Differential effects of reduced cyclic stretch and perturbed shear stress within the arterial wall

Due to the pulsatile nature of blood flow, arteries are constantly exposed to dynamic mechanical forces; the pulsatility continuously stretches the vessel wall and the flow creates a frictional force on the interior surface. These stresses, referred to as cyclic circumferential stretch and shear stress, are known to determine arterial structure and morphology; modulation of which leads to the progression of vascular diseases such as hypertension and atherosclerosis. Yet the individual contributions of cyclic stretch and shear stress, with regards to vascular disease, have yet to be revealed. In this thesis I wish to identify the role of reduced cyclic stretch in the development of endothelial dysfunction and vascular remodeling, develop an experimental model for studying the autonomous effects of shear stress and cyclic stretch and how these two stimuli individually modulate markers of vascular disease in different regions of the vascular wall. I will begin by introducing the different structural and cellular components of the vascular wall and their individual functions. From here I will introduce how hemodynamic forces transmitted to the vascular wall due to the pulsatile nature of blood flow play an essential role maintaining arterial health and function. And as such, how deviations from a physiologic hemodynamic range can have catastrophic implications for the vasculature. Next I will introduce how certain hemodynamic conditions can stimulate cellular dysfunction and how this relates to initiation and progression of vascular disease. In the first paper, we set out to determine if reduction of cyclic stretch could be a factor which induces remodeling of the arterial wall. We found that reducing compliance caused a decrease in vascular smooth muscle function, as well as inducing switch in smooth muscle cell phenotype. Arteries exposed to a reduced cyclic stretch also exhibited increased matrix degradation and cellular proliferation than those allowed to stretch physiologically. These findings accent the importance of cyclic stretch in the maintenance of a differentiated and fully functional phenotype of vascular smooth muscle cells, as well as in the regulation of migratory properties, proliferation and matrix turnover in the vascular wall. In the second paper we investigated how reduction of cyclic stretch influences endothelial dysfunction and modulation of nitric oxide bioavailability. We observed that reduced compliance significantly decreases the activity of the enzyme responsible for producing nitric oxide (eNOS). Overall production of reactive oxygen species were also increased by reducing compliance, which we were able to attribute to stimulation of the superoxide generating NAD(P)H oxidase. We found that experimentally reduced compliance also caused a significant decrease in endothelial function, as assessed with bradykinin dependent vascular relaxation. The results from this study point out how reduced arterial compliance interrupts the eNOS activation pathway and increases vascular levels of oxidative stress, which together could explain the measured decreases on endothelial functionality. In the third article we used our experimental model to investigate how shear stress and cyclic stretch independently stimulate the vascular wall. We found that both oscillatory flow and reduced stretch are detrimental to endothelial function, whereas oscillatory flow alone, dominated total endogenous vascular wall superoxide anion production. Yet when superoxide anion production was analyzed in just the endothelial region we observed that it was modulated more significantly by reduced cyclic stretch than by oscillatory shear, emphasizing an important distinction between shear and stretch mediated effects to the vascular wall. Analysis of eNOS and nitro-tyrosine, the by-product of superoxide anion and nitric oxide, proved that they too are more significantly negatively modulated by oscillatory flow, than by reduced stretch. The findings from this study point out how shear and stretch stimulate regions of the vascular wall differently, affecting NO bioavailability and contributing to vascular disease. The goal of the fourth article is to further investigate how shear stress and cyclic stretch modulate markers of vascular remodelling in different regions of the arterial wall. We demonstrated that while total superoxide production, fibronectin expression and gelatinase activation are predominantly mediated by shear stress, their expression in the endothelial region is mediated by reduced cyclic stretch, which correlates well with results from total MMP-2 expression. By plotting intensity versus radius for these markers of vascular remodeling we are able to see that superoxide production and gelatinase activity follows trends indicating their expression is in part mediated by stress distributions through out the vascular wall, while fibronectin and p22-phox were much less or not at all. Most importantly these findings, when coupled with our results from tissue reactive studies, suggest that the arterial remodeling process triggered in the endothelial region due to reduced stretch causes the most significant changes in arterial smooth muscle function. Perturbed shear stress and reduced arterial compliance have both been implicated in the initiation and progression of vascular disease: this work provides a new perspective into how these stimuli are perceived through out the vascular wall. To conclude, this body of work has shown that cyclic stretching due to the pulsatile nature of blood flow is an essential stimulus regulating arterial remodeling and endothelial viability. We have performed experiments permitting the autonomous effects of cyclic stretch and shear stress to be studied. Yet more importantly, we have shown how cyclic stretch and shear stress stimulate the vascular wall in different regions and compared this data to arterial functionality studies. These results have indicated that although reduced cyclic stretch may not stimulate the total expression of certain markers of vascular disease as much as an OSC shear stress, it does so in specific regions of the vascular wall which can in fact have a more detrimental effect on arterial function. As reduced arterial cyclic stretching is associated with the aging process, this work gives insight into the progression of vascular disease over the course of a person's life time.

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

Arterial in vitro remodeling

Gabriela Montorzi Thorell

Vascular remodeling is defined as any enduring change in the size and composition of an adult blood vessel, allowing adaptation or repair. The vascular remodeling response has been shown to depend on a variety of endogenous and environmental factors. Physiological remodeling is a tightly regulated process that mainly occurs in response to long-term changes in hemodynamic conditions. The adaptation to these hemodynamic changes implies the production of mediators that influence structure as well as function. A loss of regulation in the adaptive response underlies the pathogenesis of major cardiovascular diseases, including hypertension, atherosclerosis, restenosis and arterial aneurismal dilatation. Specialized enzymes called matrix metalloproteinases (MMPs) have been shown to have a predominant participation in the reorganization of the vessel structure, through the degradation of the extracellular matrix scaffold. The aim of this thesis is to gain insight in the biological and mechanical processes taking place in the vascular wall as an adaptive response to different biomechanical stimuli such as blood pressure and blood flow. This work proposes a new model for the study of vascular remodeling where physical factors acting on the arterial wall can be dissociated and analyzed, individually, in relation to the biological response. The results are presented in form of an introduction, three scientific papers and a conclusion section. The investigation has been designed around three different approaches: adaptation of a non-uniform artery to an in vitro environment, vascular adaptation to steady and pulsatile pressure and vascular adaptation to unidirectional and oscillatory flow. The adaptive response to each one of the variables chosen has been analyzed through biological and biomechanical remodeling indicators of the arterial wall. The introduction is an overview of the biological and biomechanical characteristics of arterial wall in relation to the remodeling process. The contribution of vascular smooth muscle cells and extracellular matrix to physiological and pathological arterial remodeling is discussed. Paper I assesses the relative remodeling of a non-axisymmetric artery in relation to its environment. The study considers the native circumferential asymmetry of the porcine right common carotid, which results from the non-homogenous mechanical and hemodynamic native environment. The adaptive response of the artery to an in vitro perfusion environment is analyzed. The study shows that in vitro perfusion leads, through remodeling, to a circumferentially uniform scleroprotein distribution and to a change in arterial compliance. This study emphasizes the link between structural changes, the biomechanical response and the enzymatic implication in the adaptive response. In paper II we investigate the role of continuous and cyclic stretch, produced by steady or pulsatile pressure acting on the arterial wall, on the remodeling response. The study shows that exposure to continuous and cyclic stretch differentially affects the relative scleroprotein content and leads to a change in arterial wall stiffness. The adaptive outcome is studied through an integrative approach taking into consideration the geometrical and structural adaptation, the biomechanical behavior and the enzymatic agents implicated in extracellular matrix turnover. Paper III analyzes the influence of different flow patterns on the arterial adaptive response. We investigate the differential effects of oscillatory flow, mimicking a plaque-prone hemodynamic environment, and unidirectional flow, to mimicking a physiologically protective (plaquefree) environment. The effect of these hemodynamic forces on the remodeling response are characterized through the study of endothelial and smooth muscle cell function as well as through assessment of agents influencing extracellular matrix turnover. The conclusions section presents a synthesis of the results and contribution of this thesis and proposes perspectives for future studies.

EPFL2004

Autonomous effects of shear stress and cyclic circumferential stretch regarding endothelial dysfunction and oxidative stress: an ex vivo arterial model

Rafaela Fernandes Da Silva, Paolo Silacci, Nikolaos Stergiopulos, Tyler Nerton Thacher

Cyclic circumferential stretch and shear stress caused by pulsatile blood flow work in concert, yet are very different stimuli capable of independently mediating endothelial function by modulating eNOS expression, oxidative stress (via production of superoxide anion) and NO bioavailability. Porcine carotid arteries were perfused using an ex vivo arterial support system for 72 h. Groups we created by combining normal (5%) and reduced (1%) stretch with high shear (6 +/- 3 dynes/cm(2)) and oscillatory shear (0.3 +/- 3 dynes/cm(2)) stress while maintaining a pulse pressure of 80 +/- 10 mm Hg. Oscillatory flow and reduced stretch both proved detrimental to endothelial function, whereas oscillatory flow alone dominated total endogenous vascular wall superoxide anion production. Yet, when superoxide anion production was analyzed in just the endothelial region, we observed that it was modulated more significantly by reduced cyclic stretch than by oscillatory shear, emphasizing an important distinction between shear- and stretch-mediated effects to the vascular wall. Western blotting analysis of eNOS and nitrotyrosine proved that they too are more significantly negatively modulated by oscillatory flow than by reduced stretch. These findings point out how shear and stretch stimulate regions of the vascular wall differently, affecting NO bioavailability and contributing to vascular disease.