Molecular Mechanisms of Sex-Differences in Vascular Reactivity of Human Internal Mammary Artery

Abstract

Background: Clinical and epidemiological evidences have revealed that females have increased post-operative mortality and worsen outcome following coronary artery bypass grafting (CABG) compared to males. The potential reason for the sex-disparity is multifactorial and may include a combination of clinical (for example, smaller vessel size), psychosocial (for example, depression) and biological factors (for example, increase platelet activity and postmenopausal status) in female compared to male. Additional biological factor could include vascular reactivity of the conduit vessels that has not been previously assessed. Previous work in our group has revealed female internal mammary artery (IMA) graft conduit hypersensitivity to phenylephrine (PE) and serotonin (5-HT) but not endothelin-1 (ET-1) and thromboxane A₂ stable mimetics U46619 when subjected to ex-vivo organ bath experiment. Presently, the mechanisms responsible for the female IMA hypersensitivity to PE and 5HT are unknown. The present study evaluates potential mechanisms responsible for IMA hypersensitivity to PE and 5-HT specifically assessing the role of: (1) the endothelium, (2) nitric oxide (NO) pathway, (3) prostaglandin (PG) pathway, and/or (4) specific vascular receptors. Methods: Human IMA segments were obtained from 160 males (with mean age 67.3 ± 5.9 years) and 99 females (with mean age 67.8± 6.0 years) undergoing CABG and subjected to the following experimental conditions. (1) Contractile responses of isolated human IMA in the presence or absence of an intact endothelium were evaluated in an organ bath preparation coupled with challenge by cumulative doses of PE (0.01 μM -300 μM), 5-HT (0.001-300 μM), ET-1 (0.01-300 μM) and U46619 (0.001-300 μM). (2) The role of nitric oxide was determined by assessing the influence of the nitric oxide synthase (NOS) inhibitor, N-w-Nitro-L-arginine Methyl Ester (L-NAME, 300 μM) on PE-and 5-HT mediated vasoconstriction. Electron paramagnetic resonance spectrometry (EPR) coupled with the NO spin trap iron (II) dithiocarbamate complexes containing diethyldithiocarbamate; Fe (DETC)₂ was used to quantify NO release. (3) The role of prostaglandins in mediating PE and 5-HTvasoconstrictor responses was assessed using the cyclooxygenase inhibitor indomethacin (10 μM). Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to evaluate prostanoids metabolite including prostaglandin E₂ (PGE₂), PGD₂, PGF₂α, 6-keto PGF1α, and TXB₂ in vessel in IMA segments in response to A23187 (10 μM) (4) Phos-tag™ western blot was used to quantify abundance and phosphorylation status of α1-adrenergic and 5-HT₂A+₂B receptors in IMA segments. Data are expressed as mean SEM. Statistical analysis of unpaired data has been made using Student t-test. Differences are considered significant at p < 0.05. Results: Compared with males, female IMA were hypersensitive to 5-HT and PE but not U46619 and ET-1. The female IMA hypersensitivity was (1) abolished by endothelial denudation, (2) unaffected by NOS inhibition, with no difference in EPR-assessed NO levels, (3) abolished by COX inhibition, with a trend in reduced 6-keto PGF1α but not PGF₂α levels in females, and (4) unrelated to the abundance and phosphorylation of α1-adrenergic and 5- HT₂A+₂B receptors. Conclusions: This data indicate that female IMA hypersensitivity is agonist specific; attributable to an endothelium-dependent COX pathway involving possible impaired 6-keto PGF1α productions in female. Thus, post-operative inotrope use or serotonin release from platelet following CABG in females will produce more IMA vasoconstriction than male, which may result in significant anterior wall ischaemia and contribute to their poorer outcomes.