risk of LAA compared to the S/S genotype patients (odds ratio [OR] 3.35, 95% confidence interval [CI] 1.98-5.67,p＜0.001). After stratification by sex, the protective effect of theHO-1 (GT)n S/S genotype was highly significant in men (OR 5.50, 95% CI 2.67-11.34,p＜0.001), whereas the association was not significant in women (OR 1.60, 95% CI 0.75-3.34,p = 0.228).
Conclusion: Short (GT)n variants inHO-1 may confer significant protection against LAA stroke in men but not in women.
Keywords: HO-1 gene, Larger-artery atherosclerosis, Stroke, Polymorphism, Oxidative stress, Sexual dimorphism
Jintao Li and Junting Chen contributed equally to this work.
Shuen Li and Zhu Shi contributed equally to this work.
Background
Stroke is the second leading cause of death and disability worldwide, with approximately 12 million deaths attributed to this condition by 2020 1) . Large-artery atherosclerosis (LAA) is one of the most frequent causes of stroke and is closely associated with a high risk of recurrent stroke 2) . Established major risk factors for LAA include hypertension, diabetes mellitus, dyslipidemia, and smoking. However, clinical data suggest that stringent control of these risk factors cannot entirely prevent the occurrence and progression of LAA 3 - 5) . The generation of oxidized-low-density lipoprotein (LDL) and reactive oxygen species (ROS) in the vascular system upon exposure to risk factors play a critical role in driving atherosclerosis 6 , 7) . A comprehensive understanding of the inflammatory responses and oxidative stress in the vascular system following exposure to risk factors, along with the identification of therapeutic targets, is essential for alleviating the burden of stroke.
Heme oxygenase 1 (HO-1) is a rate-limiting enzyme that catalyzes the breakdown of heme into carbon monoxide (CO), free iron, and biliverdin and serves as a crucial antioxidant that enhances host defense against oxidative stress. HO-1 mitigates cholesterol-induced oxidative damage in vascular endothelial cells and exerts protective effects against atherosclerosis 8 , 9) and renal and cerebral ischemic injuries 10 - 12) . The (GT)n repeat sequence in the promoter region of HO-1 exhibits high polymorphism and regulates the transcriptional activity of HO-1. Genetic epidemiological evidence suggested that longer HO-1 (GT)n repeat sequences were positively associated with an increased risk of coronary artery disease in patients with vascular risk factors 13 , 14) . However, the role of HO-1 in the development and progression of cerebral atherosclerosis remains unclear, and evidence linking HO-1 (GT)n polymorphism to the risk of LAA stroke is currently insufficient.
The incidence of cardiovascular diseases varies by sex and throughout the lifespan 15 - 17) . The increased incidence of cardiovascular disease in postmenopausal women cannot be solely attributed to menopause-related changes effects on usual cardiovascular risk factors, differences in oxidative stress pathways may also contribute to sexual dimorphism 18 - 20) . In the present study, we first examined the association between HO-1 (GT)n polymorphism and LAA stroke and then stratified the analysis by sex to evaluate the potential differences between men and women.
Population and Method
Study Population
This cross-sectional study was approved by the Institutional Review Board of Dongguan People’s Hospital (KYKT2021-013), and written informed consent was obtained from all participants. We consecutively enrolled patients admitted to the Stroke Center of the Tenth Affiliated Hospital, Southern Medical University, from January 2021 to February 2024. In addition, 600 controls matched for age and sex with no ischemic stroke history were drawn from the cohort of our earlier research 21) .
The inclusion criteria were: (1) age ≥ 18 years old; (2) ischemic stroke confirmed by brain magnetic resonance imaging (MRI) or computed tomography (CT); (3) no metal implants, claustrophobia, or other contraindications for MRI; and (4) informed consent obtained from the patient or their representative. We classified the study population according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST): LAA, small-vessel disease (SVD), and cardioembolic (CE), along with additional stroke types grouped as “Other causes,” which represented specific underlying mechanisms.
The exclusion criteria were as follows: (1) severe systemic diseases, including liver and kidney dysfunction, hematological disorders, malignancies, and radiation therapy; (2) missing clinical data including previous history, laboratory tests, and imaging information; (3) poor quality of MRI, rendering it unanalyzable; and (4) unavailability of gene testing results.
The inclusion criteria for control subjects were as follows: (1) Chinese Han ethnicity; (2) age ≥ 18 years old; (3) no history of stroke; and (4) no serious systemic diseases, such as endocrine disorders, autoimmune disease, hematologic disease, cancer, or chronic renal or hepatic disease.
Definition of Covariates
Information regarding medications and chronic diseases, such as hypertension and diabetes mellitus (DM), was obtained from medical records. Hypertension was defined as an average of 2 systolic blood pressure measurements ≥ 140 mmHg, an average of 2 diastolic blood pressure measurements ≥ 90 mmHg, or current use of antihypertensive medications. DM was defined as a fasting blood glucose level ≥ 6.0 mmol/L, hemoglobin A1c level ≥ 6.5%, or current use of antidiabetic medications. Dyslipidemia was defined as a total cholesterol level exceeding 5.2 mmol/L and/or a low-density lipoprotein (LDL) cholesterol level exceeding 4.1 mmol/L, or current use of lipid-lowering drugs. Smoking refers to individuals who currently smoke or who have smoked previously.
Lab Tests
Fasting serum samples were collected and biochemical assays were performed using an automated analyzer (Toshiba TBA-120 FR, Toshiba Medical Systems, Tokyo, Japan) to measure total bilirubin (T-BIL), direct bilirubin (D-BIL), indirect bilirubin (I-BIL), total cholesterol (TC), triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Hemoglobin A1c levels were measured using an automated glycohemoglobin analyzer (HA 8180, ARKRAY America, Inc., Minneapolis, U.S.A.).
Genetic Testing
Genomic DNA was isolated from the peripheral blood using a DNA extraction kit (TIANGEN BIOTECH Inc, Beijing, China). Specific primers were designed based on conserved sequences of the variable number tandem repeat (VNTR) in the promoter region of HO-1 (HO-1-F: 5’-GAA GGT GAC CAA GTT CAT GCT AGA GCC TGG GGT TGC TAA GT; HO-1-R: 5’-ACA GCT GAT GCC CAC TTT CT). Polymerase chain reaction (PCR) products were labeled with a 6-carboxyfluorescein forward primer (5’-GAA GGT GAC CAA GTT CAT GCT), and the fragment sizes were determined using a capillary sequencer (ABI 3730xl DNA Analyzer). To ensure accuracy and reproducibility, all samples were subjected to at least two independent PCR amplifications, and the obtained results were verified to be accurate within a ±1 GT repeat range. A genotyping analysis was performed by laboratory personnel who were blinded to the clinical and imaging data. Based on a bimodal distribution of (GT)n repeats and our previous reports 21) , we designated GT repeats ≤ 24 as short (S) alleles, those between 25 and 30 as medium (M) alleles, and repeats ≥ 31 as long (L) alleles ( Supplementary Fig.1 ) .
Supplementary Fig.1. Representative images of genetic analysis for short (S), medium (M), or long (L) classifications.
We applied the method of counting (GT)n repeats as follows:
n = (Length peak map – length connection sequence – length Flanking/conserved sequences)/2
A: Both haploid (GT) n repeats were 23 [n = (406−21−340)/2 = 23]; this figure represents the SS genotype.
B: The two haploid numbers were 23[n = (406−21−340)/2 = 23] and 30[n = (421−21−340)/2 = 30]; this figure represents the SM genotype.
C: The two haploid numbers were 30[n= (421−21−340)/2=23] and 37[n= (434−21−340)/2=30]; this figure represents the ML genotype.
Statistical Analyses
Continuous variables are presented as the mean±standard deviation (SD) if normally distributed or median (interquartile range) otherwise. Categorical variables were presented as frequencies and percentages. Chi-square tests were conducted to compare differences in clinical data and genotype frequencies between the stroke population and the control group, as well as between the LAA stroke and non-LAA stroke groups. A binary logistic regression model was applied to calculate the odds ratio (OR) and 95% confidence interval (CI) for the relationship between HO-1 genotype and LAA stroke. Such associations were further assessed using a binary logistic regression model after stratification for sex.
Statistical analyses were performed using the SPSS software program (IBM SPSS 29.0 Statistics, Chicago, IL, USA) and OriginPro 2024 (OriginLab, Northampton, MA, USA). Two-sided P-values were calculated, with P＜0.05 as statistically significant.
Results
Population Characteristics
A total of 616 consecutive patients with stroke were enrolled during the study period. Among them, 15 had comorbid serious liver and renal dysfunction, 5 did not yield successful gene sequencing results because of unsuccessful assays, and 18 lacked clinical data for an accurate TOAST analysis. In total, 578 eligible patients were included in the final stroke group. Six cases in the control group were excluded because of unsuccessful assays, leading to a final inclusion of 594 subjects. In total, 1,172 eligible patients were included in this study. The median age of the study population was 68 years old, and 727 (56.31%) were men. Among all the participants, 784 (66.89%) had a history of hypertension, 397 (33.87%) had diabetes, 404 (34.47%) had hyperlipidemia, and 323 (27.56%) were current smokers. Based on the HO-1 (GT)n repeat number (S ≤ 24, M = 25–30, L ≥ 31), the genotypes were categorized into six subtypes: S/S (n = 163, 13.91%), S/M (n = 377, 32.17%), S/L (n = 168, 14.33%), M/M (n = 224, 19.11%), M/L (n = 185, 15.78%), and L/L (n = 55, 4.69%), as previously reported 21) . Subjects in the stroke group had a higher prevalence of hypertension, diabetes mellitus, hyperlipidemia, and smoking history than those in the non-stroke group. Serum levels of LDL, HCY, Cr, and UA were significantly higher in stroke patients than in non-stroke subjects. However, there were no significant differences in TBIL, DBIL, and IDIL levels, as well as the distribution of HO-1 (GT)n genotypes between the two groups ( Table 1 and Fig.1 ) .
Table 1. Clinical characteristics and HO-1 (GT)n genotypes of study population .
| Total (n = 1172) | Stroke (578) | Non-stroke (594) | p value | |
|---|---|---|---|---|
| Age, years (median, IQR) | 68.00 (58.00, 75.00) | 69.00 (59.00, 75.00) | 68.50 (61.00, 75.00) | 0.859 |
| Men, n (%) | 727 (62.03) | 370 (64.01) | 357 (60.10) | 0.168 |
| Hypertension, n (%) | 784 (66.89) | 453 (78.37) | 331 (55.72) | ＜.001 |
| Diabetes Mellitus, n (%) | 397 (33.87) | 255 (44.12) | 142 (23.91) | ＜.001 |
| Hyperlipidemia, n (%) | 404 (34.47) | 228 (39.45) | 176 (29.63) | 0.002 |
| Smoking, n (%) | 323 (27.56) | 206 (35.64) | 117 (19.70) | ＜.001 |
| Lab tests | ||||
| TC, mmol/L | 4.50 (3.80, 5.30) | 4.54 (3.87, 5.47) | 4.44 (3.76, 5.20) | 0.141 |
| TG, mmol/L | 1.25 (0.89, 1.82) | 1.28 (0.90, 1.90) | 1.24 (0.89, 1.78) | 0.132 |
| HDL, mmol/L | 1.14 (0.97, 1.34) | 1.08 (0.92, 1.27) | 1.21 (1.01, 1.43) | ＜.001 |
| LDL, mmol/L | 2.97 (2.30, 3.74) | 3.15 (2.46, 3.95) | 2.86 (2.19, 3.48) | ＜.001 |
| Hcy, μmol/L | 11.02 (8.91, 13.61) | 11.52 (9.37, 14.16) | 10.50 (8.48, 12.90) | ＜.001 |
| TBIL, μmol/L | 12.60 (9.70, 16.50) | 12.80 (9.90, 16.90) | 12.40 (9.70, 16.10) | 0.148 |
| DBIL, μmol/L | 4.00 (2.67, 5.40) | 4.00 (2.80, 5.50) | 3.90 (2.50, 5.30) | 0.092 |
| IBIL, μmol/L | 8.90 (6.60, 11.60) | 9.00 (6.80, 11.70) | 8.65 (6.50, 11.28) | 0.273 |
| Cr, umol/L | 74.70 (61.60, 92.32) | 76.70 (63.62, 94.20) | 73.40 (60.30, 89.88) | 0.002 |
| UA, μmol/L | 363.90 (294.90, 436.85) | 367.45 (301.60, 446.45) | 358.40 (285.52, 425.70) | 0.029 |
| HO-1 (GT)n Genotype, n (%) | 0.789 | |||
| SS | 163 (13.91) | 74 (12.80) | 89 (14.98) | |
| SM | 377 (32.17) | 195 (33.74) | 182 (30.64) | |
| SL | 168 (14.33) | 82 (14.19) | 86 (14.48) | |
| MM | 224 (19.11) | 106 (18.34) | 118 (19.87) | |
| ML | 185 (15.78) | 94 (16.26) | 91 (15.32) | |
| LL | 55 (4.69) | 27 (4.67) | 28 (4.71) | |
Fig.1.
Marginal plot showed the distribution and frequency of HO-1 (GT)n repeat counts in stroke population and control
Association between HO-1(GT)n Genotypes and LAA Stroke
Of the 578 patients with stroke, 353 (61.1%) were classified as having LAA and 225 (38.9%) as non-LAA. Compared to non-LLA patients, LAA patients were more likely to be men (70.25% vs. 54.22%, p＜0.001) and had higher rates of diabetes (49.86% vs. 35.11%, p＜0.001) and hyperlipidemia (43.63% vs. 32.89%, p = 0.010), along with significantly elevated serum TC (4.79 [4.00, 5.65] vs. 4.24 [3.62, 4.86] mmol/L, p＜0.001) and LDL (3.40 [2.63, 4.24] vs. 2.85 [2.20, 3.47] mmol/L, p＜0.001). The distribution of HO-1 (GT) genotypes among LAA patients differed significantly from that of non-LAA patients, with a significantly lower frequency of the SS genotype observed in the LAA group (7.08% vs. 21.78%, p＜0.001) ( Table 2 ) .
Table 2. Clinical characteristics and HO-1 (GT)n genotypes in terms of LAA vs. non-LAA stroke .
| LAA (n = 353) | Non-LAA (n = 225) | p value | |
|---|---|---|---|
| Age, year | 69.00 (60.00, 74.00) | 69.00 (58.00, 76.00) | 0.778 |
| Men, n (%) | 248 (70.25) | 122 (54.22) | ＜.001 |
| Hypertension, n (%) | 280 (79.32) | 173 (76.89) | 0.489 |
| Diabetes, n (%) | 176 (49.86) | 79 (35.11) | ＜.001 |
| Hyperlipidemia, n (%) | 154 (43.63) | 74 (32.89) | 0.010 |
| Smoking, n (%) | 131 (37.11) | 75 (33.33) | 0.355 |
| Lab tests | |||
| TC, mmol/L | 4.79 (4.00, 5.65) | 4.24 (3.62, 4.86) | ＜.001 |
| TG, mmol/L | 1.27 (0.90, 1.89) | 1.29 (0.90, 1.97) | 0.905 |
| HDL, mmol/L | 1.08 (0.92, 1.26) | 1.09 (0.93, 1.29) | 0.670 |
| LDL, mmol/L | 3.40 (2.63, 4.24) | 2.85 (2.20, 3.47) | ＜.001 |
| Hcy, μmol/L | 11.75 (9.41, 14.42) | 11.13 (9.37, 13.84) | 0.112 |
| TBIL, μmol/L | 12.90 (9.70, 16.50) | 12.80 (10.30, 17.40) | 0.451 |
| DBIL, μmol/L | 4.00 (2.80, 5.60) | 4.00 (2.60, 5.40) | 0.615 |
| IBIL, μmol/L | 9.00 (6.50, 11.70) | 9.10 (6.90, 11.90) | 0.338 |
| Cr, umol/L | 77.60 (65.10, 97.10) | 74.90 (62.00, 91.20) | 0.168 |
| UA, μmol/L | 373.00 (302.50, 456.70) | 364.50 (300.50, 433.60) | 0.192 |
| HO-1 (GT)n Genotype, n (%) | ＜.001 | ||
| SS | 25 (7.08) | 49 (21.78) | |
| SM | 119 (33.71) | 76 (33.78) | |
| SL | 61 (17.28) | 21 (9.33) | |
| MM | 65 (18.41) | 41 (18.22) | |
| ML | 68 (19.26) | 26 (11.56) | |
| LL | 15 (4.25) | 12 (5.33) | |
| non-SS vs SS | 328 (92.92) | 176 (78.22) | ＜.001 |
Multivariable logistic regression indicated that risk factors for LAA included male sex (OR 1.81, 95% CI 1.26-2.60, p = 0.001), diabetes mellitus (OR 1.80, 95% CI 1.25-2.59; p = 0.002), hyperlipidemia (OR 1.50, 95% CI, 1.03-2.16; p = 0.032), and non-SS genotypes (OR 3.35, 95% CI 1.98-5.67, p＜0.001) ( Table 3 ) . To clarify the interaction between sex and HO-1 (GT) genotypes, we compared the genotype distribution according to sex stratification in patients with LAA and non-LAA. In the LAA population, a significant difference was observed in the ratio of SS to non-SS genotypes between men and women. In contrast, no significant differences in genotype distribution were detected between men and women in non-LAA patients ( Supplementary Table 1 , 2 ) .
Table 3. Logistic regression for risk factors of LAA stroke.
| Univariate | Multivariate | |||
|---|---|---|---|---|
| OR (95% CI) | p value | OR (95% CI) | p value | |
| Age | 1.01(0.99-1.03) | 0.869 | ||
| Men | 2.11 (1.49-2.99) | ＜.001 | 1.81 (1.26-2.60) | 0.001 |
| Diabetes Mellitus | 1.84 (1.30-2.59) | ＜.001 | 1.80 (1.25-2.59) | 0.002 |
| Hyperlipidemia | 1.58 (1.11-2.24) | 0.010 | 1.50 (1.03-2.16) | 0.032 |
| HO-1 (GT)n Genotype | ||||
| SS | Ref | Ref | ||
| SM | 3.07 (1.75-5.38) | ＜.001 | 2.96 (1.66-5.27) | ＜.001 |
| SL | 5.69 (2.85-11.37) | ＜.001 | 5.59 (2.75-11.37) | ＜.001 |
| MM | 3.11 (1.67-5.78) | ＜.001 | 2.97 (1.57-5.64) | 0.001 |
| ML | 5.13 (2.65-9.92) | ＜.001 | 4.51 (2.30-8.87) | ＜.001 |
| LL | 2.45 (1.00-6.02) | 0.051 | 2.78 (1.11-6.99) | 0.041 |
| non-SS vs SS | 2.45 (1.00-6.02) | ＜.001 | 3.35 (1.98~5.67) | ＜.001 |
Supplementary Table 1. The distribution of HO-1(GT)n genotypes according to sex stratification in LAA stroke patients .
| SS (n = 25) | SM (n = 119) | SL (n = 61) | MM (n = 65) | ML (n = 68) | LL (n = 15) | p value | non-SS (n = 328) | ＊p value | |
|---|---|---|---|---|---|---|---|---|---|
| Gender | 0.164 | 0.012 | |||||||
| Male | 13 (3.7) | 37 (10.5) | 16 (4.5) | 18 (5.1) | 16 (4.5) | 5 (1.4) | 92 (26.1) | ||
| Female | 12 (3.4) | 82 (23.2) | 45 (12.7) | 47 (13.3) | 52 (14.7) | 10 (2.8) | 236 (66.9) |
p indicated ANOVA between HO-1 (GT)n genotypes; ＊p indicated chi-square analysis between SS genotype and non-SS genotype.
Supplementary Table 2. The distribution of HO-1(GT)n genotypes according to sex stratification in non-LAA stroke patients .
| SS (n = 49) | SM (n = 76) | SL (n = 21) | MM (n = 41) | ML (n = 26) | LL (n = 12) | p value | non-SS (n = 176) | ＊p value | |
|---|---|---|---|---|---|---|---|---|---|
| Gender | 0.577 | 0.331 | |||||||
| Male | 19 (8.4) | 33 (14.7) | 10 (4.4) | 22 (9.8) | 13 (5.8) | 4 (1.8) | 82 (36.4) | ||
| Female | 30 (13.3) | 43 (19.1) | 11 (4.9) | 19 (8.4) | 13 (5.8) | 8 (3.6) | 94 (41.8) |
p indicated ANOVA between HO-1 (GT)n genotypes; ＊p indicated comparison between SS genotype and non-SS genotype.
Sexual Dimorphism on the Association between HO-1(GT)n Genotypes and LAA Stroke
After stratification by sex, the univariate analysis indicated that the risk factors for LAA stroke differed between men and women. Among male stroke patients with LAA, the incidences of diabetes mellitus, hyperlipidemia, serum triglycerides, and LDL levels were significantly higher than those in non-LAA patients. However, in female patients, triglyceride levels were marginally higher than those in non-LAA patients, although no other significant differences were detected. Notably, in male stroke patients, the distribution of six HO-1 (GT)n genotypes revealed significant differences between LAA and non-LAA stroke, with LAA stroke patients exhibiting a significantly higher frequency of non-SS genotypes (95.16% vs. 75.41%, p＜0.001). However, in female patients, no significant differences were found in genotype distribution, although the frequency of non-SS genotypes was slightly elevated among LAA patients (87.62% vs. 81.55%, p = 0.225) ( Table 4 ) .
Table 4. Clinical characteristics and HO-1 (GT)n genotypes for LAA vs non-LAA stroke patients stratified by sex .
| Men | Women | |||||
|---|---|---|---|---|---|---|
| LAA (n = 248) | Non-LAA (n = 122) | p value | LAA (n = 105) | Non-LAA (n = 103) | p value | |
| Age, year | 68.00 (59.00, 74.00) | 67.00 (56.00, 76.00) | 0.477 | 71.00 (65.00, 76.00) | 70.00 (62.50, 77.00) | 0.658 |
| Hypertension | 195 (78.63) | 92 (75.41) | 0.485 | 85(80.95) | 81 (78.64) | 0.678 |
| Diabetes | 133 (53.63) | 45 (36.89) | 0.002 | 43 (40.95) | 34 (33.01) | 0.236 |
| Hyperlipidemia | 116 (46.77) | 35 (28.69) | ＜.001 | 38 (36.19) | 39 (37.86) | 0.803 |
| Smoking | 192 (51.89) | 68 (55.74) | 0.229 | 7 (6.67) | 7 (6.80) | 0.970 |
| TC, mmol/L | 4.79 (3.98, 5.64) | 4.06 (3.50, 4.69) | ＜.001 | 4.70 (4.00.5.70) | 4.40 (3.92, 5.23) | 0.100 |
| TG, mmol/L | 1.31 (0.92, 1.91) | 1.20 (0.86, 1.79) | 0.207 | 1.18 (0.89, 1.80) | 1.35 (0.98, 2.01) | 0.193 |
| HDL, mmol/L | 1.04 (0.89, 1.20) | 1.04 (0.90, 1.24) | 0.494 | 1.19 (1.01.1.36) | 1.12 (0.95, 1.33) | 0.118 |
| LDL, mmol/L | 3.42 (2.60, 4.27) | 2.69 (2.03, 3.35) | ＜.001 | 3.38 (2.65, 4.08) | 3.01 (2.50.3.63) | 0.114 |
| TBIL, μmol/L | 13.55 (9.90, 17.07) | 12.90 (10.30, 17.48) | 0.815 | 11.80(9.10,15.40) | 12.80 (10.40, 17.10) | 0.053 |
| DBIL, μmol/L | 4.10 (3.17, 6.00) | 4.00 (2.90, 5.70) | 0.568 | 3.40(2.60,4.60) | 3.70 (2.50, 5.05) | 0.352 |
| IBIL, μmol/L | 9.20 (6.60, 11.72) | 8.60 (6.90, 11.57) | 0.804 | 8.60 (6.40, 11.10) | 9.70 (7.30, 12.35) | 0.049 |
| HO-1 (GT)n Genotype, n (%) | ＜.001 | 0.333 | ||||
| SS | 12 (4.84) | 30 (24.59) | 13 (12.38) | 19 (18.45) | ||
| SM | 82 (33.06) | 43 (35.25) | 37 (35.24) | 33 (32.04) | ||
| SL | 45 (18.15) | 10 (8.20) | 16 (15.24) | 11 (10.68) | ||
| MM | 47 (18.95) | 22 (18.03) | 18 (17.14) | 19 (18.45) | ||
| ML | 52 (20.97) | 13 (10.66) | 16 (15.24) | 13 (12.62) | ||
| LL | 10 (4.03) | 4 (3.28) | 5 (4.76) | 8 (7.77) | ||
| non-SS | 236 (95.16) | 92 (75.41) | ＜.001 | 92 (87.62) | 84 (81.55) | 0.225 |
In logistic regression analyses conducted for male and female stroke populations, the odds ratio (OR) for LAA stroke in men with HO-1 (GT)n non-S/S genotypes was 5.50 (95% CI, 2.67-11.34; P＜0.001) compared to those with S/S genotypes; in contrast, no significant relationship was found between S/S genotype and LAA stroke in female patients (OR 1.60, 95% CI, 0.74-3.44; P = 0.228) ( Table 5 , Fig.2 ) . Considering the distinct risks associated with the S/S genotype exposure, we conducted further interaction analyses. The interaction regression model revealed a significant interaction between sex and S/S genotype for LAA stroke (P interaction = 0.020), whereas no other significant interaction effects were observed (see Fig.3 ).
Table 5. Logistic regression for risk factors of LAA stroke based on sex stratification.
| Men | Women | |||||||
|---|---|---|---|---|---|---|---|---|
| Univariate | multivariate | Univariate | multivariate | |||||
| OR (95% CI) | P value | OR (95% CI) | P value | OR (95% CI) | P value | OR (95% CI) | P value | |
| Age | 1.01 (0.99-1.03) | 0.227 | 1.02 (0.99-1.04) | 0.193 | ||||
| Diabetes | 1.94 (1.27-3.09) | 0.003 | 2.06 (1.27-3.24) | 0.004 | 1.44 (0.82-2.48) | 0.236 | ||
| Hyperlipidemia | 2.18 (1.37-3.48) | ＜.001 | 2.22 (1.34-3.67) | 0.002 | 0.93 (0.53-1.63) | 0.803 | ||
| HO-1 (GT)n Genotype | ||||||||
| SS | Ref | Ref | Ref | |||||
| SM | 4.77 (2.22-10.24) | ＜.001 | 3.95 (1.81-8.64) | 0.004 | 1.64 (0.70-3.82) | 0.253 | ||
| SL | 11.25 (4.32-29.32) | ＜.001 | 10.17 (3.83-27.04) | ＜.001 | 2.13 (0.75-6.03) | 0.156 | ||
| MM | 5.34 (2.31-12.36) | ＜.001 | 4.18 (1.76-9.92) | 0.007 | 1.38 (0.53-3.60) | 0.505 | ||
| ML | 10.00 (4.05-24.70) | ＜.001 | 8.86 (3.53-22.25) | ＜.001 | 1.80 (0.65-4.97) | 0.258 | ||
| LL | 6.25 (1.64-23.84) | 0.007 | 6.86 (1.74-26.98) | 0.018 | 0.91 (0.24-3.42) | 0.893 | ||
| non-SS vs SS | ||||||||
| SS | Ref | Ref | Ref | |||||
| Non-SS | 6.41 (3.15-13.06) | ＜.001 | 5.50 (2.67-11.34) | ＜.001 | 1.60 (0.74-3.44) | 0.228 | ||
OR: odds ratio, CI: confidence interval
Fig.2. The odds ratios for large-artery atherosclerotic (LAA) stroke based on HO-1 (GT)n genotypes and sex.
The P values in parentheses reflected comparisons of each subgroup against the reference group (men with the HO-1(GT)n SS genotype).
The P values adjacent to the curve arrow showed the risk of LAA stroke in male patients with HO-1 (GT)n non-SS genotypes versus those with HO-1 (GT)n SS genotypes.
Fig.3.
Forest plot of the odds ratios (ORs) of predictors for LAA stroke, stratified by sex, diabetes mellitus and hyperlipidemia
Discussion
Our findings suggest that (1) The HO-1 (GT)n polymorphism was not associated with overall ischemic stroke but was independently linked to the occurrence of LAA stroke, a significant subtype. Specifically, the short (GT)n repeat (SS) protected against LAA stroke compared to the non-SS genotypes. (2) Furthermore, the relationship between HO-1 (GT)n genotypes and LAA showed sex-based patterns. In men, the SS homozygote was significantly associated with a lower risk of LAA stroke, whereas in women, this association was not significant.
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme metabolism, catalyzing the oxidative breakdown of heme to produce equimolar amounts of carbon monoxide (CO), Fe2+, and biliverdin, which are subsequently converted to bilirubin by cytosolic biliverdin reductase (BVR). These metabolites exert multiple beneficial effects on endothelial cells, including anti-inflammatory, antioxidant, anti-apoptotic, and anti-proliferative properties 22 , 23) . Ishikawa investigated the progression of atherosclerotic lesions in LDL receptor-knockout mice fed a high-fat diet. They induced HO-1 expression through heme injection and found that HO-1 serves as a protective factor against the formation of atherosclerotic lesions 24) . Fiorelli et al. enrolled 30 patients with coronary artery disease and used optical coherence tomography (OCT) to examine the characteristics of coronary plaques, along with their oxidative stress status. They measured HO-1 levels in monocyte-derived macrophages and identified a positive correlation between low HO-1 levels and the vulnerable characteristics of plaques observed on OCT 25) . A pathological study of carotid plaques found that the expression of HO-1 in carotid atherosclerotic plaques could stabilize carotid plaques by inhibiting oxidative stress induced by Helicobacter pylori infection 26) .
The regulation of human HO-1 gene expression involves several mechanisms, notably the upstream promoter (GT)n repeat sequence, whose length is negatively correlated with HO-1 transcriptional activity 27 , 28) . The HO-1 (GT)n short sequence exerted significant protective effects regarding the systolic dysfunction, occurrence and mortality in patients with coronary heart disease 6 , 14 , 29 , 30) . Consistently, Bai et al. investigated HO-1 (GT)n repeat length in 183 patients with ischemic stroke and found that patients with longer HO-1 (GT)n repeats exhibited a heightened risk of stroke in the context of dyslipidemia 31) . However, stroke is a heterogeneous cerebrovascular disease with significant variability in etiology and mechanism, among which LAA stroke is a major subtype associated with atherosclerotic precession, high rates of recurrence, and a poor prognosis. Therefore, we further delineated the etiology of stroke and focused on the characteristics of HO-1 (GT)n in patients with LAA stroke. Consistent with previous studies on coronary artery disease, we observed that individuals with the HO-1 (GT)n SS homozygous genotype exhibited a lower risk of LAA than those with non-SS genotypes. Pechlaner et al. found that subjects with long HO-1 (GT)n tandem repeats exhibited an increased risk for both cardiovascular disease and accelerated carotid atherosclerotic progression 29) . As carotid atherosclerosis is a major cause of LAA stroke, this finding is consistent with our results.
Another interesting finding of our study was that the association of HO-1 (GT)n polymorphism with LAA stroke (LAA) exhibited sex-based differences. In men, the SS homozygous genotype, which tended to exhibit higher HO-1 transcriptional activity, provided a significant protective benefit against LAA stroke, whereas this relationship was not significant in women. The relationship between sex dimorphism and stroke risk has long been an important topic. Large-scale epidemiological studies indicate that postmenopausal women display marked acceleration in the clinical and serological characteristics of atherosclerosis with an accelerated risk for cardiovascular disease, which tends to be higher than that in age-matched men 32 - 34) . Several hypotheses have been proposed for this gender difference, highlighting the differential downstream roles of hormones (estrogen and testosterone) in endothelial and vascular aging, variations in sex chromosomes between genders, epigenetic regulation influenced by dosage compensation, and the impact of environmental factors 35 , 36) .
Gender differences in HO-1 expression and biological effects have been observed in animal models of glucose-lipid dysmetabolism. Hosick et al. established a specific HO-1(−/−) knockout mouse model for adipose tissue and discovered that the expression of HO-1 in adipocytes offered greater protection against elevated body fat and fasting hyperglycemia in female mice than in male mice 37) . In another study involving streptozotocin-induced diabetic rats, HO-1 protein was induced in females and was associated with renal vasodilation, decreased renal nitrosative stress, and reduced albuminuria 38) . Recent studies have further indicated that with aging, significant differences arise in the regulation of oxidative stress signaling pathways between sexes, in addition to changes in hormone levels 18 , 39 - 41) . Our study primarily involved elderly participants with usual risk factors, and we observed that the protective effect of the HO-1 (GT)n SS genotype against LAA stroke exhibited significant sex differences. We suggest that these variations may be attributed to age-related influences on oxidative stress regulation. This finding underscores the importance of emphasizing personalized approaches in future stroke research, particularly regarding sex differences.
Several limitations associated with the present study warrant mention. First, this was a single-center investigation conducted within the Chinese Han population, primarily involving elderly patients who exhibited a higher prevalence of stroke risk factors. The results need to be further validated in other cohorts, and caution is advised when extrapolating these findings. Second, our study population exhibited a higher proportion of patients with LAA than those in other studies. Recent reports indicated that LAA prevalence was higher in the Chinese population than in Western cohorts 42 , 43) , likely reflecting distinctions in ethnicity and risk factor exposure. In addition, there is a possibility that atherosclerotic conditions, such as aortogenic stroke or branch atheromatous disease (BAD), might be misclassified as non-LAA. Third, our study focused solely on the HO-1 (GT)n repeat, and the observed association might be influenced by other polymorphisms linked to the (GT)n. Further research is needed to elucidate the underlying mechanisms. Finally, the observed sex dimorphism in the association between LAA stroke and HO-1 (GT)n lack long-term follow-up data. This disparity may change with advancing age, necessitating further longitudinal cohort studies for validation.
Conclusion
Short (GT)n variants in HO-1 may confer significant protection against LAA stroke in men but not in women.
Author Contributions
JL planned and designed the experiments. JL and JC performed the genetic experiments, analyzed the data, and wrote the manuscript. JW, KC, XF, and SL assisted with the genetic experiments and data analysis. JW and XF commented on this manuscript. SL and ZS conceived and supervised the project, designed the experiments, interpreted the data and wrote the manuscript.
Funding Information
Guangdong Province Basic and Applied Basic Research Grant (2021B1515120089)
Conflict of Interest Statement
The authors declare that there is no conflict of interest
Data Availability
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Clinical Trial Registry
https://www.chictr.org.cn/ ChiCTR2100045869
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Clinical Trial Registry
https://www.chictr.org.cn/ ChiCTR2100045869