Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives

Abstract

Vascular endothelial cells (ECs) are exposed to hemodynamic forces, which modulate EC functions and vascular biology/pathobiology in health and disease. The flow patterns and hemodynamic forces are not uniform in the vascular system. In straight parts of the arterial tree, blood flow is generally laminar and wall shear stress is high and directed; in branches and curvatures, blood flow is disturbed with nonuniform and irregular distribution of low wall shear stress. Sustained laminar flow with high shear stress upregulates expressions of EC genes and proteins that are protective against atherosclerosis, whereas disturbed flow with associated reciprocating, low shear stress generally upregulates the EC genes and proteins that promote atherogenesis. These findings have led to the concept that the disturbed flow pattern in branch points and curvatures causes the preferential localization of atherosclerotic lesions. Disturbed flow also results in postsurgical neointimal hyperplasia and contributes to pathophysiology of clinical conditions such as in-stent restenosis, vein bypass graft failure, and transplant vasculopathy, as well as aortic valve calcification. In the venous system, disturbed flow resulting from reflux, outflow obstruction, and/or stasis leads to venous inflammation and thrombosis, and hence the development of chronic venous diseases. Understanding of the effects of disturbed flow on ECs can provide mechanistic insights into the role of complex flow patterns in pathogenesis of vascular diseases and can help to elucidate the phenotypic and functional differences between quiescent (nonatherogenic/nonthrombogenic) and activated (atherogenic/thrombogenic) ECs. This review summarizes the current knowledge on the role of disturbed flow in EC physiology and pathophysiology, as well as its clinical implications. Such information can contribute to our understanding of the etiology of lesion development in vascular niches with disturbed flow and help to generate new approaches for therapeutic interventions.

Conflict of interest statement

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

FIG. 1

Atherosclerosis preferentially develops at arterial branches and curvatures. Schematic drawing shows a longitudinal representation of the major arterial vasculature illustrating the observed distribution of atherosclerotic plaques (gray shading) in the vasculatures of LDLR−/− mice fed a high-fat atherogenic diet. 1, Aortic sinus; 2, ascending aorta; 3, inner (lesser) curvature of aortic arch; 4, outer (greater) curvature of aortic arch; 5, innominate artery; 6, right common carotid artery; 7, left common carotid artery; 8, left subclavian artery; 9, thoracic aorta; 10, renal artery; 11, abdominal aorta; 12, iliac artery. [Redrawn from VanderLaan et al. (590).]

FIG. 2

Hydrogen bubble visualization of flow in molds of carotid bifurcation. Flow visualizations were made with flow division ratio between internal and external carotid branches of 70:30 and Reynolds number (based on the inlet flow rate and hydraulic diameter) of 400 (A) and flow division ratio of 80:20 and Reynolds number of 800 (B) to show the effects of these hemodynamic parameters on flow pattern. A: flow is rapid, laminar, and longitudinal along the common carotid artery and the inner wall of the internal carotid sinus (black arrow); a large area of flow separation is formed along the outer wall of the sinus (white arrows). B: streamlines are skewed towards the apex of the bifurcation, and complex helical flow patterns occupy the separated flow region of the sinus. The five areas indicated are as follows: A, common carotid; B, proximal internal carotid; C, midpoint of carotid sinus; D, distal internal carotid; E, external carotid. [From Zarins et al. (644).]

FIG. 3

Schematic diagram showing the generation of shear stress (parallel to the endothelial cell surface) by blood flow and the generation of normal stress (perpendicular to the endothelial cell surface) and circumferential stretch due to the action of pressure. (Shu Chien and Yi-Shuan Li, unpublished Figure.)

FIG. 4

Atherosclerotic lesions in the aortic arch. A: gross appearance of atherosclerotic lesions in the aortic arch of a 20-wk-old ApoE−/− mouse fed the chow diet. Yellowish-white lesions are observed in regions of inner curvarure of the aortic arch and at the orifices of the arch branches. The more yellowish material in the branches is adventitial fat tissue. B: thin and transparent aorta of mouse allows direct visualization of early lesions in ApoE−/− mice by dissection microscopy. Under reflected light, elevated white lesions are visible on the luminal surface of the aortic arch of a 8-wk-old ApoE−/− mouse fed the Western-type diet. Two areas of lesion development are prominent and contain opaque sites of lesion formation: the lesser curvature (on the right, between the arrows) and the orifice of the arch branches (on the left, single arrow). [From Nakashima et al. (406, 407).]

FIG. 5

Shear stress distributions in mouse aortas. A: a representative image of mouse aortas illustrated by micro-CT scan. For reference, the diameter of the descending aorta is ~1 mm. B: distributions of mean shear stress in the aortas. Velocities in the aortas are measured by Doppler ultrasound. Mean values of wall shear stress are computed by averaging wall shear stress magnitudes over the cardiac cycle. Colors are used for scaling the values in dyn/cm2, and data are shown for two different mice. The mean wall shear stress in the mouse is much higher than that in the human (553), although the inner curvature of the arch and the entrance to the orifice of the innominate artery are areas of lower shear stresses. On the other hand, lateral surfaces of the ascending aorta and the region of the arch around the branch of left common carotid artery experience higher shear stress values. [From Suo et al. (552).]

FIG. 6

Velocity of blood flow through a venous valve and forces acting on a venous valvular leaflet. A: the reduced cross-sectional area between the valvular leaflets produces a proximally directed jet of increased axial velocity. B: axial flow between the leaflets generates a pressure (Po) that tends to keep the leaflet in the open position, and vortical flow in the valve pocket generates a pressure (Pi) that tends to close the leaflet. These pressures depend on the respective flow velocities (Vvortical and Vaxial); pressure is inversely related to velocity. [From Bergan et al. (36).]

FIG. 7

The configutrations for in vitro systems for applying shear flow to ECs. A: parallel-plate flow chamber. B: cone-and-plate viscometer. C: parallel disk viscometer. D: orbital shaker. E: tubular or rectangular capillary tube. It is noted that, for ease of visualization, the ECs are not drawn all around the inner lumen of the tube.

FIG. 8

Diagram showing the parallel-plate flow chamber for vertical-step flow. The polycarbonate base plate (top), two gaskets with different open areas, and the glass slide with EC monolayer (bottom) are held together by a vacuum suction applied at the perimeter of the slide, forming a channel with a lesser depth at the entrance, creating a step. Cultured medium enters at inlet port through entrance slit into the channel and exits through exit slit and outlet port.

FIG. 9

Visualization of flow patterns created in vertical-step flow channel. A: schematic diagram of the flow channel showing the flow pattern immediately beyond the step. h and H are heights of the channel above the step and beyond the step, respectively. B, top: phase-contrast photomicrograph (top view) of experimental flow patterns in the vertical-step flow channel. Flow is from left to right and is made visible with marker microparticles (1 µm in diameter). Flow separation occurs in the region distal to the step, forming four specific flow areas: a, the stagnant flow area; b, the center of the recirculation eddy; c, the reattachment flow area; and d, the fully developed flow area. From on-line microscopic observations, the particles transported from the bulk flow along the curved streamlines with decreasing velocities towards the wall near the reattachment point (area c). While some of the particles moved forward to rejoin the mainstream with increasing velocities, others moved in a retrograde direction towards the step as recirculation eddies. These latter particles moved upstream initially with increasing velocities and decelerated when approaching the wall of the step (area a), from where the particles were carried away from the floor of the chamber by upward curved streamlines. Bottom: schematic drawing of the side view of the streamlines in the vertical-step flow channel deduced from the top view photograph. [From Chiu et al. (88).]

FIG. 10

Morphological changes induced in a confluent EC monolayer by exposure to disturbed flow in the step flow channel. A, The stagnant flow area; B, the center of the recirculation eddy; C, the reattachment flow area; D, the fully developed flow area (same designations as in Fig. 9). The mean values of wall shear stress created in areas A, B, C, and D are characterized to be 0.5, 20, 0, and 20 dyn/cm2, respectively, by measurements using micron-resolution particle image velocimetry (μPIV) and by computational simulation (88, 94). The direction of the main flow is left to right. Control photograph is from the same experimental monolayer before shearing (0 h). [Modified from Chiu et al. (94).]

FIG. 11

Fluorescence photographs of F-actin filaments (A) and microtubules (B) after exposure to disturbed flow in the step-flow channel for 24 h. a, The stagnant flow area; b, the center of the recirculation eddy; c, the reattachment flow area; d, the fully developed flow area (same designations as in Figs. 9 and 10). The direction of the main flow is left to right. Magnification: ×400. [Modified from Chiu et al. (94).]

FIG. 12

In situ observation of EC morphologies in regions of laminar and disturbed flows. A: EC alignment in unidirectional laminar flow in the descending thoracic aorta. The direction of flow is indicated by the downward white arrow near the center of the picture. B: morphological changes in ECs in regions near a branch of the aorta. ECs change their morphologies from aligned elongations to polygonal shape beyond a line of flow separation (curved arrows) that marks the boundary of a disturbed flow region where oscillating, multidirectional flow typically occurs (double-headed arrow). Scale bar in each panel = 15 µm. For details, see Davies (125). [From Davies (125).]

FIG. 13

EC proliferative rate is increased in regions of disturbed flow. The proliferation of cultured bovine aortic ECs, as assessed by the BrdU incorporation assay, is markedly elevated in the disturbed flow region near the reattachment point in the step-flow channel. Top panel shows the side view of the step flow channel. Middle panel shows BrdU incorporation into ECs in one experiment. Bottom panel shows the BrdU incorporation into the ECs in four experiments. Bars are means ± SE. Asterisk indicates significant difference in BrdU incorporation in the region of disturbed flow near the reattachment point. Not shown in the bar graph is an increase in BrdU incorporation immediately next to the step. [From Chien (86).]

FIG. 14

Laminar flow promotes EC migration compared with disturbed flow and static control. The wounded EC monolayers are kept under static conditions or are subjected to laminar or disturbed flow in a stepflow channel. The direction of laminar flow is from left to right. The left-hand side of the zone is denoted as “upstream” side, and the right-hand side is “downstream.” A: phasecontrast images of the EC migration over the 21-h period of time. White vertical lines indicate the locations of wound edges at the beginning of the experiment. B: the net migration distance as a function of time for the cells in the first row at wound edges (both upstream and downstream sides). [From Hsu et al. (259).]

FIG. 15

VE-cadherin and β-catenin staining in ECs exposed to pulsatile and reciprocating flows. Confluent monolayers of bovine aortic ECs are kept as controls (A, B) or subjected to different flow patterns in a flow chamber. VE-cadherin staining is observed by confocal microscopy. Stainings for VE-cadherin and β-catenin at intercellular junctions are discontinuous after exposing to pulsatile (C, D) or reciprocating (E, F) flow for 6 h. The distribution of these junction proteins becomes continuous around the entire periphery of the cells after 24, 48, or 72 h of exposure to pulsatile flow (G, H, K, L, O, P), but not reciprocating flow (I, J, M, N, Q, R). [From Miao et al. (379).]

FIG. 16

Enhancement of monocytic THP-1 cell adhesion to activated ECs by disturbed flow. ECs are maintained in static condition without (A) or with TNF-α (125 U/ml) treatment (B) for 4 h, and then exposed to disturbed flow in the step-flow channel with THP-1 cell suspension at a concentration of 5 × 105 cells/ml. Prior to the perfusion, THP-1 cells are labeled with calcein-AM at a concentration of 7.5 mM for 30 min to facilitate visualization of the cells under fluorescence optics against the endothelial background. Phase-contrast and corresponding fluorescent microphotographs are taken after 10 min of perfusion. [From Chiu et al. (88).]

FIG. 17

Neutrophils, peripheral blood lymphocytes, and monocytes show different migratory behaviors in EC/SMC coculture under disturbed flow. Purified neutrophils, peripheral blood lymphocytes (PBLs), and monocytes are perfused over the EC/SMC coculture in the step-flow channel, and the migration of arrested WBCs is traced for 20 min in areas A, B, C, and D (same designations as in Figs. 9–11) or under static condition after their transmigration across the EC monolayer to the underside. This figure shows the results of a representative experiment containing 8 cells migrating in area A and 15 cells migrating in areas B, C, and D, as well as under static condition. The positions of the centers of the cells are determined at 20-s intervals from 0 to 20 min, and their paths of travel are processed with a commercialized image analysis software. [From Chen et al. (76).]

FIG. 18

In vivo observations of VE-cadherin distribution and KLF-2 expression in response to disturbed versus laminar flows. A: side view of the rat abdominal aorta with a local stenosis created by using a U-shaped titanium clip, with the aim of studying the effects of flow patterns on VE-cadherin distribution and KLF-2 expression in vivo. Four weeks after the creation of stenosis, the vessel is harvested, fixed, embedded, sectioned longitudinally, and subjected to immunohistochemical staining with anti-VE-cadherin (a and b) or anti-KLF-2 (c and d) (B). Blood flow is from left to right, as indicated by the arrow. In the laminar flow region 5 mm upstream (as well as 5 mm downstream; not shown) to the clip site, VE-cadherin is highly expressed at EC borders (B–a), and the endothelium shows strong KLF-2 staining (closed arrowhead) (B–c). No detectable VE-cadherin is found at cell borders in the disturbed flow region downstream to the constriction (B–b). In this region, there is also a lack of KLF-2 staining (open arrowhead) (B–d). Bars in B–b and B–d are 30 and 10 µm, respectively. [From Miao et al. (379) and Wang et al. (600).]

FIG. 19

Both unidirectional low shear stress and disturbed flow with low and reciprocating shear stress induce the development of atherosclerotic lesions. A: schematic representation of the shear stress patterns induced by the cast. On the right is a straight segment of a mouse common carotid artery without a cast, which has laminar blood flow (indicated by parallel arrows). On the left is a a mouse common carotid artery with the cast. Upstream from the cast, shear stress is relatively low (compared with the shear stress in the control vessel) due to the flow-limiting stenosis induced by the cast. Within the cast, a gradual increase in shear stress occurs due to the tapered shape of the cast. A disturbed flow region with low and reciprocating shear stress occurs immediately downstream the cast. B: ApoE−/− mice are instrumented with the cast 2 wk after starting the atherogenic Western diet and the specimens were obtained after 6 (B–a), 9 (B–b), or 12 wk (B–c) of cast placement. Whole-mount aortic arches and carotid arteries are stained with Oil red O for atherosclerotic lesions. White lines demarcate the previous position of the cast with the increased shear stress region. It is noted that atherosclerosis is induced in regions of both 1) unidirectional low shear stress upstream the cast and 2) disturbed flow with low and reciprocating shear stress downstream the cast. For details, see Cheng et al. (82). [From Cheng et al. (82).]

FIG. 20

Enhanced expression of VCAM-1 along the inner curvature of the aortic arch and at the orifices of the arch branches. VCAM-1 protein expression in the mouse aortic arch is analyzed ex vivo by using immunohistochemical staining with an antibody against VCAM-1, followed by Qdot-bioconjugated secondary antibody, and two-photon excitation microscopy (center). Regions with enhanced VCAM-1 expression can be observed along the inner curvature of the aortic arch and at the orifices of the arch branches. This distribution correlates with the development of lesions (Fig. 4) and low values of mean wall shear stress (Fig. 5B). Panels A, B, C, and D show differential expression of VCAM-1 in different areas indicated by arrows. The lateral walls of the ascending aorta (i.e., panel C) are areas where there is less VCAM-1 expression, which coincides with a relatively high mean shear stress. [From Suo et al. (552).]

FIG. 21

RasN17 prevents restenosis of rat common carotid artery after balloon injury. The phtographs show (left to right) the wide lumen in control artery, the severe stenosis following balloon injury without AdRasN17 (with only control AdLacZ), and the marked reduction of stenosis with vessel patency following balloon injury with AdRasN17 treatment. [From Chien (86).]

FIG. 22

EC signaling, gene expression, and function regulated by disturbed flow. ↑, Upregulation or sustentation versus laminar flow with higher shear stress; ↓, downregulation versus laminar flow with higher shear stress. “???” indicates that the molecules responsible for mediating the signaling are still not known.

FIG. 23

Summary of effects of different flow patterns and associated shear stresses on endothelial and vascular biology.