Influence of hemodynamic forces on vascular endothelial function In vitro studies of shear stress and pinocytosis in bovine aortic cells

Influence of hemodynamic forces on vascular

endothelial function. In vitro studies of shear

stress and pinocytosis in bovine aortic cells.

P F Davies, … , E J Gordon, M A Gimbrone Jr

J Clin Invest.

1984;

73(4)

:1121-1129.

https://doi.org/10.1172/JCI111298

.

The relationships between fluid shear stress, a physiologically relevant mechanical force in

the circulatory system, and pinocytosis (fluid-phase endocytosis) were investigated in

cultured bovine aortic endothelial cells using a specially designed apparatus. Continuous

exposure to steady shear stresses (1-15 dyn/cm2) in laminar flow stimulated time- and

amplitude-dependent increases in pinocytotic rate which returned to control levels after

several hours. After 48 h continuous exposure to steady shear stress, removal to static

conditions also resulted in a transient increase in pinocytotic rate, suggesting that temporal

fluctuations in shear stress may influence endothelial cell function. Endothelial pinocytotic

rates remained constant during exposure to rapidly oscillating shear stress at near

physiological frequency (1 Hz) in laminar flow. In contrast, however, a sustained elevation of

pinocytotic rate occurred when cells were subjected to fluctuations in shear stress amplitude

(3-13 dyn/cm2) of longer cycle time (15 min), suggesting that changes in blood flow of

slower periodicity may influence pinocytotic vesicle formation. As determined by

[3H]thymidine autoradiography, neither steady nor oscillating shear stress stimulated the

proliferation of confluent endothelial cells. These observations indicate that: (a) alterations

in fluid shear stress can significantly influence the rate of formation of pinocytotic vesicles in

vascular endothelial cells, (b) this process is force- and time-dependent and shows

accommodation, (c) certain patterns of fluctuation in shear stress result in sustained

elevation […]

Research Article

Influence

of Hemodynamic Forces

on

Vascular Endothelial

Function

In Vitro Studies of Shear Stress and

Pinocytosis

in Bovine Aortic Cells

Peter F. Davies, C. Forbes Dewey, Jr.,

Steven R. Bussolari, Ethel J. Gordon, andMichael A.Gimbrone, Jr.

Vascular Pathophysiology Laboratory, Departmentof Pathology,

Brigham andWomen's Hospital, Harvard MedicalSchool,

Boston, Massachusetts02115;FluidMechanics Laboratory, Departmentof MechanicalEngineering, MassachusettsInstitute

of Technology, Cambridge, Massachusetts02139

Abstract.

The relationships

between

fluid

shear

stress, a

physiologically

relevant

mechanical force

in the

circulatory

system,

and pinocytosis (fluid-phase

endo-cytosis)

were

investigated in

cultured bovine aortic

en-dothelial

cells using

a

specially designed

apparatus. Con-tinuousexposureto

steady shear

stresses( 1-15

dyn/cm2)

in

laminar flow

stimulated

time-

and amplitude-depen-dent

increases

in

pinocytotic

rate

which

returnedto

con-trol

levels after several

hours.

After

48 h

continuous

ex-posureto

steady shear

stress,

removal

to

static conditions

also

resulted

in a

transient increase in pinocytotic

rate,

suggesting that temporal fluctuations in

shearstressmay

influence endothelial

cell

function. Endothelial

pinocy-toticrates

remained

constant

during

exposureto

rapidly

oscillating shear

stress atnear

physiological frequency

(1

Hz)

in laminar flow. In contrast, however, a sustained

elevation of

pinocytotic

rate occurred when cells were

subjected

to fluctuationsinshearstress

amplitude

(3-13

dyn/cm2)

of

longer cycle

time

(15

min), suggesting

that

changes

inblood flowof slower

periodicity

mayinfluence

pinocytotic vesicle formation.

As determined

by

[3H]-thymidine autoradiography,

neither

steady

nor

oscillating

shearstressstimulatedthe

proliferation

of confluent

en-dothelial cells. These observations indicate that:

(a)

al-terations in fluid shear stress can

significantly

influence

the rate offormation of

pinocytotic

vesiclesin vascular

endothelial

cells, (b)

this process is force- and

time-de-Receivedfor publication19October 1983.

pendent and shows accommodation, (c) certain patterns

of

fluctuation in shear

stress result in

sustained

elevation

of

pinocytotic rate, and (d) shear stresses

can modulate endothelial

pinocytosis independent of

growth

stimula-tion.

These

findings

are relevant to (i)

transendothelial

transport and the

metabolism of

macromolecules

in

nor-mal

endothelium and (ii) the role of hemodynamic factors

in the localization of atherosclerotic lesions in vivo.

Introduction

Inthesystemic and pulmonary circulations, vascular endothelial cellscomprise the interface between flowing blood and vessel wall, and thus, are subjected to a wide variety of hemodynamic factors. A major component of these is wall shear stress, the tractiveforceproduced by flowing blood upon the endothelial cellsurface. Thisparticularhemodynamicfactorhas been im-plicated in the atherosclerotic process because a strong correlation

exists between the location ofdeveloping arterial lesions and regions where large variations in wall shear stress occur(1, 2). Endothelial desquamation isnotrequired toinitiatethe devel-opmentoffocal atheroscleroticlesions (3, 4).Itislikely, therefore,

thatmoresubtlechanges, whichcompromisenormal endothelial function (4, 5), are involved in early focal atherogenesis. A

prominent candidate for the inductionofendothelialdysfunction (6)ishemodynamic shearstress.

Thedevelopment of

specialized

in vitroexperimentalsystems (7) has been necessary to allow more precise studies ofthe relationships between shear forces andendothelialcellbiology.

Forthis purpose,we haverecently developed acone-plate ap-paratus in which cultured endothelial cellscan beexposed to

defined shear stresses comparable with those encountered in vivo for intervals varying from minutestodays(7-9).Significant

shear-induced alterations in endothelial cell

shape

and cyto-skeletal organization have been observed inthis

experimental

system (7, 10,

11).

Inthe studies reported here, the apparatus

J.Clin. Invest.

C TheAmericanSocietyforClinicalInvestigation,Inc.

0021-9738/84/04/1121/09 $1.00

has been used to investigate the influenceof shear stressesupon

a fundamental cellular process, pinocytosis (fluid-phase endo-cytosis), which is the uptake of extracellular fluid via plasma-lemmal vesicles.

Endothelial cellshavelarge numbers ofplasmalemmal

in-vaginations and vesicles, some of which participateinthe trans-port of fluid and macromoleculesacross theendothelialbarrier

(12, 13), while others deliver exogenous substances to the

ly-sosomal degradation system of the cell (14, 15). The initial

infolding of cell membrane to form an endocyticvesicle may be influenced by the distribution ofphysical forces along the

cellsurface; thus, fluid flow may modify the rate or nature of

vesicle formation.Theobservations reportedhere indicatethat the rateof pinocytosis in cultured endothelial cells is sensitive toappliedfluid shear stressesand that the cellsappeartoundergo

acomplex process of accommodationto the shear stress. These

invitro observationssuggest that hemodynamic factors such as

shear stress can significantly influence vascular endothelial function in vivo.

Methods

Shearstress. Thetheory anddesignoftheshearstressapparatus, which utilizesthegeometry ofacone-plate viscometer,has beenfullydescribed elsewhere (7, 8). Fluid shearstresswasproducedby rotation ofashallow

cone with respect to a stationary base plate that contained 12 glass coverslips of cultured endothelial monolayers.Theunit,contained within

aplexiglasschamber,wassterilized beforeusewithethyleneoxidegas.

Stable platetemperature(370+0.30C) and controlled chamber

atmo-sphere were provided by resistance heaters and humidified gas (5% CO2:95%air), respectively.

Theconeangleused inthe shearstressexperimentsdescribedhere was0.50.Rotational speedsup to160rpm wereused with tissue culture medium having fluid viscosity of 7.6X l0-3cm/sec. This combination ofviscosity,coneangle, and rotationalspeed producedshearstressesin the range of0-15 dyn/cm2. Forexperimentsexceeding several hours, freshculturemedium wascontinuously infused (3 ml/h) tomaintain

constantosmolarity and nutrient concentrations.Shearstress wasapplied

toendothelial monolayers in three different patterns: (a) Steady shear

stress wasappliedasaconstantforce of1,5, 8,or 15dyn/cm2throughout thetimeperiodsindicated in eachexperiment. (b)Periodicshearstress

involvedthe application ofshear stressin squarewavecycles of long duration (5or 15min)betweenalow(0, 1,or3dyn/cm2)andahigher (8 or 13 dyn/cm2)amplitude. For example, cells were exposed to 3

dyn/cm2

for 15 minbefore an abrupt increaseto 13 dyn/cm2for 15 min followed by another period of exposureat the loweramplitude,

andso on.(c) Oscillating shearstress was aspecialcaseofperiodicshear stress,beingarapid sinusoidal change ofshearstress at60cycles/min

(1 Hz)between 3 and 13 dyn/cm2(time average shear stress, 8dyn/

cm2). Thisconditionwas obtained by tiltingthecone withrespectto

theaxisof rotationtoproduceaslight wobble intheconerotation.The

frequency ofthese oscillations approximated thatofthe mammalian cardiaccycle.

Endothelialcells. Bovine aortic endothelialcells(BAEC)'wereisolated from calfaortausing techniques described fullyelsewhere(16),and were

1.Abbreviations usedinthis paper: BAEC, bovine aortic endothelial cells;HRP,horseradishperoxidase.

culturedin Dulbecco's Modified Eagles Mediumwith 10%calfserum (M.A.Bioproducts, Walkersville,MD) supplementedwith 2 mM

glu-tamineand 100 U eachof penicillin and streptomycin permilliliter. Cells ofa single strain (1I-BAEC) between passages 14 and 20 were used in thesestudies.Cellsuspensionswerereplicate-platedon 12-mm

diameterglasscoverslips(Bellco No. 1, 0. 15 mmthickness;Bellco Glass, Inc., Vineland,NJ)where they grew toconfluence under standardculture

conditions. Atconfluence, the cells weregrowth-inhibited asrevealed

by low

_{[3Hlthymidine}

labelling (<6%).12coverslip cultureswereplaced

in theapparatus foreachexperiment. Thebiological compatibility of the shearstress apparatus withBAEC has beenpreviously established

(7). For each groupoftestmonolayers subjectedtoshear stress, control monolayers fromthe same batchof cellsweremaintained under static conditions(0shear).

Pinocytosis

(fluid

endocytosis). Horseradish peroxidase(HRP,

iso-enzyme Type VI; Sigma Chemical Co., St. Louis, MO)was used to measure the rateof bulk fluid uptake into endothelialpinocytoticvesicles. CellularHRP wasdeterminedspectrophotometricallyby the procedure of Steinman andCohn(17) using a-dianisidine and hydrogenperoxide

assubstrate.Theprotocol for endothelialcells was aspreviously described

(15).

HRP was injected into the apparatus at a concentration of40 mg/ml togiveafinalconcentration of2mg/ml culture medium. This concentrationwasverified by removinganaliquot of the final media from both test and control systems forassay ofHRP activity at the beginning andendof incubation. Anysmall differencesin HRP

con-centrations (range 1.91-2.17mg/ml)werecorrected by normalizingthe rateofHRP uptaketo amedium concentration of2 mg/ml. This is

validbecause HRPis distributed uniformly inthe medium anddoes notadsorbto cells

(15,

17) or tosiliconized surfaces oftheapparatus

(P. F. Davies, unpublished observations). HRPuptakewasmeasured forup to 2 h.During this time cellularaccumulation isproportional

topinocyticrate;after about 3hof continuous HRPentryinto thecell,

theuptakecurvesbegintoplateau reflectingsignificant

lysosomal

deg-radation (17, 18).

Ultrastructural cytochemicallocalizationofHRP.After exposureto shear stress in thepresence of HRP, endothelialcells werewashed five times with Hanks' balanced salt solution (HBSS, M.A. Bioproducts)

containing 0.2% bovineserumalbumin, rinsed twice with HBSS, and

thenfixedwith3%glutaraldehyde in0.1Mcacodylate buffer with 0.05%

CaCl2.

Peroxidase activitywaslocalized inthecells byamodification of the

histochemical

technique of Graham and Karnovsky (19). The

cellswerepreincubated for30 minin 50mg/100mldiaminobenzidine

in 0.05M

Tris-HCl

atpH 7.6.H202wasthenaddedto the diamino-benzidine toafinal concentration of 0.01% for 30min. Afterrinsing with cacodylate buffer, cells were postfixed in 1% OSO4, dehydrated, andembedded in Epon 812directly in thepetri dish. After polymer-ization,the cells wereremountedon Eponblanks andcuttransversely

orenface. Thinsectionswereexamined inaPhilips 201 electron

mi-croscope(Philips Electronic Instruments,Inc., Mahwah, NJ).

[3H]

Thymidineautoradiographyofendothelial cells. After exposure

to shearorstatic conditions, coverslips of endothelialcells wereremoved

to apetri dishwheretheywereincubatedinculturemedium containing 0.3gCi

[3H]thymidine/ml.

After24 hofcontinuous incubationat37°C, cellswerewashed twice with HBSS, fixed in ethanol:acetic acid (2:1,

vol/vol)for 10 min, rinsed with deionizedwater, and air dried. Each

monolayerwasoverlayed withNTB2emulsion(Eastman Kodak Co., Rochester, NY)and exposedat

4°C

for 5d.Silver grains derived from radioactive nucleiweredevelopedon the filmin situand thecell nuclei

was counted directly by lightmicroscopyat a powerofX 100.Atotal of1,000 cells werecounted in fiveregions of eachcoverslip.

Results

Applicationofshearstresses tocultured endothelial monolayers.

The movementof a viscous fluid over an endothelial cell mono-layer results in a tractive horizontal force applied to the cell surface. The force per unit surface area is defined as shear stress. Bothin vivo and under the conditions of our experiments in vitro, the shear stresses acting upon theendothelium depend

only upon the fluid viscosity and the detailed distribution of flowvelocities near the cell surface (8), as shown schematically in Fig. 1. Thein vitro approach used here allows the prolonged application of varying amplitudes of shear stress to endothelial cellsmaintained in a defined chemical milieu. Previous

mor-phological studies performed in this system have shown that 24-48-h exposure of endothelial cell monolayers to a steady shear stressof 5-8 dyn/cm2 results in shape change (polygonal toellipsoidal) and alignment of the cells in the direction of the applied force (7). As demonstrated below, alterations in pino-cytosis occur earlier than shape change and cell alignment and

atshear stressamplitudes lower than those required for detectable morphological change.

Pinocytosis is initially time- and shear-dependent. When

confluentBAEC were exposed to steady shear stresses varying in amplitude between 0 and 15dyn/cm2, there was a time- and

force-dependentincrease of fluid phaseendocytosis,asmeasured byHRPuptake, during the first hours of exposure (Fig. 2 A). Shear stress at 1 dyn/cm2, an amplitude that fails to align en-dothelial cells invitro after prolonged application, resulted in

a small elevation ofHRPuptake by 2 h. Ahigher rate of

pi-SHE AR STRESS

TEST SPECIME

nocytosis was reachedduring1-h exposure to 15dyn/cm2 than during 2 h exposure to 8 dyn/cm2. A maximum increase of

four- to fivefold over static control cells was observed during the first hour of exposure to 15dyn/cm2, whereas the second

hourofexposure toHRPresultedinonlyasmallfurther increase.

To further investigate theperiod immediately followingtheinitial increase of pinocytosis,sequential2-htimeframes wereanalyzed during 8 h of steady shear of 8 dyn/cm2. As shownin Fig. 2

B, afteran initialdoubling of HRP uptake duringthe first 2h, therewas a markedinhibitionof HRPuptaketo46%(±3) that of static controls during the period between 2 and4hofshear

stress. Thiswasfollowedduring the subsequent4 hbya return

towards control levels (4-6 h, 127%+15; 6-8 h, 85%±7).

Pi-nocytosis rates measured 40 h later confirmed asustained return

to control levels(9 1%±+11). The shape of the

stimulation-in-hibition cycle reportedin Fig. 2 B bears a striking resemblance to the relaxation-oscillation cycle of a damped mechanical system.

Step-decreaseinthemagnitude ofshearalsoresults in tran-sient elevation ofpinocytosis. As noted inFig. 2B, after48 h of exposuretosteady shear of 8 dyn/cm2, endothelial pinocytotic rates returned to control levels. When, however, such endo-thelium was returned to static conditions (0 shear stress), there was also atransient rise in the rate of pinocytosis (Fig. 3). In

cells exposedtosteady shear stress for 48hbefore introduction ofHRPforafurther2 hat 8dyn/cm2 (groupD), pinocytotic rates were not significantly different (102%±8) from control levels (cells at 0 shear stress for 50 h, group A). Step-up from

0to 8 dyn/cm2 for 2 h (group B)or step-down from 8 to 0

dyn/cm2 for 2 h (group C), however, resulted in significantly elevated levels ofpinocytosis (190%±31 and 170%±23,

respec-tively).In asimilarexperiment,step-down from 8 dyn/cm2after

Figure 1. Diagram of the essentialcomponentsofthe shear stress apparatus. A

rotatingconeapplies fluid

shear stress toendothelial

cellmonolayers maintained

onglasscoverslips. The shear stressforce(T)

de-pendsupon thefluid vis-cosity(A)and thedetailed distribution of flow veloci-tiesnearthe cellsurfaceas

PINOCYTOSISRATE

In[fluid-mgcellprot.2h41 % Change

a STEP- UP

El

ci

OJ

"*8

1

- ^~~~~~~-l

0 48 50

HOURS A

HRP

STEP-DOWN

. ,__ _ --~~

~IN

01 I

10

0 0

I-0

0 30 60

Minutes

I.~~

it

0-2 2-1 4-6 6-8 48-50

Measurement interval afteronsetof shear stress I hoursl

Figure2.(A) Influenceofsteadyshearstressuponfluid-phase endo-cytosis inconfluentendothelial monolayersasmeasuredbyHRP

up-take. 5min aftertheimpositionofshearstress,HRPwasinjected

into theculturemedium(time 0). Coverslipswerewithdrawnat in-tervalsfor theassayof cellular HRP uptake. Controlcoverslips(0 dyn * cm-2)weremaintained inplastic petridishes. Eachcoverslip

contained -2 X i01endothelial cells. Eachpoint isthe_meanof four

measurements±SD. (B)Fluid-phaseendocytosis measuredatvarious

intervals duringexposureof confluent endotheliumtoshearstress at

8dyn *cm-2.Each pointisaseparateexperimentinwhich cellular

HRP uptakewasexpressed as apercentage(±SD)ofthatofa

matched control (0dyn cm-'). HRPwasinjectedattheappropriate time(0,2, 4, 6,and 48h)after theimpositionof shearstress.Each

experimentwasterminated2 hlater,thecoverslipsremoved and

as-sayed for cellular HRPuptake.

39.1 * 9.2

20.6± 1.9

21.1 7.2

35.2_±7.3 170%(±23)ofD

48 50

A

HRP

Figure 3. Effects of 48-h exposure and step-change of shearstress

upon endothelialpinocytosis. Endothelial monolayerswere main-tained for 48 h at 0 or 8 dyn * cm-2 (groups A and D). HRPwas in-jected into the culture medium and its cellularuptakewasmeasured

over a2-h period. Step changewasinduced by either increasing shear stress from 0 to 8 dyn * cm-2 (step-up; group B) orremoving endothe-lial monolayers from the apparatus (step-down; group C). HRP up-takewasthen measured for 2 h. . Significantly different from group A, P < 0.01. tNotsignificantly different from group A. § Signifi-cantly different from group D,P <0.025.

28 halso resulted in asignificantelevation of HRPuptaketo

227%±44 ofcontrol level. These experiments indicated that following accommodationtoeither steady shearstress orstatic conditions, pinocytotic rates were equal, i.e., independent of the absolute level of shearstress, and confirmed thatchanges in steady shear stress levels resulted in transiently increased pinocytotic rates.

Slowperiodic shearstressincreasespinocytoticrate. In at-tempts to magnify the effects ofstep changes in shear stress uponpinocytoticrate, confluent endothelial cells wereincubated for 2 h with HRP under conditions where the amplitude of shear stress wasperiodically changed. Inthe first series of

ex-periments, shearstress wasabruptly changedbetween 1 and 8 dyn/cm2 every 15 minfor2 h(averageshear stressamplitude,

4.5 dyn/cm2). This resulted in a 51% increase ofpinocytosis rate compared with stationary controls (datanotshown).When

the averageshearstressamplitudewasincreasedto 8 dyn/cm2 by alternating between 3 and 13 dyn/cm2 with a periodicity of 15

min,

pinocytotic rates increased to 264%±63 ofstatic

control levelsduringthe first 2 handdid notdecline significantly (207%±5 1) duringthe subsequent 6 h(Fig. 4). Whenthe

pe-riodicitywasreduced to 5 min, however,HRP uptake by the

endothelial cells did not increase but remained near control

levels(0-2 h, 93%+21;4-6h, 11 3%±23). It appears, therefore, that eitherastepchangeofsteady shear stress,orperiodic change

ofshear stress, was sufficienttoinvoke elevation of pinocytotic rates inconfluent endothelium. As the time between change of amplitudewasshortened, however, thestimulatoryeffect upon pinocytosis diminished even thoughthe meanshearstress

am-190*/6(| 31)of A 120

-C

.

0

-E .e

: ₈₀

In

V

20-0

2C

0-a_

-6

.tn

0

10

*0

'S

u

0l

In c

lL

102%(±8)of At

90 120

Amplitude Fluid PhaseEndocytosis

Pattern ofFluid ShearStress (dyn cm-2) (S of control ± SD) 0-2h 4-6h 15-17h

Steady, continuous 8 210±46* 116±17 91±13

Periodic, 15-min cycle _L 3-13 264±63* 207±51* ND

Periodic, 5-min cycle JJl 3-13 93±21 112±23 ND

Figure 4. Duration of effects of steady and periodic shearstresses

upon fluid phaseendocytosisincultured endothelium. During

peri-odic shear stress, cells were exposed to repeated step changes (5 or 15

min cycle) of shear stress. HRP was injected and its uptake was mea-sured during the period indicated. *, Significantlydifferent from

con-trol, P < 0.05. ND, notdetermined.

plitudewas at alevel (8 dyn/cm2) that,uponsteadyapplication,

wouldstimulate HRP uptake.

Oscillations of shearstress atphysiological frequencies (I

Hz) donotstimulate pinocytosis. By the useofawobblingcone,

shearstressesoscillatingbetween 3 and 13 dyn/cm2at afrequency

of 60 cycles/min (1 Hz)wereappliedtothe endothelial mono-layers. During a 2-h period of exposure (time average shear stressamplitude, 8 dyn/cm2), there wasno significant change

inpinocytoticrate asdeterminedby cellular HRP uptake(Fig. 5) compared with static controls (0 shear stress). When the

period of exposure tooscillating shearstress wasincreased to 4, 15, or 25 h, HRP uptakeduringthefinal2 hremained within

16%of static controls(Table I).Thus, a cleardifference in the

cellular response ofendotheliumtosteady andoscillatingshear

stressexists,eventhough the timeaverageshearstressamplitude

in both cases(8 dyn/cm2)wasthesame.

Ultrastructural cytochemical localization ofHRP. The ul-trastructural distribution ofHRPin endothelial cells exposed

to steady shear stress and fixed during a period of elevated pinocytoticrate is illustrated in Fig. 6.The patternwas quali-tatively similarto that seen incontrol endothelial cells. HRP was located in membrane-bounded vesicles, endosomes, and

secondary lysosomes with no indication ofcytoplasmic distri-bution, which is consistent with previous studies (15, 17).

[3H]

Thymidine

nuclearlabeling ofendothelium exposedto

shear.Confluentendothelial cells in cultureare

growth-inhibited.

60 Figure 5. Influence of

50 j! rapidly oscillating (1 Hz)

O

_50-

/ _{laminar shear stress upon}

>.0

40/ endothelialendocytosis. A

- /? wobblingcone wasused

30 toimpose sinusoidal

os-E / - cillating shear stress

be-.0 - tween 3 and l3

iZc 1 e ,/ dyn

-cm-2(time-average

amplitude 8dyn._cm2).

0 Cellular HRPuptakewas

0 30 60 90 120 determined at various

in-Minutes tervals. Endpointis the meanof six

measure-ments. Standarddeviationswerewithin 30% ofthemean. Static

con-ditions,(-o-),0dynes cm-2; oscillatingshear stress,(-- v--).

Table I. PinocytosisinConfluent Cultured EndothelialCells

ExposedtoOscillatingShear Stressat I HzFrequency*

No.ofexperiments

Duration ofexposure (No. of measurements) Pinocytotic ratet

h %control

0.5 4(12) 83±18 1.0 5(15) 103±18 2.0 7(21) 100±15 4.0 2(12) 86±3 6.0 2(12) 85±2 15.0 1 (6) 91±17 24.0 1 (6) 108±13 * Oscillating shear stress in laminar flow. Sinusoidalfrequency, 1 Hz. Range, 3-15dyn/cm2.Time averageamplitude,8

dyn/cm2.

f Measured ascellularHRP uptake per milligram cellproteinduring final 2 h ofexposure to shearstress. Expressedaspercentage of value determinedincontrol monolayers

(0 dyn/cm2)±SD.

Incubation with

[3H]thymidine

for24 h results in a lowfrequency oflabeling ofnuclear DNA as measured byautoradiography

(18).

When a majority ofthe cells are made to divide by

dis-ruption ofthe monolayer, increased pinocytotic rates are

ob-served whicharerelatedtothe cell cycle(18,5).It wastherefore importanttodetermine whether exposuretoshearstresses altered the growth patterns ofconfluent endothelium.

As shown in Table II, over a wide range ofshear stress

conditions, the fraction of labeled endothelial cell nuclei

re-mained low(<6%). Much of this data wasobtained from the same experiments in which HRP uptake wasmeasured. Asa

positive control, thepercentage of labeled cells on a growing

subconfluentendothelial culturewas43%.Weconclude,

there-fore,thatshearstress-inducedchanges in pinocyticrateoccurred inthe absenceofany significant alterations in endothelial cell

growthstatus.

Discussion

In vivo studies conducted over many years suggest that

he-modynamic forces influence arterial wall physiology and

pa-thology in anumber ofways (for reviews, see references 20-23). It hasbeen proposed that extremesof shear stress, either

very high (1) or very low (20), can contribute to vessel wall

pathology.

Increased

endothelial permeabilityhas beenmeasured

nearvesselbranches andbifurcations (24), regions thatare

ex-posedto bothhigh and low shearstresses (25). Disruption of endothelial cell alignment has been observed in areas where flow separation is expected to occur(26, 27), and such areas

appear to bemore susceptible to endothelial damage, intimal cellproliferation, and the development ofatherscleroticlesions (28). Sites of highest shearstress arelocatednearthe flow dividers whichtend to be sparedendothelialdamage andlipid deposition.

Theseinvivo observationssuggest,therefore, that boththe

time-average magnitude ofwall shearstresses, as well as temporal

variations in magnitudeanddirection,maymodifyendothelial

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Table II. Endothelial Cell [3H]Thymidine-labeling

AfterExposure toShear Stress

Percentlabelednuclei*

Period Steady shear Steady shear Oscillating shear ofexposure 8dyn/cm2 15dyn/cm2 3-13 dyn/cm2 h

0.5 5

2.0 3 3 6

4.0 4 6

8.0 3

24.0 4 6

Controls: static(0 shear) confluent monolayer, 4±2%(SD); subcon-fluent, growingcells(0shear),43±17%(SD).

*_{Afterexposure toshear stress,coverslips of cellswereincubated}

with_{[3H]thymidine}for 24 h and processedforauto-radiography.

This paper presents evidence that the application of shear

stress tothesurface ofconfluent endothelium in vitrocandirectly influence a basic endothelial cell function, fluid-phase

endo-cytosis. Furthermore, the cellscanaccommodate quite rapidly

tothe newconditions andreturn to basalrateswithin several hoursafter the onset of shear. An interesting aspect of the ac-commodation response is the periodduringwhichpinocytosis

issignificantly inhibited,aphenomenonwhichalso appears to

bedependent upon theamplitude of shear stress in that itoccurs

within2 hwhen the cells are exposed to 15dyn/cm2 and within

4hwhen exposed to 8 dyn/cm2. The removal ofshear stress

afterlong-term exposure (48 h) also results inincreased

pino-cytosis, suggesting that change of shear ismoreimportant than the direction ofchange. This ispotentially important in view of the constantly changing flows ofvarying magnitudesin the vascular system, particularly in the major arteries (29). When the frequency ofshearstressapproximatedquasi-physiological

levels(1 Hz), nochange inpinocytosisoccurred. However, when shear stresseswereappliedtotheendotheliumperiodically, pi-nocytotic rates in the cells increased when the time between

applicationsof shearstress wasrelatively long.

The aboveobservationscanbeconsideredtobe relevantto twohemodynamic situationswhichoccurinvivo.First,cycles of systole and diastole produce arapid oscillatoryshearstress

in the arterial circulation. Maximum frequencies up to 20 cycle/s have been recorded under conditions where harmonics contributesignificantlytothewaveforms(Langille,L., personal

communication). The amplitude of such fluctuations is greatest in the large vessels and decreases in the muscular distributing arteries because of the dampening effect ofthe large elastic arteries, particularly the aorta. Our oscillating shear stress

ex-periments(1 Hz)areanalogoustothefundamental pulse

com-ponent. Second, in addition to such rapid changes ofshear stress, there are components of flow-induced shear of slower

periodicityinduced by thegeometricarrangement of blood

ves-selscombined with poorlydefined fluctuationsin blood pressure that can normally occur throughout the day. For example, at

branch arteries ofthe aorta and at bifurcations, complex he-modynamic events occur which result in a slow periodicity of change ofshear close to the branch points, and even reversal offlow direction (30). These events are superimposed upon the higher frequency pulse wave. The dynamics offlow in these regions are highlysusceptible to a number ofexternal conditions (e.g.,hypertension,exercise, smoking, and psychological stress). It is conceivable that our data with slow periodicity andsteady shear stress are relevant to hemodynamically-induced vascular changes in these regions. Over a 24-h period, major changes of blood pressure and pulse rate occur with frequencies varying from 1 h to several minutes (31). There appears, therefore, to beabroad spectrum of fluctuations in hemodynamic parameters that may influenceendothelial cell function.

All of the data in the present study refer to well-defined laminar flow conditions. In addition to laminar flow, however, turbulent flow occurs in certain regions of the major arterial vessels and suchforces may affect endothelial morphology and

function in quite different ways than does laminar flow (Re-muzzi, A., C. F. Dewey, Jr., and M. A. Gimbrone, Jr., unpub-lished observations).

Fluid-phase endocytosis is a constitutive function of all eu-karyotic cells and encompasses entry of extracellular fluid and solublemacromoleculesinto both coated and uncoated vesicles after which the vesicle contents may be processed in a number of ways (seereferences 14, 32for review). Pinocytosis is very

closely associated with the recycling of membrane to the cell

surface(33), e.g., anincrease ofpinocytotic rate will result in

faster recycling ofmembrane. In endothelial cells in vivo, en-docytosis is implicated in the movement of plasma molecules

totheinterstitialsubendothelial space (13). It is not clear whether

increased pinocytosis will result in increased transendothelial transport because (a) some vesicles are also targeted for the endothelial lysosomes where their contents are degraded (15), and (b) transport across the endothelium has not been

satis-factorilymeasuredinvitro under conditions of shear, although recent attemptstoproduce a functional endothelial barrier in vitro appear promising (34).

Figure6. Ultrastructuralhistochemical localization ofHRPin culturedBAECexposedto(A) static conditions 0dyn/cm2and

(B)steady shear stressof8dyn/cm2.Cellswereincubated with

2 mg HRP/mlfor2h.Cellularlocalization ofHRPreactionproduct

includes secondarylysosomes(L)andcytoplasmicendosomes(E) in

transit betweenthecell surface(S)andlysosomes. Thedistribution ofHRPis similarintheabsenceandpresenceof shearalthoughthe magnitude ofuptakein(B) ismorethan twicethatof(A)as deter-minedspectrophotometrically.Enfacesections.(A) X 14800,

Shear stresses mayinfluence the rate ofpinocytosis in

en-dothelium by modulating the mechanismsofpinocytotic vesicle formation. Weinbaum and Chien (35) haveelegantlymodeled the interactions between the vesicle membrane and the plasma membrane, i.e., vesicle detachment and attachment, the former of whichmaybe rate-limiting for theinternalization of vesicles.

In such amodel, it is possible that external physical forces such

as shear stress directly influence the deformability of vesicle attachment sites on the apicalendothelial surface. While this approachmaybe relevant to random vesicular movement,the

formationand movementofmostendothelial vesiclesappears tobe largely energy-dependent and related to theintegrity of the cytoskeleton. Pinocytosis of bulk fluid tracers is inhibited byup to90% by drugs which inhibitglycolysisand respiration (36). Microfilament disassembly by agents suchascytochalasins also results insignificant (50%) inhibition ofpinocytosis(37). It is reasonableto_{postulate, therefore, that shear stress indirectly}

influences HRP uptake in endothelium via other metabolic and structural systemsinadditiontoany directphysicaleffectsupon

vesicle detachment at the cell surface.

The experiments reported here measure totalendocytic

vol-ume ingested by the cell without

distinguishing

between

receptor-mediated and nonspecific endocytic mechanisms. Surface

membrane is continuously recycled through vesicular

com-partments ofthe cell (33). The presence ofspecific receptors for exogenous macromolecules on thevesicular membrane is

subject to control mechanismsthat,inallprobability,arequite separate from _{those which mediate membrane}

_recycling.

For

example, the expression oflipoprotein receptors is governed

primarilybytheavailabilityof exogenouscholesteroltothe cell (38). Itshould notbe expected apriori,

therefore,

that the

in-fluences of shear stress upon theendocytosis(and metabolism)

ofspecific ligandssuch aslowdensity lipoproteinswill be the

same as forpinocytoticrates. Itis

important,

however, to

doc-ument the changes inpinocytosisinduced by shearstresses in

order to interpret theendocytosis of

lipoproteins;

such

inves-tigations are currently underway in thislaboratory(39).

Insummary, we havedemonstratedthat abasic endothelial

cell_{function, fluid-phase pinocytosis,}isinfluencedbyfluidshear

stress under laminar flow conditions; that thefrequencyas well

asthe amplitude of the shear stress affectsthis response; and that under certain circumstances, the endothelium shows ad-aptation to newconditionsof shear stress. The studies are

rel-evant to thetransendothelial transport and/or metabolic pro-cessing ofmacromolecules, and perhaps to other aspects of

en-dothelial physiology and pathology in vivo.

Acknowledgments

We thankCathy Kerr, Susan O'Connor,and BarbaraEisenhaure for

experttechnical assistance.We are alsogratefulto Dr. LowellLangille, University ofWesternOntario, Canada for discussion concerning the rangeandperiodicity of physiological shear stress.

Thisresearch wassupportedby National Institutes ofHealth grants

HL25536andHL22602andby theWhitaker Health Sciences Fund of

theMassachusetts Institute of Technology.

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