Increased Expression of Oxidation-Specific Epitopes and Apoptosis Are Associated With Haptoglobin Genotype: Possible Implications for Plaque Progression in Human Atherosclerosis

• Published in: Journal of the American College of Cardiology 2012-07, Vol.60 (2), p.112-119
• Main Authors: PURUSHOTHAMAN, K-Raman, PURUSHOTHAMAN, Meerarani, KINI, Annapoorna, FAYAD, Zahi A, FUSTER, Valentin, SHARMA, Samin K, MORENO, Pedro R, LEVY, Andrew P, LENTO, Patrick A, EVRARD, Solene, KOVACIC, Jason C, BRILEY-SAEBO, Karen C, TSIMIKAS, Sotirios, WITZTUM, Joseph L, KRISHNAN, Prakash
• Format: Article
• Language: English
• Subjects: Aged; Alleles; Apoptosis - physiology; Atherosclerosis (general aspects, experimental research); Biological and medical sciences; Blood and lymphatic vessels; Cardiology. Vascular system; Caspase 3 - metabolism; Coronary Artery Disease - genetics; Coronary Artery Disease - metabolism; Disease Progression; DNA Fragmentation; Epitopes - metabolism; Female; Genetic Predisposition to Disease; Genotype; Haptoglobins - genetics; Humans; Immunohistochemistry; Iron - metabolism; Macrophages - metabolism; Male; Medical sciences; Middle Aged; Oxidative Stress - genetics; Plaque, Atherosclerotic - genetics; Plaque, Atherosclerotic - metabolism; Rupture, Spontaneous
• ISSN: 0735-1097; 1558-3597
• 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.