Role of ERK1/2 activation and nNOS uncoupling on endothelial dysfunction induced by lysophosphatidylcholine

Background and aims: Lysophosphatidylcholine (LPC) - a main component of oxidized LDL -is involved in endothelial dysfunction that precedes atherosclerosis, with an increased superoxide anions and a reduced NO production via endothelial NO synthase (eNOS) uncoupling. However, there is no evidence about the mechanisms involved in neuronal NOS (nNOS) uncoupling. Extracellular signal-regulated kinase (ERK) is related to the control of NO production and inflammatory gene transcription activation in atherosclerosis. Our aim was to investigate the role of nNOS/ERK1/2 pathway on endothelial dysfunction induced by LPC, in mouse aorta and human endothelial cells. Methods: Thoracic aorta from wild type mice was used to perform vascular reactivity studies in the presence or absence of LPC. Human endothelial cells were used to investigate the effect of LPC on expression of nNOS and his products NO and H2O2. Results: LPC reduced acetylcholine (ACh)-induced vasodilation in mouse aorta (Emax(CT/LPC) = similar to 95 +/- 2/ 62 +/- 3%, p = 0.0004) and increased phenylephrine-induced vasoconstriction (Emax(CT/LPC) = similar to 4 +/- 0,1/ 6 +/- 0,1 mN/mm, p = 0.0002), with a reduction in NO (fluorescence intensity(CT/LPC) = 91 +/- 3/62 +/- 2 x 10(3), p = 0.0002) and H2O2 (fluorescence intensityCT/LPC = similar to 16 +/- 0,8/10 +/- 0,7 x 10(3), p = 0.0041) production evocated by ACh. An inhibition of nNOS by TRIM (Emax(CT/CT+TRIM) = similar to 93 +/- 1/43 +/- 3%, p = 0,0048; Emax(LPC/LPC+TRIM) = similar to 62 +/- 3/65 +/- 3%) or H2O2 degradation by catalase (Emax(CT/CT+cat) = similar to 93 +/- 1/46 +/- 2%, p < 0,001; Emax(LPC/LPC+cat) = similar to 62,8 +/- 3,2/60,5 +/- 4,7%) reduced the relaxation in the control but not in LPC group. PD98059, an ERK1/2 inhibitor, abolished the increase in vasoconstriction in LPC-treated vessels (Emax(LPC/LPC+PD) = similar to 6 +/- 0,1/3 +/- 0,1 mN/mm, p = 0.0001). LPC also reduced the dimer/monomer proportion and increased nNOS(ser852) phosphorylation. Conclusions: LPC induced nNOS uncoupling and nNOS(Ser852) phosphorylation, reduced NO and H2O2 production and improved superoxide production by modulating ERK1/2 activity in human and murine endothelial cells. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Effect of intimal shear and cyclic strech on arterial segments perfused in vitro

Veronica Gambillara

Hemodynamics have crucial role in the control of vascular tone, in the regulation of arterial remodeling, and in the production of mediators that maintain arterial function. Changes in hemodynamics forces induce profound alterations in vascular cell metabolism and function, in the extracellular matrix, and in smooth muscle cell (SMC) phenotypes, all being part of vessel wall remodeling. Vessel wall remodeling plays a key role in arterial wall homeostasis but also in the initiation and development of arterial disease. This aim of this work is to study the effects of two distinct biomechanical stimuli, oscillatory shear and cyclic stretch on vessel wall function. Our 71 investigation is performed using an improved in vitro artery perfusion system, which allows to study the exact contribution of each biomechanical stimulus on isolated arterial segments, where the biological cell responses of endothelial and SMCs, and the extracellular matrix interactions are well preserved. The present thesis is divided into two main sections. The first part focuses on the effects of oscillatory shear stress on arterial segments, a mechanism presumed to be involved in the localization and initiation of atherosclerotic plaques. The second section investigates the influence of reduced cyclic stretch on the arterial wall, a procedure that is occurring in arterial stiffening. In the first and second papers, we examined the effects of three different shear stress conditions on the endothelium, SMC, and arterial wall responses, in porcine carotid arteries perfused in vitro for three days. We demonstrated that oscillatory shear stress, which is usually present in plaque-prone areas, decreased the bradykinin-induced vasorelaxation after 3 days of perfusion. The endothelial dysfunction was correlated to an impaired endothelial nitric oxide synthase gene expression and bradykinin-mediated activation. The potential lack of nitric oxide production and/or bioavailability observed in arteries exposed to oscillatory shear stress may also be the cause of the start of matrix turnover. Expression and activation of matrix metalloproteinase-2 and-9 were increased in arteries exposed to oscillatory shear, suggesting that this type of shear stress triggers a remodeling process. This part of the study provides a new insights on the role of shear stress in the control of arterial function and supports the hypothesis that oscillatory shear stress is a determining factor predisposing the arterial wall to atherosclerosis. The third paper focuses on the effects of cyclic stretch on vascular smooth muscle function, phenotype and wall remodeling. Arteries were wrapped by an external banding, which drastically decreased compliance and therefore the cyclic stretch. Arteries were perfused for one day under a unidirectional shear stress (6±3 dyn/cm2) in combination with a mean pressure of 80 mmHg and a pulse pressure of +/-10 mmHg. We demonstrated that exposure to reduced cyclic stretch induced a rapid modulation of SMC phenotype which was combined with a decrease in norepinephrine-induced vasocontraction. SMC dedifferentiation was associated to a decrease of plasminogen activator inhibitor-1 expression, which, combined with enhanced matrix metalloproteinase-2, may promote wall remodeling and SMC migration. These findings give a further approach into possible consequences of an arterial stiffening on large vessel wall patho-physiology.

EPFL2005

Plaque-prone hemodynamics impair endothelial function in pig carotid arteries

Veronica Gambillara, Sylvain Roy, Paolo Silacci, Nikolaos Stergiopulos

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 +/- 3 and 0.3 +/- 0.1 dyn/cm(2)) and oscillatory shear stress (0.3 +/- 3 dyn/cm(2)). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.

2006

Considerations for analysis of endothelial shear stress and strain in FSI models of atherosclerosis

Lydia Aslanidou, Fotios Savvopoulos

Atherosclerosis is a lipid driven chronic inflammatory disease that is characterized by the formation of plaques at predilection sites. These predilection sites (side branches, curved segments, and bifurcations) have often been associated with disturbed shear stress profiles. However, in addition to shear stress, endothelial cells also experience artery wall strain that could contribute to atherosclerosis progression. Herein, we describe a method to accurately obtain these shear stress and strain profiles. We developed a fluid-structure interaction (FSI) framework for modelling arteries within a commercially available package (Abaqus, version 6.12) that included known prestresses (circumferential, axial and pressure associated). In addition, we co-registered 3D histology to a micro-CT-derived 3D reconstruction of an atherosclerotic carotid artery from a cholesterol-fed ApoE-/-mouse to include the spatial distribution of lipids within a subject-specific model. The FSI model also incorporated a nonlinear hyperelastic material model with regionally varying properties that distinguished between healthy vessel wall and plaque. FSI predicted a lower shear stress than CFD (--12%), but further decreases in plaque regions with softer properties (--24%) were dependent on the approach used to implement the prestresses in the artery wall. When implemented with our new hybrid approach (zero prestresses in regions of lipid deposition), there was significant heterogeneity in endothelial shear stress in the atherosclerotic artery due to variations in stiffness and, in turn, wall strain. In conclusion, when obtaining endothelial shear stress and strain in diseased arteries, a careful consideration of prestresses is necessary. This paper offers a way to implement them.

ELSEVIER SCI LTD2021

Effect of intimal shear and cyclic strech on arterial segments perfused in vitro

Veronica Gambillara

EPFL2005