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Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1770-1776
doi: 10.1161/hq1001.097104
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1770.)
© 2001 American Heart Association, Inc.

Vascular Biology

Pulsatile Flow Regulates Monocyte Adhesion to Oxidized Lipid-Induced Endothelial Cells

Tzung K. Hsiai; Sung K. Cho; Srinivasa Reddy; Susan Hama; Mohamad Navab; Linda L. Demer; Henry M. Honda; Chih M. Ho

From the Division of Cardiology, Department of Medicine (T.K.H., H.M.H., S.R., S.H., M.N., L.L.D.), Department of Physiology (L.L.D.), and Department of Molecular and Medical Pharmacology (S.R.), UCLA School of Medicine, and the Department of Mechanical and Aerospace Engineering (T.K.H., S.K.C., C.M.H.), UCLA School of Engineering and Applied Sciences, Los Angeles, Calif.

Correspondence to Tzung K. Hsiai, MD, Division of Cardiology, 47-123 CHS, UCLA School of Medicine, Los Angeles, CA 90095-1679. E-mail thsiai{at}mednet.ucla.edu


*    Abstract
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Abstract—— Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), a component of minimally modified low density lipoprotein, induces monocyte adhesion to endothelial cells. It is not known whether the upstroke slopes of pulsatile flow, defined as shear stress slew rates ({partial}{tau}/{partial}t), can regulate monocyte binding to ox-PAPC-treated bovine aortic endothelial cells (BAECs). At 60 cycles per minute, ox-PAPC-treated BAECs were exposed to 3 conditions representing known vascular conditions: (1) high shear stress slew rate ({partial}{tau}/{partial}t=293 dyne · cm-2 · s-1), with time-averaged shear stress=50 dyne/cm2; (2) low shear stress slew rate ({partial}{tau}/{partial}t=71 dyne · cm-2 · s-1), with identical time-averaged shear stress; and (3) reversing oscillating flow (0±2.6 mm Hg). Reverse transcription-polymerase chain reaction and quantification were performed for monocyte chemoattractant protein-1 (MCP-1) mRNA expression. High {partial}{tau}/{partial}t reduced monocyte binding to ox-PAPC-treated BAECs by 64±3.2% compared with static conditions, and low {partial}{tau}/{partial}t reduced monocyte binding by 31±3.4%, whereas oscillating flow increased monocyte binding by 22±1.7% (P<0.005). High {partial}{tau}/{partial}t downregulated MCP-1 expression by 33±8%, and low {partial}{tau}/{partial}t downregulated MCP-1 expression by 15±4%, but oscillating flow upregulated MCP-1 by 13±5%. These results suggest that shear stress slew rates regulate monocyte binding by modulating the expression of a potent monocyte chemoattractant.


Key Words: shear stress • slew rate • oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine • monocyte adhesion • monocyte chemoattractant protein-1


*    Introduction
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Monocyte adhesion to endothelium, subsequent transmigration, and the development of foam cells are considered to be early events of atherosclerosis.1–5 Atherosclerosis develops preferentially and geometrically at the curvatures and lateral walls of vascular branching points.6–8 In these susceptible regions in which disturbed flow develops, the time-averaged shear stress ({tau}ave) is low, but the spatial shear stress gradient ({partial}{tau}/{partial}x) is high.9–12 It has been demonstrated in vitro that monocyte binding was greatest at points of flow separation, where {partial}{tau}/{partial}x is maximal.9,13 In contrast, fewer lesions develop in straight sections of arteries, where laminar flow predominates and spatial shear stress gradients are minimized.14,15 Thus, the spatial component of shear stress is important in promoting the multistage interaction between monocytes and endothelial cells in atherosclerosis.

The temporal component of shear stress ({partial}{tau}/{partial}t) also plays a role in endothelial function. Steady laminar flow has been shown to be atheroprotective through sustained upregulation of endothelial cell NO synthase, cyclooxygenase-2, and manganese superoxide dismutase and downregulation of putative atherogenic genes, including monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and platelet derived growth factor-B.16–19 In contrast, oscillating flow upregulates atherogenic but downregulates atheroprotective gene expression.14,19 Bao et al20 further demonstrated that impulse flow or step flow (where {partial}{tau}/{partial}t is maximal) but not steady shear stress (where {partial}{tau}/{partial}t=0) induced platelet derived growth factor-A and MCP-1 expression.20 Recently, we have shown that higher levels of monocyte binding to bovine aortic endothelial cells (BAECs) occurred under low shear stress and flow reversal compared with unidirectional flow.21 These findings suggest that the {partial}{tau}/{partial}t gradient is involved in modulating endothelial gene expression and function.

LDL oxidation is one of the fundamental processes in atherogenesis. LDL particles trapped within the subendothelial space undergo oxidative modification, resulting in the formation of minimally modified LDL (MM-LDL) and highly oxidized LDL (ox-LDL). MM-LDL and ox-LDL play major roles in the injury reaction leading to atherosclerosis.22–24 Specifically, MM-LDL induces the expression of connecting segment-1 on endothelial cells as well as MCP-1 expression, leading to monocyte binding and chemotaxis and subsequent transendothelial migration.25–27 Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), a biologically active component of MM-LDL present in atherosclerotic lesions, is a potent inducer of monocyte binding to endothelial cells.28 No prior studies have addressed whether pulsatile flow patterns can modulate monocyte binding to endothelial cells treated with oxidized lipids.

In the present study, we designed a device generating pulsatile flow to investigate how the upstroke slope of pulsatile flow, defined as shear stress slew rate ({partial}{tau}/{partial}t), affects monocyte binding to oxidized lipid-treated endothelial cells. We used ox-PAPC as a surrogate for MM-LDL.26,28 Shear stress at high and low slew rates downregulated monocyte binding to ox-PAPC-treated BAECs. In contrast, oscillatory flow upregulated monocyte binding. Additionally, compared with low shear stress slew rates, high shear stress slew rates exerted a greater effect on downregulating monocyte binding after exposure to ox-PAPC, despite identical magnitudes of {tau}ave and periodicity. The differential levels of MCP-1 mRNA expression in response to pulsatile versus oscillating flow suggest one of the mechanisms by which shear stress slew rates regulate monocyte adhesion to ox-PAPC-treated BAECs.


*    Methods
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 *Methods
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Design of a Novel Pulsatile Flow System
We designed a pulsatile flow channel to isolate the effects of shear stress slew rates with well-defined {tau}ave, frequency, and amplitude. Three parameters governed the design of this parallel flow channel: (1) shear stress acting on the parallel plates, (2) Reynolds number, and (3) entrance length.29 Symmetrical contractions and diffusers were connected to the inlet and outlet of the parallel channels. This unique configuration ensured velocity uniformity and the absence of flow separation across the width of the channel during flow reversal. The contraction was designed by a fifth-order polynomial at a 3:1 contraction ratio30; the diffuser was built at a half angle of 4.3° (Figure 1).



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  		Figure 1. Parallel-plate flow channel. Symmetrical contractions and diffusers were connected to the inlet and outlet of the parallel channel to provide uniform flow fields across the width of the channel.

The pulsatile flow system consisted of a stepping motor (No. KML092F07, Warner/Superior) with a driver (No. SS2000D7, Warner/Superior), a peristaltic pump (No. E-28002-60, Ismatic), and a digital-to-analog converter (No. PCI-MIO-16E-4, National Instrument). The pressure signals were recorded from a tap located on the upper wall of the parallel plate as a voltage signal via a sensor with a high frequency response of 10 kHz, allowing capture of pulsatile flow at high and low slew rates (model 143SC05D, SenSym); see Figure I, which can be accessed online at http://atvb.ahajournals.org. At various upstroke slopes of pulsatile flow, the downstroke slopes were kept essentially identical. Time-averaged flow rate was monitored downstream by an electromagnetic flowmeter (ECMO MAGFLO 1100).

The flow channel system was mounted on the inverted microscope with x10, x20, and x50 phase-contrast objectives (Nikon Eclipse TE 200). The DMEM culture medium was maintained at a temperature of 37°C. A temperature probe (Omega RTD-2-1 PT100K) in the channel was connected to the heating circulator (Fischer Isotemp 2006P). The pH of the DMEM culture medium was kept at 7.4 by using 5% CO2. The pH of the circulating culture medium was monitored by using a pH probe (Fischer Scientific Accumet AP series handheld Model AP63) and controlled by using 5% CO2.

The system allowed real-time observation, temperature control, pressure and flow rate monitoring, and the ability to deliver physiological mean shear stress from 0 to 100 dyne/cm2 and instantaneous shear stress from 0 to as high as 200 dyne/cm2 with mean Reynolds numbers of 200 to 6000.

Endothelial Cell Culture
BAECs between passages 5 and 9 were seeded on glass slides (5 cm2) coated with Cell-Tak cell adhesive (Becton-Dickinson Labware) and Vitrogen (RC 0701, Cohesion) at 3x106 cells per slide. BAECs were then grown to confluent monolayers in DMEM supplemented with 20% FBS, 0.05% amphotericin B, and 100 U/mL streptomycin for 48 hours in 5% CO2 at 37°C.

Ox-PAPC Preparation
PAPC (Sigma-Aldrich) was oxidized by transferring 1 mg in 100 µL of chloroform to a clean 16x25-mm2 glass test tube and evaporating the solvent under a stream of nitrogen. The lipid residue was allowed to auto-oxidize while being exposed to air for 24 to 48 hours. The extent of oxidation was monitored by positive electrospray ionization-mass spectrometry in the positive mode.28

Experimental Protocols
Confluent monolayers of BAECs grown on glass slides were incubated with 50 µg/mL ox-PAPC for 4 hours. The endothelial monolayers were then rinsed with DMEM and placed in the pulsatile flow channel. These ox-PAPC-treated BAEC monolayers were exposed to 3 flow conditions at 1 Hz for 4 hours: (1) oscillating flow (±5 dyne/cm2) with {tau}ave=0 dyne/cm2 (Figure 2), (2) high shear stress slew rate ({partial}{tau}/{partial}t=293 dyne · cm-2 · s-1), with {tau}ave=50 dyne/cm2, and (3) low shear stress slew rate ({partial}{tau}/{partial}t=71 dyne · cm-2 · s-1, with the same {tau}ave; Figure 3B). For oscillating flow, minimal forward flow at a mean shear stress of 0.2 dyne/cm2 was provided every hour to deliver nutrients and remove waste products from the cells.



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  		Figure 2. Absolute pressure signals experienced by BAECs in the parallel plate. Oscillating profiles with 0±2.6 mm Hg at 1 Hz and dP/dt=9.6 mm Hg/s are shown. The {tau}ave is zero. Absolute pressure signals, which denote a value at 1 point as opposed to pressure difference, cannot be used to reconstruct shear stress profiles.



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  		Figure 3. A, The voltage (V) output, which represents the pressure difference upstream from the channel, was phase-averaged and converted to flow rate for high and low slew rates. B, Pulsatile shear stress profiles for high vs low slew rates at 1 Hz from panel A. The linear horizontal line denotes the time-averaged shear stress for both flow profiles (50 dyne/cm2).

Monocyte Adhesion Assay
Monocytes were isolated by using a modification of the Recalde method, as described by Fogelman et al,31 from normal volunteers with institutional review board approval. Endothelial cells were treated with ox-PAPC for 4 hours and then exposed to flow for 4 hours. The cover slides were then rinsed with DMEM and assayed for monocyte binding activities. Endothelial monolayers were incubated with freshly isolated monocytes (105 monocytes/cm2) for 10 minutes at 37°C under static conditions. Nonadherent monocytes were washed with DMEM. The adherent monocytes were fixed with 1% glutaraldehyde. Adherent monocytes were counted in a total of 20 high-power (x400) standardized fields.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
BAECs were exposed to the flow conditions described above in Experimental Protocols. At the end of the experiment, slides were removed from the flow channel, and the monolayers were washed with PBS and then lysed. Total RNA was then isolated by using the RNeasy Total RNA kit (Qiagen). Equal amounts of RNA were reverse-transcribed and amplified with avian myeloblastosis virus reverse transcriptase (Promega) and with Thermus flavus DNA polymerase, respectively, in 1 reaction with the use of the Access RT-PCR System (Promega) according to the manufacturer’s protocol. The primers used for amplification of MCP-1 mRNA were as follows: 5'-GTG CCT GCT ACT CAC AGT AG-3' (upper) and 5'-GGA GTT TGG TTT TTC TTG TT-3' (lower). The primers used for bovine GAPDH mRNA were as follows: 5'-TGG CAA AGT GGA CAT CGT CG-3' (upper) and 5'-TTG CGT GGA CAG TGG TCA TAA GTC-3' (lower). The annealing temperatures were 52°C and 58°C for MCP-1 and GAPDH, respectively.

Quantification of MCP-1 mRNA
Equal amounts of RNA extracts (50 ng) were reverse-transcribed and amplified by using MCP-1 and GAPDH primers as described above in a thermal cycler (MJ Research) for 20 cycles. Under these conditions, the amplification was linear for GAPDH between 12.5 and 100 ng of total RNA. Polymerase chain reaction (PCR) products were analyzed by agarose gel electrophoresis containing SYBR Gold nucleic acid gel stain (No. S-19914, Molecular Probes) and by densitometric scanning of the DNA bands (ImageQuant, Molecular Dynamics). The intensities of all bands were normalized to that of GAPDH and analyzed under identical conditions.

Statistical Analysis
Data are expressed as mean±SD compared with controls and compared among separate experiments. For comparisons between 2 groups, statistical analysis was performed by using the 2-sample independent-groups t test. Comparisons of multiple mean values were made by 1-way ANOVA, and statistical significance among multiple groups was determined by using the Tukey procedure (for pairwise comparisons of means between static-like and pulsatile flow conditions). Values of P<0.05 were considered statistically significant.


*    Results
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Flow Patterns Generated by the Pulsatile Flow Device
Three flow profiles representing different sites at vascular branching points were generated: oscillatory flow and unidirectional pulsatile flow at high and low shear stress slew rates. Figure 2 illustrates the oscillatory pressure profile at a periodicity of 1 Hz in the parallel-plate channel, simulating the flow pattern seen at the reattachment points.10,14,19 This profile reflected the absolute pressure signals fluctuating between 2.6 and -2.6 mm Hg with {tau}ave=0. The absolute pressure signals, which denote a value at 1 point rather than the pressure difference, could not be used to reconstruct shear stress profiles. Figure 3A illustrates the signals of pressure difference recorded upstream from the channel for high and low slew rates. These digital signals were phase-averaged and converted to flow rates, followed by fast Fourier transform and conversion into temporal shear stress, {tau}(t) (Figure 3B). The theoretical formulation of pulsatile flow can be referred to in the Appendix, which can be accessed online at http://atvb.ahajournals.org. The {tau}ave value under high and low shear stress slew rates was 50 dyne/cm2.

Monocyte Binding in Response to Pulsatile Flow at a Low Shear Stress Slew Rate
Under static conditions, treatment of BAECs with ox-PAPC (50 µg/mL) for 4 hours markedly increased monocyte binding (static control=6±2 monocytes/HPF, ox-PAPC-treated BAECs [static]=26±2; P< 0.05). A nonsignificant decrease in adherent monocytes was observed in BAECs exposed to low a shear stress slew rate compared with the static control (control=6±2 monocytes/HPF, low shear stress slew rate alone=3±1; P>0.05). However, pulsatile flow at a low shear stress slew rate attenuated monocyte binding to ox-PAPC-treated BAECs by 31±3.4% compared with the ox-PAPC-treated BAECs under static conditions (ox-PAPC-treated BAECs plus low shear stress slew rate=19±3 monocytes/HPF, ox-PAPC-treated BAECs [static]=27±1; P<0.005; Figure 4A).



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  		Figure 4. A, Effect of low shear stress slew rates on monocyte binding in response to ox-PAPC. Incubation of BAECs with ox-PAPC markedly increased the number of adherent monocytes. Ox-PAPC-mediated monocyte adhesion was attenuated by 30% after exposure to pulsatile flow at low shear stress slew rates. There was no significant effect on low slew rate alone to monocyte binding in response to ox-PAPC. Data (mean±SD) are based on 3 separate experiments. B, Effects of high shear stress slew rate on monocyte binding in response to ox-PAPC. Ox-PAPC-mediated monocyte adhesion to BAECs was decreased by 64% after exposure to pulsatile flow at high slew rates. There was no significant effect of high shear stress slew rate alone on monocyte binding in absence of ox-PAPC. Bars represent mean±SD of 3 separate experiments.

Monocyte Binding in Response to Pulsatile Flow at a High Shear Stress Slew Rate
As observed in the low shear stress slew rate condition, a nonsignificant decrease in adherent monocytes to unstimulated BAECs was observed compared with static conditions (control=4.4±0.4 monocytes/HPF, high shear slew rate alone=2.6±0.6; P>0.05). However, pulsatile flow at high shear stress slew rate further attenuated monocyte binding to ox-PAPC-treated BAECs by 64±3.2% compared with the ox-PAPC-treated BAECs under static conditions (ox-PAPC-treated BAECs plus high shear stress slew rate=9±3 monocytes/HPF, ox-PAPC [static]=26±1; P<0.005; Figure 4B). Thus, pulsatile flow at high compared with low slew rates exerted a stronger effect in decreasing the number of adherent monocytes to ox-PAPC-treated endothelial cells.

Monocyte Binding in Response to Oscillating Flow Conditions
Oscillating flow alone tended to accentuate endothelial monocyte adhesion; however, this did not reach statistical significance (control=4.3±0.7 monocytes/HPF, oscillatory flow alone=6.8±0.4; P>0.05). In contrast to unidirectional pulsatile flow, oscillating flow further promoted monocyte binding to ox-PAPC-treated BAECs by 22±1.7% compared with the ox-PAPC-treated BAECs under static conditions (oscillatory flow=29±1 monocytes/HPF, static conditions=24±2 monocytes/HPF; P<0.05; Figure 5).



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  		Figure 5. Effects of oscillating flow on monocyte binding in response to ox-PAPC. Incubation of BAECs with ox-PAPC markedly increased the number of adherent monocytes. Ox-PAPC-mediated monocyte adhesion to BAECs was increased by 30% after exposure to oscillatory flow at {tau}ave of zero. There was no significant effect of oscillating flow alone on monocyte binding in absence of ox-PAPC. Data (mean±SD) are based on 3 separate experiments.

MCP-1 mRNA Expression in Response to Pulsatile Versus Oscillatory Flow Conditions
Ox-PAPC treatment alone upregulated MCP-1 mRNA expression compared with expression under control conditions (Figure 6). Introduction of pulsatile flow at high and low shear stress slew rates ({partial}{tau}/{partial}t) downregulated MCP-1 mRNA expression compared with no-flow conditions by 44±8% and 22±4%, respectively (MCP-1 to GAPDH density ratio: at high {partial}{tau}/{partial}t, 0.99±0.37; at low {partial}{tau}/{partial}t, 1.36±0.24; under no flow condition plus ox-PAPC, 1.75±0.26; P<0.05 [n=5]; Figure 6). In contrast, compared with no-flow conditions, reversing oscillating flow upregulated MCP-1 expression by 15±4% (MCP-1 to GAPDH density ratio: 2.02±0.25, P<0.05 [n=5]; Figure 6). The intensity of MCP-1 bands is closely related to the numbers of monocyte binding (see Figure II, which can be accessed online at http://atvb.ahajournals.org). The intensity of all bands was normalized to that of GAPDH with a dose-dependent linear relation (see Figure III, which can be accessed online at http://atvb.ahajournals.org). Despite the same {tau}ave and periodicity as in low shear stress slew rate flow, high shear stress slew rate flow further downregulated MCP-1 mRNA expression in ox-PAPC-treated BAECs.



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  		Figure 6. Semiquantitative reverse transcription (RT)-PCR amplification of ox-PAPC-induced MCP-1 expression in BAECs. Total RNA (50 ng) from BAECs, cultured under different flow conditions, was RT-PCR-amplified by using primers specific for bovine MCP-1 and GAPDH and subjected to agarose gel electrophoresis. MCP-1 and GAPDH bands were quantified by densitometry and represented as ratios (MCP-1/GAPDH). The ratio was 1.26±0.36 in response to high {partial}{tau}/{partial}t, 1.6±0.24 in response to low {partial}{tau}/{partial}t, and 0.854±0.12 under the no-flow condition plus ox-PAPC (P<0.05, n=5). In response to reversing oscillating flow, the MCP-1/GAPDH density ratio was 2.12±0.246 (P<0.05, n=5).


*    Discussion
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Predilection sites for arterial atherosclerosis appear to be determined in large part by the flow pattern to which the endothelium is exposed.10,32–35 These sites are characterized by low {tau}ave, but they also have large spatial shear stress gradients ({partial}{tau}/{partial}x [dyne/cm3])8,11,36 as well as large temporal shear stress gradients ({partial}{tau}/{partial}t [dyne · cm-2 · s-1]) because of the oscillating flow that fluctuates in time with the cardiac cycle.14,19 Thus, either spatial or temporal shear stress gradients or both may be the key factor(s) in predilection sites for atherosclerosis.

One of the earliest steps in atherosclerosis is the binding and transmigration of monocytes into the subendothelial space. MM-LDL specifically induces the adhesion of monocytes but not neutrophils to endothelial cells.25,26 In addition, oxidized phospholipids isolated from MM-LDL and ox-PAPC mimic the biological activity of MM-LDL37 by inducing endothelial expression of MCP-1, P-selectin,38 and monocyte adhesion molecule connecting segment-1.27

The role of shear stress in monocyte-endothelial interaction has been previously investigated. Chappell et al14 showed that exposure of endothelial monolayers to oscillatory shear stress upregulated E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1, thereby enhancing monocyte binding. Barber et al9 demonstrated that monocyte transient arrests occur more frequently at the attachment site, where the endothelium is exposed to oscillatory shear stress with {tau}ave values approaching zero. Conversely, exposure of endothelial cell monolayers to laminar shear stress (>15 dyne/cm2) suppresses adhesion molecule expression and monocyte binding.39,40

The unique channel configuration in our flow device with symmetrical contractions and diffusers ensures absence of flow separation and, thus, velocity uniformity across the width of the flow channel. Therefore, we were able to isolate the effects of different upstroke slopes or shear stress slew rates while maintaining the downstroke slopes, periodicity, amplitude, mean flow rate, and, therefore, the {tau}ave of pulsatile flow.

In these experiments, we exposed endothelial cells to a {tau}ave value of 50 dyne/cm2 for 4 hours. Physiological {tau}ave levels are in the range of 20 to 30 dyne/cm2 in the human arterial circuit.41 However, extreme variations in shear stress exist in regions of branching7 and stenotic arteries.42,43 In the bifurcation of the left main coronary artery into the left anterior descending and the left circumflex coronary arteries, the mean shear stress on the flow divider is estimated to be in excess of 50 dyne/cm2, whereas the mean shear stress on the outer lateral wall is near zero.7,10,33 The peak shear stress just upstream from the stenosed arteries with diameters ranging from 0.084 to 0.159 cm varied from 520 to 3349 dyne/cm2.42,43 With pulsatile flow, the peak shear stress level on the flow divider may be in excess of 100 dyne/cm2.7 We chose a magnitude of mean shear stress that has not commonly been reported in the literature to simulate shear stress on the inner wall distal to the arterial bifurcation. Therefore, the {tau}ave value adopted in our study at 50 dyne/cm2 reflects the dynamic range in the physiological flow profiles.

The 4-hour time point was selected as an optimal duration because the assay requires changes in protein expression for monocyte binding.44,45 Previous studies examining the effects of fluid flow and monocyte binding have also used 4-hour exposure to flow.46 In addition, one mechanism by which shear stress slew rates can be protective against atherosclerosis may be through the production of NO, which downregulates MCP-1 expression.45,46 Because these studies have shown that a 2- to 5-hour minimal exposure to NO is required before any MCP-1 mRNA expression, 4-hour flow exposure is also optimal for other gene expression from endothelial cells.

We did not directly compare oscillatory versus pulsatile flow patterns at the same magnitude of mean shear stress. However, we have previously studied a closely related issue and showed that higher levels of monocyte binding to BAECs occurred under low shear stress and flow reversal compared with unidirectional flow.21 In the present study, we closely simulated the expected flow patterns encountered at arterial bifurcations. First, pulsatile flow at a mean shear stress of 50 dyne/cm2 was delivered to simulate shear stress on the inert wall distal to the arterial bifurcation. Second, oscillatory flow of -5 and 5 dyne/cm2 at a mean shear stress of zero simulated the reattachment point where flow separation occurs at the outer wall of bifurcation.33,47

To our knowledge, the present study is the first to examine whether flow patterns can modulate the biological effect of oxidized lipids on endothelium. Although the absolute changes in monocyte binding would be considered small and high shear stress slew rates did not completely abolish the effects of ox-PAPC on monocyte bindings, high shear stress slew rates did decrease monocyte binding by 60%. In addition, we have elucidated the important role of shear stress slew rates in regulating monocyte-endothelial interaction in response to oxidized lipids. The present study demonstrated that shear stress at high and low slew rates inhibited monocyte binding to ox-PAPC-treated endothelial cells. In contrast, oscillatory flow that is seen at predilection sites for atherosclerosis upregulated monocyte binding to ox-PAPC-treated endothelial cells. Furthermore, compared with low shear stress slew rates, high shear stress slew rates exerted a greater inhibitory effect on monocyte binding to ox-PAPC-treated endothelial cells, despite identical {tau}ave in both conditions. Therefore, shear stress slew rates with a high mean shear stress are protective against the binding of monocytes to endothelial cells in response to oxidized lipids and may be an important factor in the localization of atherosclerotic lesions in the vascular tree.

These in vitro results cannot be directly extrapolated to the in vivo condition because the biochemical environment and time of exposure to biomechanical forces are different. However, these results suggest 1 mechanism by which shear stress slew rates may decrease monocyte binding to the activated endothelial cells, namely, regulation of MCP-1. We have previously shown that the induction of monocyte binding to endothelial cells exposed to MM-LDL and ox-PAPC was mediated by MCP-1. In parallel with the results on monocyte binding, we found that shear stress at high and low slew rates decreased the induction of MCP-1 by ox-PAPC, whereas oscillatory flow magnified the increase in MCP-1 in response to ox-PAPC. The effect of shear stress slew rates was graded in the sense that compared with pulsatile flow at low shear stress slew rates, pulsatile flow at high shear stress slew rates was more effective in decreasing monocyte binding and MCP-1 mRNA expression.

From the clinical standpoint, the concept of shear stress slew rate suggests a mechanism by which increased cardiac contractility48 and increased shear stress during physical activity49,50 may be protective against atherosclerosis-associated clinical events. The progression of atherosclerotic lesions can be inhibited and in some cases reversed in the patients who modify their cardiovascular risk factors and engage in regular aerobic exercise.51,52 In a numerical model, exercise changed local wall shear from an oscillating flow pattern to a predominantly pulsatile laminar flow pattern.53 One mechanism by which pulsatile flow is protective may be the production of NO and prostacyclin production in response to temporal shear stress gradients.54 We suggest that another mechanism may be through decreased monocyte binding to endothelium exposed to the oxidized lipids by decreased MCP-1 expression in response to 4 hours of pulsatile flow. Because NO downregulates MCP-1 expression within 2 to 5 hours,45 these 2 mechanisms may be interdependent.

In conclusion, we developed a unique pulsatile flow system to study endothelial cell responses to well-defined pulsatile flow profiles. Our investigation revealed that the induction of MCP-1 and monocyte binding by ox-PAPC was modulated by pulsatile flow. Compared with low shear stress slew rates, high shear stress slew rates were more effective in downregulating the induction of monocyte binding to BAECs by ox-PAPC. In contrast, oscillating flow promoted the induction of MCP-1 and monocyte binding by ox-PAPC. Therefore, flow patterns are critical modulators of the biological activity of ox-PAPC on endothelial cells.


*    Acknowledgments
 
T.K.H. was supported by National Institutes of Health training grant NRSA HL-07895. S.K.C. was partly supported by the Korea Science and Engineering Foundation. The authors are indebted to Dr Alan Fogelman and Drs James Weiss, Judith Berliner, and Joy Frank of the UCLA STAR Fellowship Committee. The authors would also like to express gratitude to Dr Alan Garfinkel for his advice on statistics. T.K.H. would also like to express gratitude to Dr Shu Chien for his advice and encouragement.

Received July 12, 2001; accepted August 1, 2001.


*    References
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 *References
 

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