Hemodynamics influences vascular peroxynitrite formation: Implication for low-density lipoprotein apo-B-100 nitration

Abstract

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherogenesis. Superoxide anion (O 2 − ) reacts with nitric oxide ( NO) at a rapid diffusion-limited rate to form peroxynitrite (O 2 − + NO → ONOO −). Immunohistostaining of human coronary arterial bifurcations or curvatures, where OSS develops, revealed the presence of nitrotyrosine staining, a fingerprint of peroxynitrite; whereas in straight segments, where PSS occurs, nitrotyrosine was absent. We examined vascular nitrative stress in models of oscillatory (OSS) and pulsatile shear stress (PSS). Bovine aortic endothelial cells (BAEC) were exposed to fluid shear stress that simulates arterial blood flow: (1) PSS at a mean shear stress (τ ave) of 23 dyn cm − 2 and a temporal gradient (∂τ/∂t) at 71 dyn cm − 2 s − 1, and (2) OSS at τ ave = 0.02 dyn cm − 2 and ∂τ/∂t = ± 3.0 dyn cm − 2 s − 1 at a frequency of 1 Hz. OSS significantly up-regulated one of the NADPH oxidase subunits (NOx4) expression accompanied with an increase in O 2 − production. In contrast, PSS up-regulated eNOS expression accompanied with NO production (total NO 2 − and NO 3 −). To demonstrate that O 2 − and NO are implicated in ONOO − formation, we added low-density lipoprotein cholesterol (LDL) to the medium in which BAEC were exposed to the above flow conditions. The medium was analyzed for LDL apo-B-100 nitrotyrosine by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). OSS induced higher levels of 3-nitrotyrosine, dityrosine, and o-hydroxyphenylalanine compared with PSS. In the presence of ONOO −, specific apo-B-100 tyrosine residues underwent nitration in the α and β helices: α-1 (Tyr 144), α-2 (Tyr 2524), β-2 (Tyr 3295), α-3 (Tyr 4116), and β-2 (Tyr 4211). Hence, the characteristics of shear stress in the arterial bifurcations influenced the relative production of O 2 − and NO with an implication for ONOO − formation as evidenced by LDL protein nitration.