Zena and Michael A. Weiner Cardiovascular Institute, Marie-Josée and Henry R. Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, NY 10029, USA.
J Am Coll Cardiol. 2012 Jul 10;60(2):112-9. doi: 10.1016/j.jacc.2012.04.011.
The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype.
Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions.
Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perl's staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively.
In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p < 0.0001) along with expression of oxidized phospholipid- (78.9 ± 5.8 vs. 38.8 ± 3.8; p < 0.0001), and malondialdehyde-like OSEs (93.9 ± 7.9 vs. 54.7 ± 3.9; p < 0.0001). The total percentages of apoptotic cells (11.9 ± 0.44 vs. 3.5 ± 0.28; p < 0.0001), nuclear fragmentation (11.8 ± 0.50 vs. 3.3 ± 0.26; p < 0.0001), nuclear condensation (10.9 ± 0.58 vs. 3.4 ± 0.20; p < 0.0001), chromatin margination (14.2 ± 0.57 vs. 6.5 ± 0.37; p < 0.0001), cytoplasmic blebs (1.6 ± 0.28 vs. 0.8 ± 0.14; p < 0.002), and eosinophilia (10.8 ± 0.74 vs. 4.2 ± 0.27; p < 0.0001) were increased in Hp2-2 plaques. Furthermore, DNA fragmentation (119.9 ± 1.40 vs. 57.5 ± 0.80; p < 0.001), and active caspase-3 density (84.7 ± 7.62 vs. 50.6 ± 7.49; p < 0.004) were increased in Hp2-2 plaques. Logistic regression analysis identified correlation between the percentage of apoptotic cells and the density of OSEs (r = 0.56; p < 0.003).
These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.
本研究旨在验证下述假说，即氧化应激的增加与载脂蛋白 Hp 2-2 基因型的人类斑块中的细胞凋亡有关。
斑块内出血会释放游离血红蛋白（Hb）。Hb 清除受损会导致氧化应激，从而促使斑块进展。Hp 与 Hb 结合可减弱铁诱导的氧化反应。
对 26 个人主动脉斑块进行了 Hp 基因分型。将 Hp2-2 斑块（n = 13）与对照组（Hp1-1/2-1）（n = 13）进行比较。采用 Perl 染色法测定铁含量。通过免疫组织化学染色检测氧化特异性表位（OSEs），反映氧化的磷脂和丙二醛样表位。定量检测凋亡细胞的百分比和凋亡形态特征。通过原位末端标记和免疫组化分别检测 DNA 片段化和活性 caspase-3。
在 Hp2-2 斑块中，铁含量增加（1.22 ± 0.15 对 0.54 ± 0.08；p < 0.0001），同时还增加了氧化的磷脂（78.9 ± 5.8 对 38.8 ± 3.8；p < 0.0001）和丙二醛样 OSEs（93.9 ± 7.9 对 54.7 ± 3.9；p < 0.0001）的表达。总凋亡细胞百分比（11.9 ± 0.44 对 3.5 ± 0.28；p < 0.0001）、核碎片（11.8 ± 0.50 对 3.3 ± 0.26；p < 0.0001）、核浓缩（10.9 ± 0.58 对 3.4 ± 0.20；p < 0.0001）、染色质边缘化（14.2 ± 0.57 对 6.5 ± 0.37；p < 0.0001）、细胞质泡状突起（1.6 ± 0.28 对 0.8 ± 0.14；p < 0.002）和嗜酸性粒细胞增多（10.8 ± 0.74 对 4.2 ± 0.27；p < 0.0001）在 Hp2-2 斑块中增加。此外，Hp2-2 斑块中 DNA 片段化（119.9 ± 1.40 对 57.5 ± 0.80；p < 0.001）和活性 caspase-3 密度（84.7 ± 7.62 对 50.6 ± 7.49；p < 0.004）增加。逻辑回归分析确定了凋亡细胞百分比与 OSE 密度之间的相关性（r = 0.56；p < 0.003）。
这些发现提供了有关氧化应激遗传易感性以及 OSEs 与巨噬细胞凋亡之间关系的见解，这可能解释了人类 Hp2-2 斑块中晚期动脉粥样硬化的发生机制。
