john m. tarbell - semantic scholar · john m. tarbell the city college of new york/cuny, new york,...

3 Jun 2003 14:5 AR AR191-BE05-04.tex AR191-BE05-04.sgm LaTeX2e(2002/01/18)P1: IKH10.1146/annurev.bioeng.5.040202.121529

Annu. Rev. Biomed. Eng. 2003. 5:79–118doi: 10.1146/annurev.bioeng.5.040202.121529

Copyright c© 2003 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on March 19, 2003

MASS TRANSPORT IN ARTERIES AND THE

LOCALIZATION OF ATHEROSCLEROSIS

John M. TarbellThe City College of New York/CUNY, New York, New York 10031;email: [email protected]

Key Words LDL, shear stress, hypoxia, permeability, tight junction

■ Abstract Atherosclerosis is a disease of the large arteries that involves a char-acteristic accumulation of high-molecular-weight lipoprotein in the arterial wall. Thisreview focuses on the mass transport processes that mediate the focal accumulation oflipid in arteries and places particular emphasis on the role of fluid mechanical forcesin modulating mass transport phenomena. In the final analysis, four mass transportmechanisms emerge that may be important in the localization of atherosclerosis: bloodphase controlled hypoxia, leaky endothelial junctions, transient intercellular junctionremodeling, and convective clearance of the subendothelial intima and media. Furtherstudy of these mechanisms may contribute to the development of therapeutic strategiesfor atherosclerotic diseases.

CONTENTS

INTRODUCTION AND OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80FLUID-PHASE RESISTANCE TO TRANSPORT. . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Transport to the Entire Endothelial Cell Surface. . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Transport to Endothelial Junctions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Concentration Polarization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Solutes Limited by the Fluid Phase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

ENDOTHELIAL/INTIMAL RESISTANCE TO TRANSPORT . . . . . . . . . . . . . . . . . 89Starling’s Law Revisited. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Pathways for Transport Across the Endothelium. . . . . . . . . . . . . . . . . . . . . . . . . . . 91Intercellular Junction Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Leaky Junctions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97Fluid Mechanical Effects on Endothelial Transport. . . . . . . . . . . . . . . . . . . . . . . . . 98

MACROMOLECULAR TRANSPORT AND ATHEROSCLEROSIS. . . . . . . . . . . . 105Focal Nature of Macromolecular Uptake. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

MASS TRANSPORT MECHANISMS IN ATHEROGENESIS. . . . . . . . . . . . . . . . . 107Fluid-Phase Controlled Hypoxia RegulatesEndothelial Permeability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Leaky Junctions Control Endothelial Permeability. . . . . . . . . . . . . . . . . . . . . . . . . . 108

1523-9829/03/0815-0079$14.00 79

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80 TARBELL

Transient Intercellular Junction Remodeling ControlsEndothelial Permeability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Convective Clearance Alters Intimal/Medial Accumulation. . . . . . . . . . . . . . . . . . . 109

INTRODUCTION AND OVERVIEW

Atherosclerosis is a disease of the coronary, carotid, and other proximal arteriesthat involves a distinctive accumulation of low-density lipoprotein (LDL) and otherlipid-bearing materials in the arterial wall (1). The disease tends to be localized inregions of curvature and branching in arteries where fluid shear stress (shear rate)and other fluid mechanical characteristics deviate from their normal spatial andtemporal distribution patterns in straight vessels (2). Because of the association ofdisease with regions of altered fluid mechanics, the role of blood flow in the local-ization of atherosclerosis has been debated for many years (3, 4). Among the firstmechanisms proposed to relate blood flow to the localization of atherosclerosiswas one in which the fluid (blood)-phase resistance to transport of LDL or otheratherogens was controlled by the local wall shear rate (3). Studies by Caro & Nerem(5), however, suggested that the uptake of lipid in arteries could not be correlatedwith fluid-phase mass transport rates, leading to the conclusion that the wall (en-dothelium) and not the blood was the limiting resistance to transport. This impliedthat fluid-flow effects on macromolecular transport were mediated by direct me-chanical influences on the transport systems of the endothelium. Somewhat later,attention was drawn to the fact that accumulation of macromolecules in the arterialwall depends not only on the ease by which materials enter the wall, but also onthe hindrance to passage of materials out of the wall offered by underlying layers(6, 7). This brought into focus the possibility that the subendothelial intima andmedial layers could be important structures contributing to local macromolecularuptake patterns.

In keeping with this brief historical outline of mass transport processes in rela-tion to atherosclerosis, the present review focuses first on the possible role of fluid(blood)-phase mass transport phenomena in the localization of arterial disease; itthen directs attention to the arterial wall, discussing at length the processes that me-diate transport across the endothelium. Throughout these initial sections describingbasic transport processes and systems, the possible influences of fluid mechanics,particularly shear stress, are emphasized. Next, a review of macromolecular uptakepatterns in relation to the localization of atherosclerosis is presented. The uptakepatterns are then interpreted in light of basic mass transport processes and asso-ciated fluid mechanical influences in a final synthesis section in which four masstransport mechanisms that may play a role in the localization of atherosclerosisare described.

FLUID-PHASE RESISTANCE TO TRANSPORT

Solutes that are transported from the bulk fluid (blood) phase to the endothelialsurface will encounter a variety of surface boundary conditions depending on the

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MASS TRANSPORT IN ARTERIES 81

nature of the molecule being transported. For example, a species may be consumedover the entire endothelial surface by enzyme-catalyzed surface reactions (e.g., thehydrolysis of ATP to ADP), it may be transported across the entire endothelial sur-face because it is soluble in the plasma membrane (e.g., oxygen and other bloodgases), or it is solubilized in vesicles that can be transported across the entire sur-face (e.g., albumin and LDL may be transported in part by this mechanism). On theother hand, many hydrophilic solutes that cannot cross the plasma membrane willbe confined to transport through interendothelial junctions that are typically 20 nmin width (at the wide part of the junction) in regions of undamaged endothelium andoccupy only a small fraction of the total endothelial surface area (8). Hydrophilicsolutes below the size of albumin (7.0-nm molecular diameter) are transported pri-marily through this intercellular junction pathway. There is also a dominant trans-port pathway for large molecules (including albumin and LDL) through “leakyjunctions” associated with cells in a state of mitosis or apoptosis (9). These leakyjunctions have dimensions on the order of 30–1000 nm and occupy a very smallfraction of the total surface area. Because of these diverse surface boundary con-ditions, we consider two cases in describing fluid-phase transport: (a) transport tothe entire endothelial cell surface and (b) transport to discrete cellular junctions.

Transport to the Entire Endothelial Cell Surface

Here, we discuss three common situations for transport that utilize the entire en-dothelial surface: the reactive surface, the permeable surface, and the reactive wall.We then develop simple analytical criteria that allow us to estimate the importanceof fluid-phase transport relative to other transport processes.

REACTIVE SURFACE Referring to Figure 1, we assume that the species of interestis transported from the blood vessel lumen, where its bulk concentration is Cb, tothe blood vessel surface, where its concentration is Cs, by a convective-diffusivemechanism that depends on the local fluid mechanics and can be characterized bya fluid-phase mass transfer coefficient kL. The species flux (mass flow rate dividedby surface area) in the blood phase is given by

Js = kL(Cb− Cs). (1)

At the endothelial surface, the species may undergo an enzyme-catalyzed surfacereaction, which can be modeled using classical Michaelis-Menten kinetics, with arate given by

V = VmaxCs

km+ Cs, (2)

where Vmaxis the maximum rate (high Cs) and km is the Michaelis constant. WhenCs¿ km, as is often the case, then the reaction rate is pseudo–first order and

V = krCs, (3)

with the rate constant for the surface reaction given by kr=Vmax/km.

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82 TARBELL

Figure 1 Schematic diagram of arterial wall transport processes showing the con-centration profile of a solute that is being transported from the blood, where its bulkconcentration is Cb; to the surface of the endothelium, where its concentration is Cs;then across the endothelium, where the subendothelial concentration is Cw; and finally,to a minimum value within the tissue, Cmin. Transport of the solute in the blood phaseis characterized by the mass transport coefficient, kL; consumption of the solute at theendothelial surface is described by a first-order reaction with rate constant, kr; move-ment of the solute across the endothelium depends on the permeability coefficient, Pe;and reaction of the solute within the tissue volume is quantified by a zeroeth orderconsumption rate,Q. (Adapted from Reference 10).

At steady state, the transport to the surface is balanced by the consumption atthe surface so that

kL(Cb− Cs) = krCs. (4)

It will be convenient to cast this equation into a dimensionless form by multiplyingit by d/D, where d is the vessel diameter and D is the diffusion coefficient of thetransported species in blood or the media of interest. Equation 4 then becomes

Sh(Cb− Cs) = DarCs, (5)

where

Sh= kLd

D(6)

is the Sherwood number (dimensionless mass transfer coefficient) and

Dar = krd

D(7)

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is the Damkholer number (dimensionless reaction rate coefficient). Solving Equa-tion 5 for the surface concentration, one finds

Cs/Cb = 1

1+ Dar/Sh. (8)

When Dar¿ Sh,

Cs = Cb, (9)

and the process is termed “wall-limited” or “reaction-limited.” On the other hand,when DarÀ Sh,

Cs = (Sh/Dar)Cb, (10)

and the process is termed “transport-limited” or “fluid-phase-limited.” It is in thistransport-limited case that the surface concentration, and in turn the surface reac-tion rate, depend on the fluid mechanics, which determine the Sherwood number(mass transfer coefficient). It will therefore be useful to compare the magnitudesof Dar and Sh to determine whether fluid mechanics plays a role in the overalltransport process of a surface reactive species.

PERMEABLE SURFACE Many species will permeate the endothelium without re-acting at the luminal surface (e.g., albumin, LDL) and their rate of transport (flux)across the surface layer can be described by

Js = Pe(Cs− Cw), (11)

where Pe is the permeability coefficient and Cw is the wall concentration beneaththe endothelium (Figure 1). If the resistance to transport offered by the endotheliumis significant, then it is reasonable to assume

Cw ¿ Cs. (12)

So that at steady state when the fluid and surface fluxes balance,

kL(Cb− Cs) = PeCs. (13)

Multiplying Equation 13 by d/D to introduce dimensionless parameters and thensolving for the surface concentration leads to

Cs/Cb = 1

1+ Dac/Sh, (14)

where Sh was defined in Equation 6 and

Dae = Ped

D(15)

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84 TARBELL

is a Damkholer number based on endothelial permeability. Equation 14 showsthat when Dae À Sh, fluid mechanics again becomes important in limiting thetransport.

REACTIVE WALL Oxygen is transported readily across the endothelium, but unlikemost proteins, it is rapidly consumed by the underlying tissue. In this case, it isfair to neglect the endothelial transport resistance (assume Cw=Cs), and then byequating the rate of transport to the wall with the (zeroeth order) consumption ratewithin the wall, we obtain

kL(Cb− Cs) = QT, (16)

whereQ is the tissue consumption rate and T is the tissue thickness (distancefrom the surface to the minimum tissue concentration; see Figure 1). For thespecific case of O2 transport, it is conventional to replace concentration (C) withpartial pressure (P) through the Henry’s law relationship, C=KP, where K is theHenry’s law constant. Invoking this relationship and rearranging Equation 16 intoa convenient dimensionless form, we obtain

Ps/Pb = 1− Daw/Sh, (17)

where Sh was defined in Equation 6 and Daw is another Damkholer number basedon the wall consumption rate

Daw = QTd

KDPb. (18)

Clearly, when Daw ¿ Sh, the process is wall limited. But, as Daw→Sh, theprocess becomes limited by transport in the fluid phase (Ps→ 0) and fluid mechan-ics plays a role. Because we are treating the tissue consumption rate as a zeroethorder reaction, the case Daw > Sh is not meaningful (Ps< 0). In reality, as Sh isreduced, the tissue consumption rate must fall due to the lack of oxygen supplyfrom the blood.

DAMKHOLER NUMBERS FOR IMPORTANT SOLUTES In each of the cases outlinedabove, the transport becomes fluid-phase-limited, and the fluid mechanics may beinfluential when the Sherwood number is reduced below the Damkholer number.Clearly, species with a high Damkholer number are most likely to be limited bythe fluid phase. As described in detail elsewhere (10), Dae= 0.02–1.0 for LDLand 0.027–0.10 for albumin, assuming that the entire surface is available for thetransport of these species. In reality, only a fraction of the transport of albuminor LDL is likely to cross the entire surface (vesicular transport fraction), so thevalues of Daementioned above should be considered upper bounds for the vesiculartransport pathway. Species with much higher Damkholer numbers that are trulyassociated with the entire endothelial cell surface are ATP (Dar= 17.7) and oxygen(Daw= 10.8–49.0). So it is clear that oxygen and ATP are more likely than albumin

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and LDL to be fluid-phase limited. Because albumin and LDL are also transportedthrough intercellular junctions, the possibility that transport to the junctions isfluid-phase-limited is considered in a later section.

FLUID MECHANICAL (GEOMETRICAL) EFFECTS ON TRANSPORT TO THE ENTIRE

SURFACE For smooth, cylindrical tubes (a model of straight blood vessels) withwell-mixed entry flow, the classical Graetz solution (11) shows that for fully de-veloped transport, where the Sherwood number is minimum, Sh= 3.66 (constantwall concentration) or Sh= 4.36 (constant wall flux). It is shown elsewhere (10)that for parameters characteristic of the human aorta treated as a straight tube, thefollowing values of the Sherwood number are predicted at the end of the aorta:Sh= 31.1 (O2), 41.8 (ATP), 79.2 (albumin), 114 (LDL). These straight tube es-timates of Sh suggest that O2 and ATP might be fluid-phase-limited (Sh< Da),whereas albumin and LDL would not.

Nonuniform geometries associated with the localization of atheroscleroticplaques in arteries induce fluid mechanical features that can affect fluid-phasetransport (Sherwood number), as described in detail by Tarbell & Qiu (10). Flowthrough a sudden expansion (e.g., at the anastomosis of a vascular graft), a stenosisor flow constriction (e.g., vessel with an atherosclerotic plaque), or a bifurcation,may induce axial flow separation. In such geometries, Sh is reduced near the sep-aration point, where radial velocity away from the wall impedes transport, andincreased near the reattachment point, where radial velocity toward the wall en-hances transport. These separation zones are also regions of low fluid wall shearstress, which is identically zero at points of separation and reattachment.

In curved vessels and bifurcations, secondary flows (flows in planes perpendic-ular to the principal flow direction) can also alter fluid-phase transport rates. Forconditions characterizing the curvature of coronary arteries over the surface of theheart and the transport of oxygen, Qiu & Tarbell (12) computed Sh= 2 at the innerwall (closest to the center of curvature where atherosclerotic plaques form) andSh= 55 at the outer wall, a ratio of more than 25. Under the same flow conditions,the mean (time-averaged) wall shear stress was less than two times greater on theoutside wall than on the inside wall. In the curved geometry, the secondary flow isdirected away from the wall at the inner curvature, inducing a reduction in Sh, andtoward the wall at the outer curvature inducing an elevation in Sh. Further stud-ies of oxygen transport in an anatomically realistic right coronary artery modelaccounting for out-of-plane curvature showed a similar drastic reduction in theSherwood number at the inner wall (13, 14). Because Sh¿ Daw for oxygen atthe inner wall of these coronary artery models, hypoxia is predicted to occur atthe inner wall where atherosclerotic plaques occur. For similar reasons, hypoxiamight be expected to occur on the outer wall of a bifurcation (wall opposite theflow divider where atherosclerotic plaques are localized), and this has in fact beenobserved in experiments in the dog carotid bifurcation (15). The role of hypoxiain atherogenesis will be discussed further in a later section.

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86 TARBELL

Figure 2 Schematic diagram of fluid-phase solute transport to a vessel wall. Theendothelial cells are shown conceptually, aligned in the longitudinal direction of theflow field with intercellular clefts elongated in the direction of flow. The concentrationprofile of a solute being transported from the blood is shown, where Cb is its bulkconcentration. The fluid-phase mass transport to the cleft is characterized by the mass-transfer coefficient kL. The intercellular clefts are assumed to be the only route for thesolute uptake. (Adapted from Reference 8).

Transport to Endothelial Junctions

When transendothelial transport is confined to intercellular junctions, the fluid-phase transport problem involves a boundary surface that is impermeable almosteverywhere except at the discrete junctions. To visualize this, in Figure 2 the inter-cellular junctions are exaggerated and idealized as slits between cells that are eitherparallel (sides of the cells) or perpendicular (ends of the cells) to the flow direc-tion. This emphasizes the fact that endothelial cells are elongated in the directionof flow (16) so that most of the intercellular junction is in the parallel configuration.The problem of transport to a rectangular slit in the perpendicular configuration athigh Peclet number (Pe= γa2/D; γ is the wall shear rate, a is the slit half-width,and D is the solute diffusivity) has a well-known solution due to L´eveque (17)that predicts the surface flux (to a sink at zero concentration) to be proportionalto Pe1/3. The Leveque solution is accurate for Pe> 100, but for tight junctions(a= 10 nm) and reasonable shear rates ( ˙γ = 1000 s−1), Pe< 1 for a broad range ofsolutes, including LDL (D= 2.06× 10−7 cm2/s). Ackerberg et al. (18) presentedexperimental data for the heat transfer analog of the perpendicular slit problemand extended the range down to physiologically relevant Peclet numbers (10−4).Hodgson & Tarbell (8) solved the parallel slit configuration problem numericallyfor both specified concentration and specified flux conditions at the slit surface.There was little influence of the slit boundary condition on the Sherwood number(Sh= kca/D; kc=mass transfer coefficient based on the slit area, Ac), which forthe fixed concentration boundary condition was well predicted by the followingcorrelation of numerical results for Pe< 10:

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Sh= Sh0+ 0.220 Pe0.261, (19)

where Sh0(= 0.299) is the Sherwood number at zero flow. The value of Sh0 waswithin 2.26% of the theoretical prediction of Saito (19) for the problem of diffusionto an infinitely long slit. As shown in detail by Hodgson & Tarbell (8), the Sherwoodnumber for slits parallel to the shear direction is lower than the Sherwood numberfor perpendicular slits at all Pe> 0. The two solutions are, of course, identical atPe= 0, where Sh=Sh0. For a sink having the shape of a circular disk of radiusa, at Pe= 0, Saito (19) showed that Sh0= 1.27, but the dependence on shear rate(Pe) has not been determined for the circular disk sink.

To determine whether fluid-phase transport to intercellular junctions (modeledas slits) can limit overall transport, it suffices to compare the lowest possible valueof the Sherwood number (when Pe= 0) to the appropriate Damkholer numberfor the solute of interest. Because the Damkholer numbers described previouslywere all based on the total surface area, it is necessary to multiply the slit masstransfer coefficient (kc) by Ac/Atotal to generate the kL used in the definition ofthe Sherwood number (Equation 6). For cylindrical tubes with longitudinal slits,Ac/Atotal= 2a/w, where w is the width of an endothelial cell (order 10µm) and ais the slit half-width. Now the Sherwood number (Equation 6) is given by

Sh= Sh0(2d/w). (20)

It is interesting to note that this Sherwood number is proportional to the vesseldiameter (d) and does not depend on the junction dimension (a). In the largest artery(d= 2.5 cm), Sh= 1495, and in the smallest capillary (d= 5 µm) Sh= 0.299.Because the Damkholer numbers for albumin and LDL are at most 0.1 and 1.0,respectively, it is clear that fluid-phase transport of these solutes to intercellularjunctions is not a limiting factor in arteries, but may be important in capillaries.

The above considerations of the discrete nature of endothelial junctions astransport sinks did not include the effects of convective flux of solute into thejunction associated with pressure-driven flow across the wall. The volume flux(Jv) or superficial velocity in arteries is typically of the order 10−6 cm/s (20) basedon the filtration flow rate divided by the total surface area of the vessel (Atotal).But, the transmural flow is confined primarily to the intercellular junctions suchthat the fluid velocity into the junction (v) is given by

v = Jv(Atotal/Ac). (21)

Because the area ratio (Atotal/Ac) may be as high as 103, the local velocityentering the intercellular junction could reach 10−3 cm/s. This locally elevatedfluid velocity might be expected to convect solute into the junction at a substantialrate. Although this convective flux seems to not have been investigated thoroughlyin the literature, Hodgson & Tarbell (8) have estimated that it may be significantfor large solutes such as LDL. This additional fluid-phase-transport mechanismreinforces the conclusion of the preceding paragraph that fluid-phase transport tointercellular junctions is not a limiting transport process in arteries.

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88 TARBELL

Concentration Polarization

The convective flux of plasma proteins to and within the intercellular junctionassociated with the enhanced velocity described by Equation 21 could induce“concentration polarization”—the accumulation of solute on the surface or withinthe intercellular junctions because of the high resistance to transport offered by theendothelium (21–23). Elevated protein concentration at the surface could lead tohigher protein transport across the endothelium by diffusive mechanisms as wellas to reduced volume flux (Jv) because of the resistance of the concentrated proteinlayer and the locally elevated osmotic pressure at the endothelial surface. If signifi-cant concentration polarization layers exist on endothelial surfaces, then increasesin the fluid shear stress of blood flow would be expected to reduce the polarizationlayer (reduce the maximum protein concentration at the surface), leading to eleva-tion of Jv and reduction of the diffusive component of Js. Furthermore, these shearstress effects would be rapidly reversed after returning shear stress to its originallevel.

Lever et al. (24) measured the change in Jv across rabbit common carotid arteriesperfused with solutions containing 1% or 4% albumin when the lumenal flowshear stress was increased from zero to a level in the range of 0.57–1.65 dyne/cm2.There was an approximately 30% increase in Jv associated with shear stress thatwas reversible within 30 min after removal of shear stress. However, there wasno correlation between the Jv elevation and the shear stress level, and the proteinconcentration had no effect on the degree of Jv elevation either. An analysis based onconcentration polarization in endothelial junctions suggested that this mechanismcould not completely account for the observed changes in Jv with shear stress.

Sill et al. (25), using bovine aortic endothelial cells cultured on porous supports,observed that a step change in shear stress from 0 to 10 dyne/cm2 induced a 2.16-fold increase in hydraulic conductivity (Lp= Jv/1P), which occurred graduallyover a period of 60 min. When shear stress was removed, Lp remained elevatedfor an additional 120 min. Other tests were performed to show that the endothelialmonolayers were not damaged by shear stress and that Lp could be reversed byaddition of chemical agonists. Chang et al. (26), using the same cell culture model,showed a gradual 4.7-fold increase in Lp over a 3-h period of exposure to a stepchange in shear stress from 0 to 20 dyne/cm2. The increase in Lp in response toshear stress could be blocked completely by preincubating the cells with a nitricoxide synthase (NOS) inhibitor. These cell culture studies suggest that increases inLp in response to shear stress are associated with mechano-chemical transductionof shear stress by endothelial cells and not concentration polarization.

Williams (27) measured hydraulic conductivity in capillaries of the frog mesen-tery perfused with 1% albumin and observed that a change in shear stress fromabout 9 to 18 dyne/cm2 led to about a 5-fold increase in Lp of venular capillaries, a2.5-fold increase in Lp of true capillaries, and no increase at all in Lp of arteriolarcapillaries. The differential behavior of these capillaries in response to increasesin shear stress suggests endothelial-dependent mechano-chemical transduction asthe controlling mechanism.

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It should also be noted that most studies of the permeability of the endotheliumto macromolecules have shown that increases in blood flow (shear stress) lead toan increase in permeability, not a decrease as would be predicted by concentrationpolarization. For example, Caro & Nerem (5) showed a weak positive dependenceof endothelial permeability to lipoprotein-linked cholesterol in excised dog carotidarteries, and similar results were shown for albumin in the same model (28). Yuanet al. (29) showed that increases in flow through coronary venules of pigs increasedalbumin permeability by a nitric oxide–dependent mechanism.

There have been several computational modeling studies of concentration po-larization of LDL at the luminal surface of an artery [e.g., (30)] that have beenreviewed by Ethier (14). In these theoretical studies, transendothelial volume fluxand solute flux were assumed to be distributed over the entire surface of the vesselwall. By setting Jv sufficiently high and the LDL diffusion coefficient sufficientlylow it was possible to demonstrate conditions under which the wall concentrationof LDL would be elevated above the bulk concentration. But, as suggested above,this type of model is not consistent with most observations of the effect of increas-ing shear stress on endothelial permeability and hydraulic conductivity. A morerealistic model of the concentration polarization process would confine the volumeflux to intercellular junctions and leaky junctions and distribute the LDL flux intoa fraction associated with vesicular transport that would access the entire surfaceand a fraction associated with leaky junctions that would access only a very smallfraction of the surface. Studies of this type remain to be investigated.

Solutes Limited by the Fluid Phase

In a brief summary of the considerations of this section, it appears that oxygen,and possibly ATP, low-molecular-weight species that are consumed readily bythe arterial wall and that access the entire endothelial surface, are the most likelysolutes to be limited by fluid-phase transport. This gives rise to the predictionof hypoxic regions in arteries where there is flow separation or secondary flowthat correspond to locations where atherosclerotic plaques are often localized.The mechanisms by which hypoxia can induce atherosclerosis are discussed in asubsequent section. High-molecular-weight materials such as LDL and albuminand other hydrophilic solutes that are transported through intercellular junctionsand leaky junctions and only access a small fraction of the endothelial surface, arenot limited by the fluid-phase, but by the endothelium.

ENDOTHELIAL/INTIMAL RESISTANCE TO TRANSPORT

A number of recent review papers have treated endothelial transport (31–33) andmay be consulted for additional information. The endothelium lines all bloodvessels from the largest arteries and veins down to the smallest capillaries. Muchof what is known about the nature of transendothelial transport derives from studiesof the microcirculation. But atherosclerosis is a disease of the larger arteries, andthus endothelial transport in arteries is the focus of this section.

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To characterize transport across the endothelium, the following membranetransport equations are usually employed (34):

Jv = Lp(1P− σ1π ) (22)

Js = P01C+ (1− σ )JvC, (23)

where Jv is the volume flux across the endothelium, Js is the solute flux,1P isthe pressure differential across the endothelium,1π is the corresponding osmoticpressure differential,1C is the solute concentration differential,σ is the reflectioncoefficient, Lp is the hydraulic conductivity, P0 is the diffusive permeability, andC is the mean intramembrane solute concentration, which depends in a complexway on the relative importance of convection (second term in Equation 23) anddiffusion (first term in Equation 23).1P and1π are driving forces for volumetransport (water flow), which convects solute;1C is the driving force for diffusivesolute transport; and P0, Lp, andσ are transport properties. It is conventional tothink of membrane transport properties as constant, but a remarkable feature ofthe endothelium is that its transport properties are sensitive to both the chemicaland mechanical environment within which it resides.

The more rigorous form of Equation 23 was developed by Patlak et al. (35) andcan be represented as follows:

Pe = Js/Cs = P0Z+ (1− σ )Jv. (24)

In Equation 24, Peis the apparent permeability that is usually measured experimen-tally, and the assumption has been made that the concentration on the bottom sideof the endothelium (Cw in Figure 1) is much less than on the top (Cw¿Cs). The firstterm is the diffusive contribution and the second term is convective. The relativeimportance of diffusion to convection is determined by the factor Z, defined as

Z = NPe/[exp(NPe)− 1], (25)

where NPeis a Peclet number defined as

NPe= Jv(1− σ )/P0. (26)

Starling’s Law Revisited

Equation 22, withσ = 1, is often referred to as “Starling’s law,” as it expressesStarling’s pioneering hypothesis that fluid movement across microvascular wallsis determined by the transmural difference in hydrostatic and oncotic pressures(36). It has become a general principle of cardiovascular and renal physiologythat as capillary pressure drops below plasma oncotic pressure (about 25 mm Hg),the net driving force for volume flux changes from positive (filtration) to negative(reabsorption). Studies by Intaglietta & Zweifach (37), however, challenged thenotion that significant reabsorption occurs at the venous end of the microcirculationwhere the osmotic pressure exceeds the capillary pressure. Their investigations in-dicated that fluid reabsorption was demonstrable in short-term experiments, where

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protein does not attain a steady state distribution in the extravascular space, butnot on a long-term or steady state basis. This concept was demonstrated moreclearly by Michel & Phillips (38) who performed experiments on isolated, per-fused, frog mesenteric microvessels and observed transient reabsorption over aperiod of 15–30 s after the capillary pressure was dropped below the oncotic pres-sure, as expected from Starling’s law. When the measurements were extended for2–5 min after the drop in pressure, however, there was no evidence of reabsorp-tion, only a small filtration flow. Additional experiments using frog mesentericmicrovessels (39) and bovine aortic endothelial cells in vitro (40) have providedfurther support for the transient reabsorption/steady state filtration concept.

The first plausible model that could predict steady state filtration when the Star-ling forces (1P−1π ) predict reabsorption was presented by Michel & Phillips(38). They developed a steady state, one-dimensional, transport model based onclassical equations (Equations 22 and 24, above), but with the novel assumptionthat the pericapillary concentration, Cw, that determinesπw in Equation 22, wasgiven by a mixing condition expressed as

Cw = Js/Jv, (27)

where Js is the flux of the oncotic solute. In general, Cw differs from the globaltissue concentration that is fixed by bathing solutions or external reservoirs intypical experiments. The resulting volume flux equation,

Jv = Lp(Ps− Pw)− Lpσ2πs · 1− exp(−Npe)

1− σ exp(−Npe), (28)

only admits positive solutions for Jv (filtration only).A much more detailed microstructural model of the endothelial transport barrier

accounting for the surface glycocalyx layer and the fine structure of the interen-dothelial junction has provided further insights into the Starling mechanism (9,41). This model predicts that the Starling forces are determined by the local dif-ferences in hydrostatic and oncotic pressures across the surface glycocalyx layer.We return to a discussion of this model in a later section.

Pathways for Transport Across the Endothelium

The basic membrane transport equations presented in the preceding section canbe used to define the transport properties (Lp, Pe, σ ) based on experimental mea-surements of transendothelial fluxes in the presence of known driving forces (1P,1π , 1C). In principle, they are based on the assumption that water and solutesshare a single pathway across the endothelium. When this assumption is not valid,inconsistencies in the analysis of experimental data may arise. For example, Dullet al. (42) used Equation 24 to fit measurements of Jv and Js for albumin acrossBAEC monolayers in vitro and found that the equation could only be satisfied forσ < 0. This contradiction suggests at least two pathways for transport across theendothelium.

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In the early 1950s, Pappenheimer et al. (43) formulated the pore theory of capil-lary permeability that predicted the transport of small hydrophilic solutes throughwater-filled channels or pores of radius∼4 nm. The pores were assumed to bemorphologically represented (in continuous endothelia) by the clefts or junctionsbetween endothelial cells. Later, Grotte (44) presented evidence for a two-porebarrier partitioning blood from lymph. Grotte observed that large-molecular-sizedextrans appeared in canine leg lymph in a concentration that decreased rapidly asa function of increasing molecular size for molecules<4.5 nm in radius. Largermolecules still appeared in lymph, but at concentrations that were only slightlyaffected by molecular size. Grotte (44) therefore suggested that a few “large pores”or “capillary leaks” (1/30,000 of the small pores) with a radius of 25–60 nm wouldaccount for the transport of plasma proteins. The nature of these pores or pathwaysacross the endothelium still has not been completely resolved, although consider-able progress has been made as described below.

Intercellular Junction Transport

As suggested by Pappenheimer et al. (43), transport of water and hydrophilicsolutes below the size of albumin are believed to be dominated by the junctionbetween endothelial cells. In this section, the molecular structure and architectureof these junctions are described. Michel & Curry (33) provide a more extensivereview of the developments leading up to our current understanding of the structureand function of the interendothelial junction and may be consulted for additionaldetails.

MOLECULAR STRUCTURE OF THE INTERCELLULAR JUNCTION Figure 3 is a sche-matic diagram of the intercellular junction showing details of the molecular struc-ture of the tight junction and the adherens junction. Several protein componentsof the tight junction have been identified in recent years, and although the list isalmost certainly incomplete, the structure and organization of the tight junction isbecoming clearer (45, 46). In transmission electron micrographs, the tight junctionappears as a series of very close points of cell-to-cell contact, sometimes referred toas “kisses,” that circumscribe the cell. The first transmembrane protein identified inthe tight junction was the 65-kDa protein occludin (47), which is believed to play akey role in providing the tight junction seal. The protein has two extracellular loopsthat interact with the extracellular domain of molecules anchored to the apposingcell membrane to contribute to the tight junction seal. The cytoplasmic domain ofoccludin binds ZO-1, a cytoplasmic plaque protein that plays an important role inorganizing the paracellular seal. ZO-2 is another well-characterized protein in thecytoplasmic plaque that links the tight junction to cytoskeletal filaments includingactin. In 1998, the first members of the claudin family of tight junction proteinswere identified (48). These 23-kDa integral membrane proteins, with four trans-membrane domains, bear no sequence similarity to occludin, but are incorporatedinto the tight junction strand.

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Figure 3 The arrangement of the tight junction (zonula occludens) and the adherensjunction. The integral membrane proteins occludin and claudin, which form the tightjunction, are displayed along with peripheral membrane proteins associated with thetight junction, such as ZO-1 and ZO-2. The adherens junction involves adhesion of thetransmembrane protein VE-cadherin. Cadherins link to the cytoskeleton via cateninsand plakoglobin. (Adapted from Reference 31).

The adherens junction is a cell-cell adhesive junction that binds cells togetherand connects the cytoskeleton (actin) to the plasma membrane (49). Adherensjunctions may be considered epicenters for signal reception, transduction, and re-sponse to local patterning cues. The adhesive components of adherens junctionsare formed by type I cadherins (calcium-dependent adhesive proteins) that belongto the larger cadherin superfamily. Type I cadherins, of which VE-cadherin is theprototype, consist of five tandem extracellular 110–amino acid repeats, a trans-membrane domain, and a highly conserved cytoplasmic region. Cadherins mediatehighly specific homophilic adhesion with cadherins from apposing cells.

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Although adhesive associations of the ectodomain occur spontaneously, thecytoplasmic domain is required to support sustained adhesion. The cytoplasmicdomain governs the clustering of cadherins into a cooperative zipperlike arrange-ment and the tethering of cadherins to the cytoskeleton. The cytoplasmic domainof VE-cadherin forms a stable complex with major proteins: the catenins (al-pha, beta, gamma) and plakoglobin. The cadherin-catenin complexes are bound toalpha-actinin, vinculin, radixin, and the actin cytoskeleton.

GLYCOCALYX The plasma membrane of endothelial cells is covered by a thinlayer of macromolecules commonly referred to as the glycocalyx. This surfacelayer is also believed to fill the entrance to the intercellular junction as suggestedin Figure 3. The major molecular components of the glycocalyx are carbohydratesand glycoproteins, such as glycosaminoglycans (GAG), proteoglycans (PG), andglycolipids (50). A significant component of the proteoglycans of the endothelialglycocalyx are glucidic residues, as evidenced by the binding of lectins to theendothelium (51). The two most prevalent GAG carriers on the endothelial cellsurface are syndecans and glypicans. Syndecans, the most common GAG carrier,contain a transmembrane core protein that is directly attached to the membranephospolipid (52). On the other hand, glypicans are attached to the membrane viaa glycosyl phosphatidyl inositol linkage. Syndecan-1, the most prevalent of foursyndecan families on the vascular endothelium, has multiple forms based on thetypes of heparan sulfate GAG attached to the extracellular domain, but can alsocarry chondroitin sulfate (53, 54). The length of heparan sulfate is variable andtypically depends on the abundance of N-acetylglucosamine residues integrated inthe chain (55). There have been attempts to define the location of heparan sulfateand chondroitan sulfate GAGs along the core protein, but the location of eitherchain is not highly predictable (56).

The thickness of the glycocalyx layer has been controversial. As reviewed byPries et al. (57), several electron microscopy studies that are subject to artifactsindicate a thickness of less than 100 nm. Indirect measurements in capillaries basedon flow resistance measurements after heparinase treatment, plasma labeling, andhematocrit measurements indicate surface layer thicknesses of 0.5µm–1.1µm.

CONCEPTUAL MODEL OF THE INTERCELLULAR JUNCTION Figure 4 (adapted fromReference 58) provides a conceptual view of the interendothelial junction that cap-tures the major features and translates the molecular descriptions of the precedingsections into a more quantitative form suitable for mathematical modeling of trans-port rates. In this three-dimensional view, the upper and lower planes separatedby a distance 2B represent the surfaces of two adjacent endothelial cells and theintervening structures constitute the intercellular junction or “cleft.”

The lumen side of the channel is filled with fiber matrix (bars perpendicular tothe channel walls) representing the glycocalyx that extends into the channel butnot all the way to the “tight junction.” The fibers have a radius of 0.6 nm andare spaced 7-nm apart (close to the size of albumin) in a regular array. The tight

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Figure 4 Three-dimensional sketch of a single periodic unit of width 2D, showingcentral orifice of height 2B, and narrow slit of height 2bs in the junction strand. Thebreak in the junction strand has width 2d. (Adapted from Reference 58.)

junction is represented by the parallel walls in the middle of the channel that areseparated by an open slit of width 2bs. The slit in the tight junction that allowspassage of smaller molecules can be replaced by a set of small pores that mightmore realistically depict small channels in the tight junction strand composed ofpairs of proteins or oligomers of proteins (59). The model also incorporates breaksin the tight junction of width 2d, which repeat over a distance 2D as the tight junc-tion forms a belt around the endothelial cell. The model treats the tight junctionas a single belt with breaks, whereas freeze fracture electron microscopy studiesreveal several discontinuous belts in close proximity (33). Typical values for theparameters in this model have been developed based on data from frog mesen-teric capillaries (39): 2B= 20 nm, 2bs= 1.5 nm, 2d= 150 nm, 2D= 4300 nm.The fiber matrix in the entrance region extends 150 nm, and the distance from theend of the fiber matrix to the tight junction is at least 25 nm. The actual thicknessof the tight junction is of the order 10 nm, but is neglected in the mathematicaltreatment. The channel beyond the tight junction toward the tissue side may bethought of as representing the “adherens junction,” with protein structures that areimportant in maintaining the spacing between cells (2B), but offering little resis-tance to transport. Clearly, there are many parameters in this geometric model ofthe interendothelial junction that would be expected to vary with vessel type andspecies.

The model predicts that most of the water flow (volume flux) and larger so-lute flux (up to the size of albumin) pass through the breaks in the tight junction(2d× 2B) and not the high resistance slits in the tight junction (2bs). The glycoca-lyx serves as the primary molecular sieve. It offers little resistance to small solutes(<1-nm radius), but as the solute size increases to approach the fiber spacing of7 nm, the matrix resistance increases to become the major resistance to largersolutes the size of albumin. Whereas the tight junction provides a very high resis-tance to water flow, in the region of a break in the tight junction, the glycocalyxaccounts for a substantial resistance to water flow (up to 80%). The model alsodemonstrates that the oncotic gradient across the entire capillary wall (πs–πw)

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is not the determinant of water flow as suggested by Equation 22 and Starling’slaw. Rather, it is the oncotic gradient (albumin concentration gradient) across thesurface glycocalyx. The albumin concentration just beyond the glycocalyx layeris largely uncoupled from the concentration on the tissue side by convective flowthrough the breaks in the tight junction that prevents albumin back diffusion towardthe glycocalyx layer.

Leaky Junctions

Intercellular junctions, as described in the preceding section, would not normallyallow significant passage of LDL (diameter∼23 nm) even through breaks in thetight junction because the wide part of the cleft is expected to be on the order ofthe LDL dimensions. Thus, normal intercellular junctions would not constitute a“large pore.” Weinbaum et al. (60), building on earlier experimental observationsby Gerrity et al. (61), proposed that the large pore was an infrequent, transientlyleaky interendothelial cleft associated with a tiny fraction of cells (one cell in 103–104) that were in the process of cell turnover; i.e., either cell division or cell death.These cells had leaky junctions because they were either dying and being sloughedoff by healthy neighboring cells or had poorly formed junctions because they werein the process of dividing. Chien and coworkers, in a series of studies in the rat aorta(62) reported that approximately one cell in 3000 had a leaky junction and thisleakage would last on average about one hour before a well-formed new junctionwas established. In the rat aorta, cells in mitosis accounted for about 26% of allleakage sites, and dead or dying cells for 37% of leakage sites. The dimensions ofthe leaky junctions were estimated from HRP staining under electron microscopyto have a minimum width of 80 nm and a maximum width of 1330 nm for mitoticcells, and a minimum width of 15 nm and a maximum width of 1000 nm arounddying or dead cells (62).

In the rabbit aorta, however, mitotic cells accounted for only about 10% ofenhanced permeability sites, and the distribution of replicating endothelium didnot correlate with the distribution of sites of elevated LDL permeability or earlylesions (63). In the rabbit aorta, 31% of the sites of elevated LDL permeabilitywere associated with subendothelial white cells, suggesting a possible associationof these cells with leaky junctions. It appears, however, that these studies in therabbit aorta did not assess the contribution of dying cells to enhanced permeability.

Another possible mechanism for the generation of a transient large pore is thetransient disruption of endothelial cell plasma membranes (64). Cells that havehad their plasma membrane disrupted (wounded cells) but that are not dead arecapable of resealing their membranes and continuing vital function. Such woundedendothelial cells have been observed in the aortas of rats and varied significantlybetween 1.4% and 17.9% of the total aortic endothelial cell population. Woundedcells were heterogeneously distributed, being found in distinct clusters often inthe shape of streaks aligned with the axis of the vessel or in the shape of partialor complete rims surrounding bifurcation openings, such as the ostia of the inter-costal arteries. In fact, 80% of mitotic cells were identified as wounded. Although

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the relationship of these wounded cells to sites of enhanced permeability has notbeen studied directly, the pattern of cell wounding in the aorta bears a superficialresemblance to the pattern of enhanced permeability (e.g., concentrated aroundostia). The association with mitotic cells also suggests an association with en-hanced permeability. The fraction of wounded cells (6.5% on average in the aorta),however, is remarkably high—orders of magnitude higher than the fraction of cellsin mitosis or apoptosis. So it is not at all clear what fraction of wounded cells mightbe leaky and whether or not they would leak through their intercellular junctionsor across their abluminal membranes.

Although tight junctions are generally regarded as forming nearly continuousbelts around endothelial cells, electron microscopic freeze-fracture studies showthat tight junctions are discontinuous at tricellular corners where the borders ofthree cells converge. It has been hypothesized that tricellular corners are potentialsites for the transient opening and closing of the paracellular pathway and possibleavenues through which white cells migrate during inflammatory reactions (65). Astrong association between migrating neutrophils and tricellular corners has beendemonstrated in some studies, suggesting that these sites may also constitute a largepore pathway for macromolecules. Gaps ranging in size from 0.25 to 2µm havebeen observed in human umbilical vein endothelial cell (HUVEC) monolayers invitro. Thus, tricellular corners could also contribute to a large pore system.

Vesicles

Clathrin-coated vesicles are well-characterized endocytic organelles that providean efficient pathway for taking up specific macromolecules from the extracellularfluid, a process known as receptor-mediated endocytosis (66). The macromoleculesbind to complementary cell-surface receptors, accumulate in coated pits, and enterthe cell as receptor-macromolecule complexes in endocytotic vesicles. Becauseextracellular fluid is trapped in coated pits as they invaginate to form coated vesi-cles, substances dissolved in the extracellular fluid are also internalized by theprocess of fluid-phase endocytosis. The uptake of cholesterol bound to LDL intoendothelial cells occurs via the binding of LDL molecules to LDL receptors inclathrin-coated pits. After shedding their clathrin coats, the endocytic vesicles de-liver their contents to endosomes and the receptors are returned to the plasmamembrane. Because these vesicles can transport LDL into the cell, they couldconstitute a “large pore” system if they were likely to deposit their contents onthe abluminal side of the cells. This seems unlikely to be the case, at least forLDL, because a number of studies have shown that the endothelial permeabilityto unmodified LDL is the same as to modified forms of LDL that do not bind tothe LDL receptor (31).

Capillary endothelium is rich in homogeneously sized (60–80 nm) membrane-bound vesicles, which have been termed plasma-lemmal vesicles or caveolae (67).Most capillary caveolae are individual structures that invaginate from either theluminal or abluminal plasma membrane. Sometimes caveolae form aggregates oftwo or three interconnected vesicles or appear as free structures in the endothelial

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cell cytoplasm without connection to the plasma membrane. Caveolae lack a visiblecytoplasmic coat by electron microscopy, but a 21-kD integral membrane protein,caveolin-1, is associated with their cytoplasmic face. Caveolae may be responsiblefor transcytosis, the process by which plasma proteins are transported across theendothelium. Transcytosis is an active process in which caveolae that open to thevascular lumen take on plasma proteins as cargo then separate from the plasmamembrane, pass across the endothelial cytoplasm, and fuse with the abluminalmembrane where they discharge their contents. Persuasive evidence for the in-volvement of these vesicles in macromolecular transport comes from studies usingthe cholesterol scavenger, filipin, to inhibit caveolae. Schnitzer et al. (68) showedthat not only did filipin remove caveolae from cultured pulmonary microvascularendothelial cells, but it also inhibited transport of albumin across monolayers ofthese cells. Other studies with N-ethylmaleimide (NEM), which inhibits the fusionof vesicles, have demonstrated inhibition of albumin transport (69). Whereas LDLbinds to a surface receptor in clathrin-coated pits, albumin binds to the surface ofendothelium through specific albumin-binding proteins (gp60, gp30, and gp18),gp60 (albondin) being associated with transcytosis via noncoated plasma-lemmalvesicles (68). Albondin is well expressed in microvasclar beds, but its abundancein arterial endothelium has not been described.

Fluid Mechanical Effects on Endothelial Transport

Intercellular junctions are the principal pathway for the transport of water andhydrophilic solutes below the size of albumin. Albumin itself likely traverses theendothelium through a variety of pathways, including intercellular junctions, leakyjunctions, and vesicles. LDL and high-molecular-weight materials have limited ac-cess to the normal intercellular junction and must utilize leaky junctions or vesiclesto cross the endothelium. Each of these transport pathways can be influenced byfluid mechanical forces, particularly shear stress, acting on endothelial cells. In thissection, the overall effects of fluid mechanical forces on endothelial transport ratesare reviewed first, and then the specific influences of fluid mechanics on individualtransport pathways are considered.

The first unambiguous demonstration of a direct effect of fluid shear stress onendothelial transport was reported by Jo et al. (70) using bovine aortic endothelialcell (BAEC) monolayers cultured on a porous substrate mounted on the wall of aparallel plate flow chamber. This study demonstrated a tenfold increase in albumindiffusive permeability (P0) within 1 h after the onset of 10 dyne/cm2 steady shearstress. The permeability returned to preshear baseline levels within 2 h after theremoval of shear stress. Sill et al. (25) demonstrated a direct effect of shear stress onthe hydraulic conductivity (Lp) of BAEC monolayers, demonstrating a significantincrease in Lp after one hour of exposure to 20 dyne/cm2 steady shear stress.

More recent studies using the same BAEC model have shown that the Lp re-sponse is mediated by nitric oxide (NO) production in response to steady shearstress (26), whereas the albumin P0 response to steady shear stress is independent

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of NO (71). These findings suggest that albumin has access to different physicalpathways across the endothelium than water and that these pathways are controlledby distinct biochemical signaling mechanisms in BAECs. It must be remembered,however, that P0, the diffusive permeability, is measured in the absence of waterflux. Whether albumin transport coupled to water flux is enhanced by shear-inducedincreases in Lp has not been determined. It is known from static experiments (noshear stress) in BAECs, in which water and albumin flux were measured simul-taneously, that albumin Pe increases as Jv increases, but with a negative reflectioncoefficient (s), suggesting that water and albumin do completely share a commonpathway (42).

McIntire et al. (72) exposed bovine brain microvascular endothelial cell mono-layers grown on porous polycarbonate filters to steady shear stress of 1 or10 dyne/cm2 for up to 72 h and monitored the P0 of dextrans up to a molecu-lar weight of 2 million. Maximal increases in P0, as high as 76-fold for the largestdextran, were observed between 10 and 30 h with a return nearly to baseline after48 h.

The most recent study of BAEC monolayer transport properties in response toshear stress showed that when BAECs were exposed to steady shear stress of 20dyne/cm2 or oscillatory shear stress of 10± 10 dyne/cm2, they displayed a similarLp response: 3- to 3.5-fold increase in Lp after 3 h of exposure. But when theshear stress amplitude was increased so that a reversing oscillatory shear stressof 10± 15 dyne/cm2 was imposed, there was no increase in Lp above baselineduring 3 h of exposure (73). This surprising behavior seemed to be mediated bya dramatic upregulation of NO production under the reversing oscillatory shearconditions that suppressed the Lp increase. It therefore appears that for BAECmonolayers there is a biphasic response of Lp to NO: At low levels, increases inNO induce Lp increases, whereas at high levels of NO, increases in NO suppress Lp.Such biphasic behavior may help to explain why seemingly opposite responses ofvascular transport properties to changes in shear stress have been observed in someanimal studies. For example, Kurose et al. (74) and Baldwin et al. (75) showedthat when NO production was suppressed by blocking NOS, venules of the ratmesentery became more leaky to albumin.

It is also interesting to note that in vitro studies of transport using cell typesother than BAECs have shown diverse responses. Lakshminarayanan et al. (76),using a bovine retinal microvascular endothelial cell (BREC) model, showed adramatic increase in Lp in response to 20 dyne/cm2 steady shear stress that couldbe completely blocked by a NOS inhibitor—similar to the BAEC response. On theother hand, the same experiments with a HUVEC model showed a small reductionin Lp in response to the same shear stress stimulus (77), even though it is wellknown that HUVECs produce abundant NO in response to steady shear stress(78). This diversity of responses to shear stress was paralleled by the response ofthese same in vitro models to vascular endothelial growth factor (VEGF). Changet al. (79) observed that Lp and albumin P0 were increased significantly after 3h of exposure to 100 ng/ml VEGF in BAEC and BREC monolayers, but were

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unaffected by the same stimulus in HUVEC monolayers. It is therefore clear, andnot surprising, that the transport properties of endothelial monolayers depend onthe cell source.

Since 1991, there have been a number of additional reports of flow- or shear-dependent endothelial transport properties based on studies using intact vesselsand in vivo preparations. Lever et al. (24) observed that the Lp of rabbit carotidarteries in an ex vivo flow loop increased by 30% after 20 min of exposure to astep change in shear stress of about 1 dyne/cm2. Although this relative level ofincrease in Lp in response to shear stress is much less than described above forBAEC monolayers in vitro, it must be remembered that the medial layer of thearterial wall, which is not present in the in vitro models, is believed to contributeabout 50% of the overall resistance to volume flux in an artery (20).

Although it had been hypothesized earlier (80), only recently have several stud-ies reported an effect of flow on capillary permeability. Shibata & Kamiya (81)measured local capillary permeability of Cr-EDTA (MW 341) in the rabbit tenuis-simus muscle at various capillary blood flow levels using an indirect tissue clear-ance method. They observed a significant positive correlation between capillary Pe

and the mean capillary red cell velocity (a tripling of Pe as red velocity varied from0.5 to 2.5 mm/s). Yuan et al. (29) measured Pe of albumin in isolated cannulatedcoronary venules at various intraluminal perfusion velocities and observed a 47%increase in Pe when velocity increased from 7 to 13 mm/s. Caldwell et al. (82)determined the uptake of the fatty acid iodophenylpentadecanoic acid (IPPA) in thecoronary microcirculation of dogs exercising on a treadmill. The data could bestbe fit by a model in which the capillary Pe was taken to be a positive linear func-tion of the local flow rate. Kajimura & Michel (83) observed a positive correlationbetween flow velocity and potassium ion permeability in single perfused venulesof rats that was mediated by nitric oxide. Williams (27) measured Lp after stepchanges in shear stress in arterioles, capillaries, and venules of the frog mesenteryusing the modified Landis technique. The response of vessels was graded acrossthe capillary bed, with arteriolar capillaries demonstrating no response, true cap-illaries a moderate response, and venular capillaries a strong response (fivefoldincrease) to a step change in shear stress. This diversity of responses is reminis-cent of the differences between BAECs/BRECs and HUVECs observed in vitroas described above. It will be important in the future to determine the intracellularsignaling mechanisms and molecular components of the intercellular junction thatcontrol these diverse responses.

The studies described above showing shear stress alterations of Lp and Pe werenot designed to determine the specific transport pathway being affected by shearstress. However, there have been a number of studies specifically addressing theeffects of shear stress on tight junctions, adherens junctions, vesicles, and the leakyjunction–related processes of cell turnover and cell death. Most of these studies,described below, did not determine transcellular transport rates simultaneously, andso a direct association between altered transport pathways and altered transportrates can only be inferred.

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SHEAR STRESS EFFECTS ON TIGHT JUNCTIONS DeMaio et al. (84) determined theeffect of steady shear stress on the occludin and ZO-1 content of BAEC monolay-ers over a 3-h period. Immunofluorescence staining showed that these two proteinswere predominantly localized to the cell boundaries as expected for tight junctionproteins. Three hours of exposure to 10 or 20 dyne/cm2 shear stress had no ef-fect on ZO-1 content, but induced a 44% or 50% reduction in occludin content.Parallel transport experiments showed that 20 dyne/cm2 shear stress induced a4.7-fold increase in Lp after 3 h. Occludin expression and Lp were dependent ontyrosine kinase activity because erbstatin A attenuated both the shear-induced de-crease in occludin content and the increase in Lp. Shear stress increased occludinphosphorylation after only 5 min of exposure, and continued to induce phospho-rylation out to 3 h. The shear-induced increase in occludin phosphorylation wasattenuated with dibutyrl (DB) cAMP, a reagent that has been shown to reverse theshear-induced increase in Lp (26). It appears that alterations in occludin contentand phosphorylation are mechanisms by which shear stress alters BAEC Lp. Thephosphorylation event occurs earliest and is associated with alterations in Lp beforethere is a change in occludin content, suggesting that the phosphorylation event iscentral to the transport response.

In a similar study, it was demonstrated that VEGF increases occludin phos-phorylation in BREC monolayers within 15 min (85) and increases Lp after30 min (86). In a related transport study, DeMaio et al. (87) measured BREC Lp

and Peof 70-kDa dextran simultaneously before and after administration of VEGF.VEGF induced a 3.5-fold increase in both water and solute flux after 120 min,but the reflection coefficient (σ , Equation 22) remained constant (about 0.7) whilethe transport fluxes were increasing. Taken together, these studies of the effect ofVEGF on BREC transport suggest that the tight junction disassembles in responseto VEGF by increasing either the number of breaks or the length of breaks in thetight junction (recall Figure 4) as occludin is phophorylated. When this happens,there is increased water flux and associated solute flux through the breaks, but theglycocalyx, which serves as the solute filter and determines the reflection coeffi-cient, remains unaffected. Considering the strong parallels between shear stressand VEGF in altering tight junction proteins and transport, it is plausible that shearstress alters the tight junction in a similar manner.

SHEAR EFFECTS ON ADHERENS JUNCTIONS Schnittler et al. (88) exposed HUVECmonolayers to 20 dyne/cm2 steady shear stress and examined the junctional inten-sity of VE-cadherin, plakoglobin and platelet endothelial cell adhesion molecule1 (PECAM 1), which is a Ca2+-independent transmembrane adhesion molecule.Depletion of extracellular Ca2+ caused the disappearance of both VE-cadherin andplakoglobin from junctions, but did not affect the distribution of PECAM-1. Cellsstayed fully attached in low-Ca2+media, but began to dissociate from neighboringcells after 15 min of 20 dyne/cm2 steady shear stress. Stability to shear stress wasrestored with addition of Ca2+ in cells with normal plakoglobin content, but notin plakoglobin-deficient cells. This study shows that maintenance of intercellular

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adhesion under shear stress requires the junctional location of cadherins associatedwith plakoglobin. Beta-catenin cannot functionally compensate for the junctionalloss of plakoglobin.

It is well known that endothelial monolayers maintained in static culture as-sume a cuboidal, cobblestone morphology, whereas they elongate and align in thedirection of flow over a 24–48-h period when exposed to moderate or high steadyshear stress (89, 90). During this morphological remodeling period, it is expectedthat the intercellular junctions remodel as well. To investigate this, Noria et al.(91) exposed confluent monolayers of porcine aortic endothelial cells (PAECs)to 15 dyne/cm2 steady shear stress for 0, 8.5, 24, and 48 h and performed im-munostaining and Western blotting for VE-cadherin, alpha and beta catenin, andplakoglobin. Under static conditions, staining for all proteins was intense and pe-ripheral, forming a nearly continuous band around the cells at cell-cell junctions.After 8.5 h of shear stress, staining was punctate for all proteins, suggestive ofa partial disassembly of the adherens junction. By 48 h, when cell shape changewas completed, intense staining was recovered for all proteins, suggesting that theadherens junctions had been reassembled. Western blot analysis indicated that pro-tein levels of VE-cadherin, alpha catenin, and plakoglobin decreased, whereas betacatenin increased after 8.5 h of shear. At 48 h, all protein levels were upregulatedexcept plakoglobin, which remained below control levels.

Because intercellular adhesion at adherens junctions is thought to be a prereq-uisite for the assembly of other junctional complexes, including the tight junction(92), it is likely that tight junctions break down or are pulled apart as the adherensjunction disassembles and then reform as the adherens junction reassembles. Thein vitro transport study of McIntire et al. (72) that showed maximal increases inendothelial transport after 10–30 h of steady shear stress exposure with a return tobaseline values by 48 h is consistent with a transient breakdown and reassemblyof intercellular junctions. This is also consistent with a hypothesis of Friedman &Fry (93) suggesting that an adaptive response of the endothelium to changes inhemodynamics (shear stress) leads to a transient increase in uptake of atherogenicmolecules during the period of adaptation. But it should be remembered that thetight junction can be modified very quickly after a change in shear stress. The studyby DeMaio et al. (84), described previously, showed occludin phosphorylation asearly as 5 min after exposure to shear stress, suggesting that tight junction remod-eling may be initiated before the adherens junction is altered. Further studies arerequired to assess the dynamics of adherens junction remodeling at short times.

The preceding discussion has focused on the response of the intercellular junc-tion to shearing forces on the apical surface of the endothelial cell. The intercellularjunctions are also exposed directly to fluid shear stresses on their apposing surfacesthat are induced by transmural flow (Jv) passing through the intercellular conduit.Tarbell et al. (94), using the dimensions of the adherens junction and typical val-ues for Jv, have estimated these shear stresses to be of the order 25 dyne/cm2,comparable to the shear stresses of blood flow on the apical surface. Changes intransmural pressure drive changes in this intercellular shear stress, not only be-cause transmural pressure affects transmural flow (Equation 22), but also because

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transmural flow affects Lp, as demonstrated with BAEC monolayers in vitro byTarbell et al. (94). In addition, transmural flow imposes shear stress on the basalsurface of endothelial cells as fluid moves laterally in the subendothelial intimabetween the basal endothelial surface and the internal elastic lamina (IEL) towarda fenestration in the IEL. Tada & Tarbell (95) estimated this shear stress to be ofthe order 10 dyne/cm2, again a level comparable to the shear stress of blood flowon the apical surface.

SHEAR STRESS EFFECTS ON LEAKY JUNCTIONS Leaky junctions have been associ-ated with cell proliferation or turnover (mitosis) and cell death (apoptosis) as de-scribed earlier. Both of these processes are affected by fluid shear stress. A numberof studies have demonstrated that increasing levels of steady shear stress reduce therate of endothelial cell proliferation (78, 96). Increased cell turnover in regions ofseparated or recirculating flow has been implicated in the process of early athero-genesis. The fluid mechanical mechanism driving this increase has been attributedto elevated spatial gradients of shear stress in the separation zone (e.g., Reference97) and to increased temporal gradients of shear stress in the same region (98).

Several studies have shown that a reduction in the rate of blood flow or shearstress induces an increase in the rate of endothelial cell apoptosis, whereas anincrease in shear stress exerts the opposite effect (99, 100). Studies in HUVECsindicate that the apoptosis inhibitory effect of shear stress is mediated by upregu-lation of NO synthesis and that this mechanism is impaired in aged cells that donot display the antiapoptotic effect of shear stress (101). NO contributes to protec-tion against endothelial cell death via S-nitrosylation of caspases. Freyberg et al.(102) have shown that irregular flow conditions, such as turbulence or unsteadylaminar vortex flow, increase apoptosis rates and that an autocrine loop involv-ing thrombospondin-1 and the alpha(v)beta(3) integrin/integrin-associated proteincomplex is the molecular coupling device between flow and apoptosis. Analysis ofhuman atherosclerotic plaques from carotid arteries shows a systematic preferen-tial occurrence of elevated apoptosis in the downstream regions of plaques, wherelow flow and low shear stress prevail in comparison to the upstream regions (103).

Taken together, these studies suggest that endothelial cell turnover rates andapoptosis rates, and by association the prevalence of leaky junctions, will be greaterin regions of low mean shear stress (near separation and reattachment points) wherespatial gradients and temporal oscillations of shear stress are colocalized (104).

These fluid mechanical features are often associated with the localization ofatherosclerosis. It should be noted that hypoxia induces endothelial cell apoptosisas well (105, 106), implying that fluid mechanical conditions that produce localhypoxia will induce leaky junctions. As described in an earlier section, hypoxia ismost likely to occur in regions of separated and secondary flow that are associatedwith atherosclerotic plaque formation.

SHEAR STRESS EFFECTS ON VESICLES Davies et al. (107) determined the effect ofshear stress on pinocytosis (fluid-phase endocytosis) in BAECs by monitoringHRP uptake into cells. They found that continuous exposure to steady shear stresses

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(1–15 dyne/cm2) in laminar flow stimulated time- and shear stress level–dependentincreases in pinocytotic rate that returned to control levels after several hours ofapplication. A sudden removal of steady shear stress also resulted in a transientincrease in pinocytotic rate. Oscillating laminar shear stress at 1 Hz did not in-fluence pinocytotic rates, but changes over longer cycle time (15 min) did elevatepinocytotic rates. Elevated steady shear stress has also been shown to increase LDLendocytosis via increased expression of cell surface lipoprotein receptors (108).Kudo et al. (109) showed that after 48 h of exposure to steady shear stress, porcineaortic endothelial cells displayed an increased intracellular uptake of albumin thatpeaked at 10 dyne/cm2 and then decreased with increasing shear stress out to 80dyne/cm2. None of these studies of vesicle rates measured the overall transportrates across the endothelium, and therefore, we can’t be sure that an increase inpinocytotic rate can be equated with an increase in transendothelial transport. Fur-ther studies are required to clarify this point. The study by Davies et al. (107) doessuggest that changes in shear stress might elevate vesicular transport transiently,and this would be consistent with the transient adaptation hypothesis of Fry &Friedman (93) mentioned previously.

PRESSURE EFFECTS ON THE ENDOTHELIUM AND THE INTIMA When transmuralpressure increases as a result of an increase in vascular pressure, there is an in-crease in transmural flow (Jv) associated with the increased driving force (Equation22), but several studies have demonstrated that the Lp for the entire arterial wallactually decreases with increasing pressure in the range 50–180 mmHg (20, 110).These same studies show that when the artery is denuded of endothelium, Lp in-creases (by about twofold), but is no longer pressure dependent. The Lp of thedenuded artery is dominated by the medial layer of the wall, and the observationof pressure-independent Lp is consistent with recent theoretical models of aorticmedia predicting that aortic volume and Lp are relatively insensitive to pressure(111). Huang et al. (112) proposed that the reduction in Lp of the intact artery withincreasing pressure is a result of compaction of the arterial intima, specifically theextracellular basement matrix layer between the endothelial cells and the inter-nal elastic lamina. Subsequent experiments in perfusion-fixed rat aortas providedelectron microscopic evidence that the intima was compressed from a thicknessof 0.62 to 0.12µm as the pressure was increased from 0 to 150 mmHg (113). Thedeformable intima would also be expected to influence macromolecular transport,as the properties of the intimal matrix change with pressure, but this aspect hasnot yet been investigated.

The direct effects of hydrostatic pressure changes on endothelial cell functionhave received very little attention in the literature, although Schwartz et al. (114)showed that a sustained hydrostatic pressure of only 4-cm H2O could stimulate aselective increase in the expression of integrin subunit alpha (v) within 4 h and in-creased cell proliferation within 24 h in HUVECs in vitro. Changes in pressure aremore often associated with the induced changes in strain (stretch) of the endothelialcell layer that is supported by the elastic medial layer of the artery. Stretch effects

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on endothelial cells have been investigated quite extensively in vitro using cellssupported on deformable substrates (e.g., References 115, 116). There have notbeen any reports of the effects of stretch on transendothelial transport, apparentlybecause deformable substrates used to support endothelial cells in stretch exper-iments are not porous enough to be used in transport experiments and poroussupports are not sufficiently compliant for stretch experiments. A highly porousand deformable material would be useful for studies of the effects of stretch ontransport.

MACROMOLECULAR TRANSPORTAND ATHEROSCLEROSIS

The accumulation of lipoproteins in the arterial intima is a hallmark of atheroscle-rosis (117, 118). LDL is the most abundant atherogenic lipoprotein in plasma andhigh plasma levels of LDL are causally related to the development of atheroscle-rosis (119). The flux of LDL into the arterial wall depends on the plasma concen-tration and the permeability (Pe; Equation 24), which is similar for the aorta ofhumans, monkeys, rabbits, and pigeons—lying in the range 1.1× 10−9–2.6× 10−8 cm/s. In experimental animals, the focal sites of predilection for eitherspontaneous or dietary-induced atherosclerosis can be detected before plaques be-come visible macroscopically. Such lesion-prone areas are delineated by their invivo uptake of the protein-binding azo dye, Evans Blue. The Evans Blue model hasbeen studied extensively and characterized both morphologically and biochemi-cally (120). The lesion-prone (blue) areas display increased endothelial perme-ability to and intimal accumulation of plasma proteins, including albumin (3.5-nmradius), fibrinogen (5.5-nm radius), and LDL (11-nm radius). Thus, a correlationbetween enhanced endothelial permeability to macromolecules and the localiza-tion atherosclerotic plaques is well established.

Focal Nature of Macromolecular Uptake

The focal nature of plaque formation is a striking feature of atheroscleosis (121),and studies in rabbits support the idea that LDL permeability is increased focally atsusceptible branch sites of the aorta compared to adjacent atherosclerosis resistantnonbranch sites (7, 122, 123). Using horseradish peroxidase (HRP), Stemermanet al. (124) detected foci in the normal rabbit aorta with a luminal surface area ofabout 0.1 mm2 that had an LDL permeability up to 47 times greater than surround-ing areas and yet were covered by an apparently intact endothelium. The numberof high permeability foci per unit area was ten times higher in the atherosclerosis-susceptible areas, such as the ostia of aortic branches, than in nearby resistantareas. Detailed analysis of the distribution of high LDL permeability sites aroundintercostal and coeliac arteries of the rabbit aorta showed that high permeabilitysites occurred most frequently at the distal and lateral edges of the orifices, whichare the most susceptible sites for atherosclerosis (123).

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NITRIC OXIDE INFLUENCES THE UPTAKE PATTERN The distal (downstream) pre-dominance of LDL uptake around intercostal ostia in the rabbit model describedabove has also been observed for albumin in young animals, but attention hasrecently been drawn to the fact that this distribution pattern is reversed to one ofupstream predominance in mature animals (125). These patterns of transport cor-relate with the distribution of lipid deposits in the human aortic wall. Lesions occurmost frequently downstream of intercostal ostia in fetuses, neonates, and infants(126), but upstream in adult vessels (127). Forster & Weinberg (125) shed lighton the mechanism of this distribution pattern by observing that treatment of aortaswith the NOS inhibitor L-NMMA for only 25 min could reverse the uptake patternin mature animals to the juvenile pattern (reverting from upstream to downstreampredominance of uptake). Because age, hypercholesterolemia, and inhibition ofNOS all induce the upstream pattern, Staughton et al. (128) have speculated thatthey all act via influences on NO synthesis.

FLUID MECHANICS INFLUENCE THE UPTAKE PATTERN In a subsequent study, aneffect of blood flow pattern (and indirectly, fluid shear stress pattern) on albuminuptake around rabbit intercostal ostia was demonstrated by comparing the uptakepattern around normal ostia with the pattern around others that had the intercostalartery occluded to prevent flow into the side branch (128). In mature animals, thenormal pattern of upstream predominance was converted to the immature down-stream pattern around ostia that had been occluded for only 35 min. Because thisaltered pattern required only 35 min of altered hemodynamics, it seems unlikelyto have been induced by significant remodeling of the endothelium. Because thealtered uptake patterns induced by inhibiting NO synthesis and manipulating hemo-dynamics were similar, they suggest that the hemodynamic effect was mediatedby altered NO release. This is consistent with several studies described previouslythat showed acute influences of altered fluid shear stress on NO release by theendothelium along with altered transport rates (73).

The fluid mechanics around the intercostal ostia are undoubtedly complex andhave not been described in detail. However, pulsatile flow around the aorto-coeliacjunction has been simulated, and the associated wall shear stress features have beencompared to LDL uptake patterns in the rabbit (129). The downstream dominantuptake pattern characteristic of young rabbits was associated with high mean wallshear stress without reversal of shear stress direction around the downstream flowdivider and low mean wall shear stress with reversal around the upstream region.This is the generic shear stress pattern one would expect to find around a bifurcation,as revealed in studies of other arterial bifurcations (e.g., References 130, 131).By occluding the intercostal artery, Staughton et al. (128) very likely reducedthe significant differences in the wall shear stress patterns between the upstreamand downstream sides of the junction. All of this suggests that differences inNO production rates between low mean (reversing) shear patterns and high mean(nonreversing) shear patterns may be important in explaining uptake patterns anddisease patterns around arterial bifurcations. Further studies are required to clarifythese issues.

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INFLUENCE OF THE MEDIA The fluid phase, endothelium, and intima have gen-erally been viewed as the most influential structures regulating uptake of macro-molecules by the artery wall, and they have been discussed in detail in this review.But, as suggested by Caro et al. (3), the contribution of the medial layer shouldnot be overlooked. The importance of the media was emphasized by Lever et al.(132), who showed that the pulmonary artery and the inferior vena cava, vesselsnot normally associated with atherosclerotic plaques, had much higher endothelialpermeability to albumin and fibrinogen than did the atherosclerosis-prone aorta,carotid artery, and renal artery. Based on these observations, they suggested thatthe accumulation of plasma components during the development of atheroscleroticlesions is not determined simply by the rate at which macromolecules enter thetissue. The degree of accumulation of material in the intima may also be influencedby hindrance to outward passage of the components through the media. In supportof this view, Lever & Jay (133) observed that the pulmonary arteries and veinshave a significantly higher distribution volume (porosity) for albumin than do thearteries that are susceptible to disease. This higher porosity of the media of thevena cava and pulmonary artery may permit the easy drainage of macromolecules,preventing their accumulation in the tissue and thereby contributing to the lowsusceptibility of these vessels to disease.

Drainage or clearance of macromolecules by transport across the wall will alsobe influenced by the transmural water flux (Jv) that can flush macromolecules out ofthe wall by convection. This will be an important mechanism for molecules havinga permeability coefficient (Pe—Equation 24) that is small relative to the volumeflux (Jv; Equation 22). This condition is certainly satisfied by molecules the size ofalbumin or larger because albumin Pe for arteries is of order 0.25–1.0× 10−7 cm/sand Jv for arteries is typically 1.0–5.0× 10−6 cm/s (31). The importance of thisflushing mechanism appears to have been demonstrated by Lever et al. (132)in experiments in which collars were placed around rabbit carotid arteries andmacromolecular tracer uptake was measured. Steady state levels of fibrinogenand LDL in the media of vessels with collars were 20 to 50 times higher thanin uncollared arteries. An appealing interpretation of these observations is thatthe collar greatly reduced Jv, and as a result, solute in the media beyond the highresistance intima was not flushed out. This is consistent with the discussion in Leveret al. (132) and a simple, one-dimensional, steady state, convective-diffusion modelof medial transport with a boundary condition at the endothlelial (intimal) surfacethat matches the solute flux across the endothelium (Equation 11) to the combinedconvective/diffusive flux in the media. This simple model shows that the steadystate medial uptake is inversely proportional to Jv.

MASS TRANSPORT MECHANISMS IN ATHEROGENESIS

The preceding sections have provided a review of the basic mechanisms of masstransport in arteries and the patterns of macromolecular uptake in relation toatherosclerosis. In this final section, a synthesis of these basic mass transport pro-cesses in relation to patterns of uptake is presented in the form of several plausible

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mechanisms of atherogenesis in which mass transport plays a central role. Al-though several of these mechanisms have been proposed in the literature previ-ously, they have not been described in the context of general arterial mass transportphenomena.

Fluid-Phase Controlled Hypoxia RegulatesEndothelial Permeability

As pointed out in the section on fluid-phase resistance to transport, high-molecular-weight species, such as LDL and albumin, are not limited by the fluid phase. Oxy-gen transport, however, may be fluid-phase limited in regions of low fluid-phasemass transfer rates (Sherwood number—Sh), such as the outer walls of bifur-cations and the inner walls of curved vessels where enhanced LDL uptake andatherosclerotic lesions localize. Hypoxia in such regions has been confirmed bydirect measurements in the carotid bifurcation (15), around vascular graft anasto-moses (134), and in other vessels (135).

Hypoxia in the arterial wall has for many years been implicated in the develop-ment of atherosclerosis (136). Local hypoxia can affect the uptake of LDL and othermacromolecules by the arterial wall through several mechanisms: (a) Hypoxia canbreak down the endothelial barrier and form interendothelial gaps leading to in-creased macromolecular transport (137–139). Hypoxia also induces endothelialcell apoptosis (105, 106, 140), which can increase LDL transport through leakyjunctions. (b) Hypoxia can upregulate VEGF release by vascular cells and affectendothelial permeability. Many cell lines produce increased amounts of VEGFwhen subjected to hypoxic conditions, as do normal tissues exposed to hypoxia,functional anemia, or localized ischemia (141). VEGF is a multifunctional cy-tokine that acts as an important regulator of angiogenesis and as a potent vascularpermeabilizing agent (142). VEGF is believed to play an important role in thehyperpermeability of microvessels in tumors, the leakage of proteins in diabeticretinopathy, and other vascular pathologies (79, 86, 143). Thus, a plausible sce-nario for the increase in lipid uptake is the following: Hypoxia upregulates theproduction of VEGF by cells within the vascular wall and VEGF in turn perme-abilizes the endothelium, allowing increased transport of lipid into the wall. Insupport of this view, several recent studies have shown that VEGF is enriched inhuman atherosclerotic lesions (144–146).

Leaky Junctions Control Endothelial Permeability

The uptake of LDL is controlled by the endothelium, not the fluid phase, and leakyjunctions, not tight junctions, would appear to constitute the principal pathwayfor transport of LDL across the endothelial layer. Leaky junctions are associatedwith cells in a state of turnover (mitosis) or death (apoptosis), and these processesare affected by local fluid mechanics. As described previously, elevated steadyshear stress tends to suppress both mitosis and apoptosis, whereas low shear stressand separated or disturbed flow increase these processes. Therefore, it is expected

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that leaky junctions would be more prevalent in regions of low shear stress andseparated flow than in regions of higher, unidirectional shear stress. These areprecisely the regions where atherosclerotic plaques tend to be localized at thecarotid bifurcation (131), in coronary arteries (147–149), and the aortic bifurcation(132, 150, 151).

Transient Intercellular Junction RemodelingControls Endothelial Permeability

Friedman & Fry (93) and Friedman et al. (152) suggested that macromolecularuptake by arteries is enhanced during transient adaptation of the endothelium tochanges in blood flow (shear stress). Such changes occur on a diurnal scheduleas well as in response to many physiological stimuli. As described in detail in anearlier section of this review, virtually all transport pathways respond to changesin shear stress: intercellular junctions (tight junctions and adherens junctions)remodel, vesicle formation rates are altered, and rates of mitosis and apoptosisthat modulate leaky junctions change. The timescales for these responses to shearstress vary from minutes for the phosphorylation of tight junction proteins (84)to days for the reestablishment of the adherens junction (91). Whether “changes”in the local hemodynamic environment are more influential than the “average”environment integrated over long periods of time in determining arterial walluptake is not known. Most mass transport mechanisms in atherosclerosis, includingthe other three mentioned in this section, are based on the notion that the “average”environment controls the long-term behavior, even though dynamic processes, suchas pulsatile flow (shear stress), contribute to the “average” environment.

Convective Clearance Alters Intimal/Medial Accumulation

For large macromolecules, such as LDL, that have a low endothelial permeability(Pe) relative to volume flux (Jv), an increase in Jv with fixed Pe will reduce the ac-cumulation of solute beyond the endothelial layer (intima/media) by convectivelyclearing (flushing) out the region beyond the high resistance endothelial barrier.If we assume that a macromolecule crosses the endothelium primarily throughleaky junctions as discussed above, and that volume flux (primarily water flux) iscontrolled principally by the intercellular junctions that have a much greater totalarea than the leaky junctions, then factors that affect Jv but not Pe can influencethe accumulation of macromolecules within the wall. For example, as discussedpreviously, this could explain why collars around arteries led to an increase inmacromolecule accumulation (132). This would be expected if the collar reducedJv but did not alter Pe greatly, as seems plausible.

There are other examples that might be explained by this mechanism. Staughtonet al. (128) observed that the albumin uptake pattern around intercostal ostiaof rabbits could be transformed from one of upstream predominance to one ofdownstream predominance after only 35 min of flow alteration induced by tyingoff the intercostal artery. Alteration of the flow (wall shear stress) pattern is not

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likely to have influenced endothelial permeability to albumin within 35 min if leakyjunctions (controlled by mitosis and apoptosis) provided the dominant transportpathway. On the other hand, it is known that an alteration in wall shear stresspattern can significantly affect hydraulic conductivity (Lp) within 30 min (73).

Most studies have shown that endothelial Lp, which is controlled by the intercel-lular junction, is higher when fluid shear stress is elevated. Therefore, it is expectedthat the convective clearance mechanism will contribute to lower macromolecularaccumulation in regions of high shear stress. Because macromolecular permeabil-ity, if controlled by leaky junctions, is expected to be lower when the fluid shearstress is elevated (discussed previously), convective clearance is a mechanism thatfurther reinforces low accumulation in high shear regions.

It is hoped that further study of these mass transport mechanisms of atherogene-sis will lead to new understanding of the overall atherosclerotic disease process andmay contribute to the development of therapeutic strategies based on regulation ofmass transport phenomena.

ACKNOWLEDGMENTS

This work was supported by NIH NHLBI Grants HL35549 and HL57093 andNASA Grant NAG3-2746.

The Annual Review of Biomedical Engineeringis online athttp://bioeng.annualreviews.org

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P1: FRK

June 3, 2003 14:52 Annual Reviews AR191-FM

Annual Review of Biomedical EngineeringVolume 5, 2003

CONTENTS

BIOMATERIALS FOR MEDIATION OF CHEMICAL AND BIOLOGICALWARFARE AGENTS, Alan J. Russell, Jason A. Berberich,Geraldine F. Drevon, and Richard R. Koepsel 1

STRUCTURAL, FUNCTIONAL, AND MOLECULAR MR IMAGING OF THEMICROVASCULATURE, Michal Neeman and Hagit Dafni 29

SELECTED METHODS FOR IMAGING ELASTIC PROPERTIES OFBIOLOGICAL TISSUES, James F. Greenleaf, Mostafa Fatemi,and Michael Insana 57

MASS TRANSPORT IN ARTERIES AND THE LOCALIZATION OFATHEROSCLEROSIS, John M. Tarbell 79

TEMPORAL DYNAMICS OF BRAIN ANATOMY, Arthur W. Togaand Paul M. Thompson 119

MODELING TOTAL HEART FUNCTION, Peter J. Hunter, Andrew J. Pullan,and Bruce H. Smaill 147

THE ENGINEERING OF GENE REGULATORY NETWORKS, Mads Kærn,William J. Blake, and J.J. Collins 179

COCHLEAR IMPLANTS: SOME LIKELY NEXT STEPS, Blake S. Wilson,Dewey T. Lawson, Joachim M. Muller, Richard S. Tyler, and Jan Kiefer 207

MULTIAXIAL MECHANICAL BEHAVIOR OF BIOLOGICAL MATERIALS,Michael S. Sacks and Wei Sun 251

ENGINEERED NANOMATERIALS FOR BIOPHOTONICS APPLICATIONS:IMPROVING SENSING, IMAGING, AND THERAPEUTICS, Jennifer L. Westand Naomi J. Halas 285

NEURAL TISSUE ENGINEERING: STRATEGIES FOR REPAIR ANDREGENERATION, Christine E. Schmidt and Jennie Baier Leach 293

METABOLIC ENGINEERING: ADVANCES IN THE MODELING ANDINTERVENTION IN HEALTH AND DISEASE, Martin L. Yarmushand Scott Banta 349

BIOMONITORING WITH WIRELESS COMMUNICATIONS,Thomas F. Budinger 383

BLOOD VESSEL CONSTITUTIVE MODELS—1995–2002, Raymond P. Vitoand Stacey A. Dixon 413

v

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P1: FRK

June 3, 2003 14:52 Annual Reviews AR191-FM

vi CONTENTS

THE TISSUE ENGINEERING PUZZLE: A MOLECULAR PERSPECTIVE,Viola Vogel and Gretchen Baneyx 441

TIME-REVERSAL ACOUSTICS IN BIOMEDICAL ENGINEERING,Mathias Fink, Gabriel Montaldo, and Mickael Tanter 465

INDEXESSubject Index 499Cumulative Index of Contributing Authors, Volumes 1–5 519Cumulative Index of Chapter Titles, Volumes 1–5 522

ERRATAAn online log of corrections to Annual Review of BiomedicalEngineering chapters (if any) may be found athttp://bioeng.annualreviews.org/

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john m. tarbell - semantic scholar · john m. tarbell the city college of new york/cuny, new york,...

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