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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:996-1003

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:996-1003.)
© 1999 American Heart Association, Inc.

Original Contributions

Temporal Gradient in Shear But Not Steady Shear Stress Induces PDGF-A and MCP-1 Expression in Endothelial Cells

Role of NO, NF{kappa}B, and egr-1

Xuping Bao; Chuanyi Lu; John A. Frangos

From the Department of Bioengineering, University of California, San Diego, La Jolla

Correspondence to Dr John A. Frangos, Department of Bioengineering, University of California, San Diego, 6407 Engineering Bldg Unit 1, 9500 Gilman Dr, La Jolla, CA 92093-0412. E-mail frangos{at}ucsd.edu


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Three well-defined laminar flow profiles were created to distinguish the influence of a gradient in shear and steady shear on platelet-derived growth factor A (PDGF-A) and monocyte chemoattractant protein-1 (MCP-1) expression in human endothelial cells. The flow profiles (16 dyne/cm2 maximum shear stress) were ramp flow (shear stress smoothly transited at flow onset), step flow (shear stress abruptly applied at flow onset), and impulse flow (shear stress abruptly applied for 3 s only). Ramp flow induced only minor expression of PDGF-A and did not increase MCP-1 expression. Step flow increased PDGF-A and MCP-1 mRNA levels 3- and 2-fold at 1.5 hours, respectively, relative to ramp flow. In contrast, impulse flow increased PDGF-A and MCP-1 expression 6- and 7-fold at 1.5 hours, and these high levels were sustained for at least 4 hours. These results indicate that a temporal gradient in shear (impulse flow and the onset of step flow) and steady shear (ramp flow and the steady component of step flow) stimulates and diminishes the expression of PDGF-A and MCP-1, respectively. NO synthase inhibitor NG-amino-L-arginine (L-NAA) was found to markedly enhance MCP-1 and PDGF-A expression induced by step flow, but decrease their expression induced by impulse flow, in a dose-dependent manner. NO donor spermine-NONOate (SPR/NO) dose-dependently reduced the MCP-1 and PDGF-A expression induced by impulse flow. Moreover, impulse flow was found to stimulate sustained (4 hours) I{kappa}B-{alpha} degradation and egr-1 mRNA induction. L-NAA prevented I{kappa}B-{alpha} degradation, whereas SPR/NO increased I{kappa}B-{alpha} resynthesis 2 hours after impulse flow. Both L-NAA and SPR/NO inhibited the impulse flow inducibility of egr-1 4 hours after the flow stimulation. The results show that both NO induced by steady shear and NO donor inhibit temporal gradient in shear-induced MCP-1 and PDGF-A expression by downregulation of their respective transcription factors NF{kappa}B and egr-1, whereas NO induced by impulse flow stimulates MCP-1 and PDGF-A expression by upregulation of the transcription factors. The above findings suggest distinct roles of temporal gradient in shear and steady shear in atherogenesis in vivo.


Key Words: shear stress • gene expression • nitric oxide • transcription factor • atherosclerosis


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Because of the focal distribution of atherosclerotic plaques, hemodynamic forces including fluid shear stress have long been suggested to potentiate atherogenesis.1 2 The exact nature and influence of local shear involved in endothelial dysfunction leading to susceptibility for atherogenesis, however, remains unclear. Endothelial cells (EC) throughout the vasculature experience a variety of flow environments both with spatial variances and with temporal gradients in wall shear stress. In the venous system wall shear stress is lower and minimal gradients in shear stress exist because of the nonpulsatile nature of the blood flow. In the arterial system the flow conditions are generally assumed to be laminar and to present the endothelium with a high mean wall shear stress in addition to large temporal gradients in shear stress. At arterial bifurcations and curvatures, locations known to be highly prone to atherogenesis, disturbed flow patterns may develop that result in low mean wall shear stress, but still present the EC with large temporal gradients in shear stress.3 These observations, combined with other in vitro evidence,4 5 6 suggest that gradients in shear and steady shear represent different biomechanical stimuli that differentially regulate local endothelial function by distinct signaling pathway, and thus contribute to the characteristic distribution pattern of atherosclerosis.

A host of endothelial genes exhibit differential responses to shear stress stimuli that may be involved in the focal localization of atherogenic plaques.7 The application of step shear on EC in vitro has been shown to induce an initial transient expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic agent for monocytes, and platelet-derived growth factor A (PDGF-A), a potent mitogen and chemotactic agent for smooth muscle cells, giving a peak response at 1.5 hours followed by sustained downregulation.8 9 MCP-1 and PDGF-A are believed to participate in the early events of atherosclerosis.10 11 12 In contrast, three potential antiatherogenic genes—manganese superoxide dismutase (Mn SOD), cyclooxygenase-2 (COX-2), and endothelial nitric oxide synthase (eNOS)—were demonstrated to be continuously upregulated by steady laminar shear but not turbulent shear.13 Although the pathways and biochemical factors that control the dynamic balance of expression levels of these various genes have not been completely characterized, it is clear that imbalanced regulation of these effector molecules occurs at lesion-prone sites. Given that the onset of fluid shear in vitro can be decomposed into two separate physical stimuli, an initial step-change in shear (or a large temporal gradient in shear) followed by steady shear, we hypothesized that the induction of those atherogenesis-related genes (eg, PDGF-A and MCP-1) is associated with the temporal gradient in shear but not steady shear.

In response to fluid shear stress, EC elaborate various bioactive substances that may be involved in the regulation of these atherogenesis-related genes, such as NO.14 15 The loss of endothelium-derived NO control of vasomotion precedes the onset of atherogenesis.16 In addition to being a potent vasodilator, NO is also an inhibitor of platelet aggregation and smooth muscle cell proliferation, properties that have been shown to be antiatherogenic.17 Exposure of EC to fluid shear also stimulates the activation of transcription factors, such as egr-1,18 AP-1,19 and NF{kappa}B.19 20 Activated egr-1 binds to the proximal PDGF-A promoter by displacing sp-1 from their overlapping recognition element, and the egr-1 binding site has been identified as a cis-element for the induction of PDGF-A by fluid shear.18 The promoter of MCP-1 gene contains both NF{kappa}B and AP-1 binding sites, which may coordinately modulate shear inducibility of MCP-1.20 21 22 NO may affect expression of these genes by activation or inactivation of their transcription factors.

The present study focuses on the differential responses of two atherogenesis-related genes, PDGF-A and MCP-1, to steady shear and temporal gradient in shear in human umbilical vein EC (HUVEC). To separate these two distinct mechanical stimuli, various flow regimens were used that included impulse, step, and ramp flow profiles. By creating these simplified flow profiles, we can better characterize the responses of EC to physiological flows by deconstructing the shear stress profile into two components: steady (or slowly transiting) shear stress and impulse (or temporal gradient) in shear stress, as exists in rapidly fluctuating flow. The latter profile was chosen not to simulate physiological flow, but rather to subject the cells to one component of physiological flow. We demonstrate, for the first time, that the rate of change in shear stress rather than steady shear stress is a potent stimulus for the expression of such atherogenesis-related genes as PDGF-A and MCP-1. By using NO synthase inhibitors and NO donors, and by measuring expression of the transcription factors NF{kappa}B and egr-1, a dual role for NO in regulating the expression of MCP-1 and PDGF-A is also demonstrated in this study.


*    Methods
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 *Methods
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Cell Culture and Experimental Preparation
Primary HUVEC isolation was performed as previously described.23 Briefly, cells were harvested from human umbilical veins by collagenase (Boehringer Mannheim) treatment and were then seeded onto glass microscope slides (cell area, 16 cm2). The cells were grown to confluence within 3 to 4 days in ATP-free M199 medium supplemented with 20% FBS (Hyclone), and with 2 mmol/L L-glutamine, 0.5 U/mL penicillin, and 0.05 mg/mL streptomycin (Sigma). Before shear exposure, the confluent cells were serum-starved for 48 hours in 0.5% FBS-supplemented M199 to establish quiescence in the monolayers. For NO synthase inhibitor and NO donor studies, cell monolayers were preincubated with NG-amino-L-arginine (L-NAA; Alexis), spermine-NONOate (SPR/NO; Alexis), or spermine (SPR; Alexis) at different concentrations for 1 hour before being subjected to shear. All cell cultures were maintained in a humidified 5% CO2/95% air incubator at 37°C.

Flow Experiments
Cell monolayers on glass slides were subjected to well-defined laminar flow for various times in a parallel-plate flow chamber, in which perfusing medium was driven by a syringe pump (pump 44, Harvard Apparatus) or a constant head flow loop as described previously.23 The computer-controlled syringe pump was programmed to generate different flow profiles. The flow rate (and hence shear stress) was changed in 1-s microsteps with volume/flow increments determined from the slope of the overall increase in shear stress. Four well-defined laminar flow profiles were generated to which EC were exposed (Figure 1Down). These profiles were designed and used to separate the effect of different dynamic flow stimuli presented to EC. The following profiles were applied: (1) step flow (instantaneous shear stress increase from 0 to 16 dyne/cm2, followed by steady shear for a sustained period); (2) ramp flow (shear stress smoothly transited from 0 to 16 dyne/cm2 during 10 minutes, and then sustained for a desired period); (3) impulse flow (a 3-s impulse of 16 dyne/cm2); and (4) reverse impulse flow (a step increase to 16 dyne/cm2, sustained for 3 s, followed by a ramped decrease to 0 dyne/cm2 during 10 minutes).



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  		Figure 1. Laminar flow profiles: step flow, ramp flow, impulse flow (3 s), and reverse impulse flow. The maximal shear stress in each profile is 16 dyne/cm2. The duration of the upward and downward ramp for ramp and reverse impulse flow, respectively, was 10 minutes.

The entire flow device was placed in an air curtain incubator and maintained at 37°C throughout the experiment. The fluid used to shear the cells was the same as the medium used to preincubate the cells before the shear exposure, and was placed in the incubator for 2 to 3 hours before the flow experiments. Dextran (1.25%, 2 000 000 MW; Sigma) was added to the medium. This addition increased the media viscosity from 1 to 3 cP. The shear stress was calculated as previously described.24 After impulse flow exposure, slides were removed from the flow chamber, placed back into Petri dishes, and kept in the incubator for the appropriate matched period. Time-matched stationary (no-flow) controls were performed in Petri dishes.

RNA Isolation and Northern Blotting
Slides were removed from the flow chamber or Petri dishes, and cell monolayers were washed twice with PBS and then lysed. Total RNA was then isolated using an RNAeasy Total RNA Kit (Qiagen). After quantification by measuring absorbance at 260 nm, equal amounts of total RNA were loaded and electrophoresed on 1% formaldehyde-agarose gels, transferred to nylon membranes (Micron Separation), and then fixed by ultraviolet irradiation. The following cDNA fragments were labeled with [32P]dCTP (ICN Radiochemical) and used as hybridization probes: for PDGF-A, a 1.3-kb EcoRI fragment of PDGF-A-13.125 ; for MCP-1, a 0.7-kb EcoRI/BamHI fragment of MCP-1 (ATCC); for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an 0.8-kb XbaI/PstI fragment of pHcGAP (ATCC); and for egr-1, a 3.1-kb EcoRI/BamHI fragment of egr-1.26 Nylon filters with uniformly transferred RNA were prehybridized for 2 to 4 hours and then hybridized at 42°C in hybridization buffer overnight with the appropriate 32P-labeled DNA probes. The membranes were then washed and exposed to Kodak X-omat film at -70°C for autoradiography. For some membranes the bound probes were stripped off by washing the membranes in stripping buffer (1 mmol/L TrisCl, pH 8; 1 mmol/L EDTA, pH 8; and 0.1x Denhardts) at 75°C for 2 hours. The same membranes were subsequently rehybridized with other probes. Densitometry was performed using a model IS-10000 image analyzer (Alpha Innotech).

Protein Preparation and Western Blotting
Cells were immediately washed with ice-cold PBS and lysed at 4°C in lysis buffer (20 mmol/L sodium HEPES, pH 7.4; 1 mmol/L EDTA; 2 mmol/L MgCl2; 10 µg/mL PMSF; 2 µg/mL leupeptin; 2 µg/mL aprotinin), followed by scraping, sonication, centrifugation (15 minutes at 14 000g), and boiling (5 minutes at 100°C). Total cell lysates (50 µg/protein) were separated by SDS-polyacrylamide gel electrophoresis (10% running, 4% stacking). The protein was then transferred to nitrocellulose filters (Millipore). Blocking was performed overnight at 4°C in Tris-buffered saline (TBS) plus 5% nonfat dry milk. The blots were incubated with affinity-purified rabbit antibody to I{kappa}B-{alpha} (1:1000; Santa Cruz) overnight in buffer containing TBS with 0.05% Tween-20 and 5% nonfat dry milk (Blotto), followed by incubation with the horseradish peroxidase–conjugated goat anti-rabbit antibody (1:2000; New England) in Blotto for 45 minutes. Immunodetection was accomplished using chemiluminescence (Super Signal CL-HRP, Pierce Chemical Corp).

NO Determination
Confluent EC were incubated in modified Eagle's minimum essential medium (MEM, Sigma) containing 2% FBS overnight, and then incubated for 4 hours in serum-deprived modified MEM before exposure to shear. Two aliquots of 0.5 mL of perfusing media were obtained immediately before and 4 hours after flow shear treatment. NO levels (NO2- + NO3-) in the perfusing media were measured by an NO chemiluminescence analyzer (Sievers 270B).

Statistics
The results are presented as mean±SE compared with controls and among separate experiments. Significance was determined by unpaired Student's t test (P<0.05).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effects of Temporal Gradient in Shear and Steady Shear Stress on Gene Expression of MCP-1 and PDGF-A
To differentiate the effects of temporal gradient in shear stress versus steady shear stress on endothelial MCP-1 and PDGF-A expression, confluent HUVEC were subjected to ramp, step, or impulse laminar flow at a maximal shear stress of 16 dyne/cm2 for 1.5 hours. The level and duration of shear stress was selected because an average shear stress of 16 dyne/cm2 is within the physiological range in arterial vasculature and has been demonstrated to induce significant increases in MCP-18 and PDGF-A9 mRNA levels in HUVEC, with the maximal response at 1.5 hours. Ramp flow consistently elicited only minor increases in MCP-1 and PDGF-A expression relative to the static levels. Step flow produced a 2-fold increase in MCP-1 mRNA levels greater than that induced by ramp flow. Impulse flow resulted in a 7-fold increase in MCP-1 expression relative to ramp flow. For PDGF-A expression, step flow elicited a 4-fold increase in mRNA level, whereas impulse flow produced a 6-fold increase in mRNA level relative to ramp flow (Figure 2Down).



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  		Figure 2. A, Northern blot showing the effect of ramp, step, impulse, and reverse impulse flow on mRNA expression of MCP-1 and PDGF-A in HUVEC. Cells were exposed to ramp or step flow for 1.5 hours. Cells subjected to reverse impulse and impulse flow were statically incubated for 1.5 or 4 hours as indicated. GAPDH mRNA levels (bottom) were used to normalize those of MCP-1 and PDGF-A. B, Bar graph demonstrating the relative levels of MCP-1 and PDGF-A mRNA (relative to their respective static controls) under various flow regimens as mentioned above. Data points are presented as mean±SE (n=4). *P<0.05 versus ramp flow; #P<0.01 versus ramp flow; and +P<0.05 versus impulse flow at 1.5 hours.

To account for the possible effect of small differences in the duration of steady flow caused by linear ramp on MCP-1 and PDGF-A mRNA levels, HUVEC were subjected to two different ramp flows with the shear stress smoothly transited from 0 to 16 dyne/cm2 during 10 minutes, and then continuing for 80 minutes (Figure 2Up) or 90 minutes (data not shown) at 16 dyne/cm2. No significant differences in gene expression were observed between these two ramps.

To account for possible stimulation of cells by the manipulation during assembly of the cell-covered slide on the chamber, the slide was placed gently on the chamber without flow for 10 minutes, and then placed back in the Petri dish for another 80 minutes. This procedure of manipulating slides did not induce significant gene expression (data not shown).

Because in every case ramp flow elicited minor increases in gene expression relative to the static condition (Figure 2Up), the effect of the small increments in flow rate during the ramp period was investigated. Cells were subjected to ramp shear stress, which increased from 0 to 0.4 dyne/cm2 at the same increments (0.03 dyne/cm2 per increment) and time steps (1 increment/s) as the ramp flow, and then maintained at 0.4 dyne/cm2 for 90 minutes. This low level of shear stress induced MCP-1 gene expression at a level equivalent to that produced by ramp flow (data not shown). The result indicates that the small steps of 0.03 dyne/cm2 used to ramp the flow are themselves sufficient to stimulate MCP-1 expression, albeit not at the level of impulse flow, suggesting that further refinement in smoothly applying ramp flow should lead to a reduction of this ramp flow-induced gene expression.

To further evaluate the effects of gradient in shear stress, cells exposed to impulse flow were placed under static incubation for a longer time period. The levels of MCP-1 and PDGF-A mRNA induced by impulse flow at 4 hours were significantly higher than the mRNA levels at 1.5 hours, indicating sustained and increasing expression (Figure 2Up). The high levels of expression for PDGF-A and MCP-1 induced by impulse flow were sustained even at 8 hours (data not shown). It appears, therefore, that the induction of gene expression by impulse flow is not as transient as that by step flow, which has been shown to return to or even fall below basal levels after 4 hours.8 9

Impulse flow can be further decomposed to two stimuli: step increase and step decrease in shear. To elucidate the possible effect of a step decrease on gene expression, the responses to reverse impulse flow (see Figure 1Up) were analyzed. The levels of MCP-1 and PDGF-A mRNA induced by reverse impulse flow were equivalent to that induced by impulse flow at 1.5 hours (Figure 2AUp). The result suggests that either the step decrease in flow has no effect or the stimulus of the step increase in flow saturated the response.

Effect of NO on the Temporal Gradient in Shear-Induced Endothelial MCP-1 and PDGF-A Expression
The potential role of steady flow-associated production of NO14 in MCP-1 and PDGF-A expression induced by the temporal gradient in shear was investigated by using L-NAA, a NOS inhibitor, in cells subjected to step flow for 4 hours. L-NAA (100 µmol/L) significantly increased step flow-induced MCP-1 and PDGF-A mRNA expression (0.6- and 3.2-fold, respectively) (Figure 3ADown and 3BDown), and correspondingly decreased step flow-induced NO production by 82% (inset, Figure 3BDown) compared with that induced by step flow without treatment with L-NAA.



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  		Figure 3. A, Northern blot demonstrating the effects of step and impulse flow on endothelial expression of MCP-1 and PDGF-A in the absence or presence of L-NAA (300 µmol/L) or SPR/NO (100 µmol/L) for 4 hours. B, Bar graph showing the corresponding effect of L-NAA (100 µmol/L) on endothelial mRNA expression of MCP-1 and PDGF-A as well as NO production (inset) induced by step flow. The relative levels of MCP-1 and PDGF-A mRNA were normalized to GAPDH mRNA. Data are presented as mean±SE (n=3). *P<0.05 versus the control without L-NAA (white bar). C, Graph showing the dose-dependent effects of SPR/NO on the endothelial mRNA expression of MCP-1 and PDGF-A induced by impulse flow. MCP-1 and PDGF-A mRNA levels are expressed as a percentage of that induced by impulse flow stimulation (control). Data are presented as mean±SE (n=3).

To determine the effect of exogenous NO, cells subjected to impulse flow were treated with SPR/NO or SPR at various concentrations for 4 hours. The NO donor downregulated impulse flow-induced MCP-1 and PDGF-A expression in a dose-dependent manner (Figure 3AUp and 3CUp). SPR/NO (1, 10, and 100 µmol/L) decreased impulse flow-induced MCP-1 mRNA by 11%, 36%, and 80%, and PDGF-A mRNA by 23%, 50%, and 57%, respectively, compared with impulse flow alone. No significant effect on mRNA levels was observed with SPR alone.

Because impulse flow stimulates a burst in production of NO,4 14 we further investigated whether this burst in NO is involved in impulse-flow-induced gene expression. L-NAA (50, 100, and 300 µmol/L) dose-dependently decreased the impulse flow-induced MCP-1 mRNA by 18%, 22%, and 41%, and PDGF-A mRNA by 34%, 74%, and 76%, respectively, compared with expression induced by impulse flow alone (Figure 4Down). Similar results were observed with another NOS inhibitor, NG-nitro-L-arginine (data not shown). Thus it appears that impulse flow-induced gene expression is mediated by NO production.



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  		Figure 4. Northern blot demonstrating the dose-dependent effects of L-NAA on endothelial mRNA expression of MCP-1 and PDGF-A 4 hours after being subjected to impulse flow. GAPDH mRNA levels (bottom) were used to normalize those of MCP-1 and PDGF-A.

Effect of NO on I{kappa}B-{alpha} Expression Induced by Temporal Gradients in Shear
The promoter of the human MCP-1 gene contains functionally active NF{kappa}B binding sites.27 The activation of NF{kappa}B by tumor necrosis factor-{alpha} has been shown to occur within 15 minutes and coincides with phosphorylation and degradation of I{kappa}B-{alpha}, which is inhibited by NO after 2 hours.28 To determine how exogenous NO regulates the activation of NF{kappa}B induced by impulse flow, we investigated the fate of impulse flow-stimulated I{kappa}B-{alpha} expression in the presence and absence of SPR/NO (100 µmol/L). Exposure to impulse flow resulted in sustained (from 15 minutes to 4 hours) 6- to 7-fold I{kappa}B-{alpha} degradation. SPR/NO initially (15 minutes) reduced impulse flow-induced I{kappa}B-{alpha} expression, but increased its expression by 4- to 5-fold at 2 and 4 hours (Figure 5ADown). This indicated that NO inhibits NF{kappa}B activation not by stabilization of I{kappa}B-{alpha} but rather by increasing I{kappa}B-{alpha} resynthesis.



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  		Figure 5. Immunoblots of total cell lysates showing the effect of impulse flow on I{kappa}B-{alpha} levels with or without SPR/NO (100 µmol/L; A) at the indicated time points and L-NAA (300 µmol/L; B) at 15 minutes. The amount of protein loading correspond to the nonspecific bands (NS) in each column. Two separate experiments yielded similar results.

To determine whether endogenous NO is involved in impulse flow-induced I{kappa}B-{alpha} degradation, EC were treated with L-NAA (300 µmol/L) 1 hour before subjecting to impulse flow. L-NAA inhibited impulse flow-induced I{kappa}B-{alpha} degradation by 3-fold at 15 minutes (Figure 5BUp), but had no significant inhibitory effect on the expression of I{kappa}B-{alpha} in static cells. The inhibitory effect of L-NAA on impulse flow-induced I{kappa}B-{alpha} degradation was also observed at 4 hours (data not shown). All data presented were normalized by nonspecific bands, which correspond to the amount of the protein loading.

Effect of NO on egr-1 mRNA Expression Induced by Temporal Gradient in Shear
Because the egr-1 binding site in the proximal PDGF-A promoter is responsible for its shear stress inducibility,18 we investigated the effect of NO on egr-1 mRNA expression. EC were exposed to impulse flow in the presence or absence of L-NAA and SPR/NO for 4 hours. L-NAA (100 µmol/L) almost completely abolished impulse flow-induced egr-1 mRNA expression. On the other hand, addition of SPR/NO (10 µmol/L) also significantly decreased the impulse-flow induced egr-1 mRNA expression. SPR alone did not cause a significant change in egr-1 mRNA levels (Figure 6Down).



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  		Figure 6. Northern blot demonstrating the effect of impulse flow on endothelial egr-1 mRNA levels in the absence or presence of L-NAA (100 µmol/L) or SPR/NO (10 µmol/L) for 4 hours. GAPDH mRNA levels (bottom) were used to normalize that of egr-1. This is a representative assay from 2 separate experiments.


*    Discussion
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 *Discussion
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Hemodynamic shear stress influences the physiology of EC and participates in the development and progression of arterial diseases.29 30 31 In addition to steady shear stress, recent attention has also focused on gradients in shear stress as a contributing factor that may link disturbed fluid dynamics to atherosclerosis.4 5 6 Exposure of endothelial cells to fluid flow in vitro has been used as a simplified means of understanding the precise role of fluid shear in endothelial function. The concept that fluid flow provides two distinct stimuli—temporal gradient in shear and steady shear—to the endothelium has been previously suggested in prostacyclin production,23 and demonstrated in a number of studies, including NO and cGMP production4 14 and pinocytosis.32 This study, for the first time, directly compared the induction of certain atherogenesis-related genes (PDGF-A and MCP-1) in EC exposed to temporal gradient in shear and steady shear, using three well-defined laminar flow profiles: impulse, step, and ramp flow.

The present experiments demonstrate the enhanced expression of MCP-1 and PDGF-A mRNA at 1.5 hours in EC exposed to impulse flow relative to step flow and even more pronounced elevations with respect to ramp flow (Figure 2Up). In addition, impulse flow was shown to further increase expression of these two genes after 1.5 hours as observed at 4 hours after shear stimulation (Figure 2Up). This is in sharp contrast to the effect of step flow at 4 hours, when gene expression returns to basal or sub-basal levels.8 9 Both step and impulse profiles contain a sharp gradient in shear stress. However, a component present in both step and ramp profiles—steady shear stress—is absent in impulse flow. The contrasting abilities of step and impulse flow, and the inability of ramp flow to elicit significant gene expression, suggest that temporal gradient in shear but not steady shear stimulates the expression of these atherogenesis-related genes.

Temporal gradients in shear in the absence of steady shear stress induced a larger and sustained expression. Thus, one can hypothesize that the continued exposure of EC to steady shear promotes a downregulation of these atherogenesis-related genes. This hypothesis is supported by the observation that steady shear downregulates the expression of vascular cell adhesion molecule-1 (VCAM-1), another potential proatherogenic leukocyte adhesion molecule.33 Together with the observation of the selective and sustained upregulation of three endothelial antiatherogenic genes (eNOS, COX-2, and MnSOD) by steady shear,13 our findings suggest that steady shear may exert a coordinated influence on endothelial function, the net effect of which is antiatherogenic. In contrast, temporal gradients in shear, such as may be found in areas with disturbed hemodynamic flow, induce endothelial atherogenesis-related gene expression, and thus serve as a biomechanical risk factor.

The present study focused on a physiological level of steady shear of 16 dyne/cm2 that in vivo approximates the average wall shear typically encountered in lesion-protected areas of the vasculature. We and others have shown, however, that exposure of EC to prolonged steady low shear (2 dyne/cm2) induces sustained increases in endothelin-1 (a mitogen for vascular smooth muscle cells) release, as well as NF{kappa}B, MCP-1, and VCAM-1 expression.20 34 35 These observations suggest that steady low shear may be another biomechanical risk factor. Taken together, the coordinated induction of these atherogenic factors may occur at the lesion-prone area, where the disturbed hemodynamic flow patterns—low mean shear levels associated with temporal gradients in shear—prevail, and thus may be one of the mechanisms contributing to the focal distribution of early atherosclerotic lesions.

Until now the role of NO in regulation of gene expression has been controversial because both stimulatory and inhibitory effects have been observed. For example, NO has been shown to inhibit cytokine-induced expression of VCAM-1, ICAM-1, and MCP-136 37 38 39 through cGMP-independent pathways, whereas NO can stimulate the expression of certain immediate early genes through cGMP-dependent pathways.40 41 42 43 Here we observed the effects of NO induced by different flow profiles, as well as those of exogenous NO, on gene expression. We found that NO induced by impulse flow stimulated MCP-1 and PDGF-A expression (Figure 4Up), whereas sustained NO release, generated by steady shear (or NO donors), inhibited the expression of these atherogenesis-related genes (Figure 3Up). This dual action of NO is further supported by the activation of NF{kappa}B (Figure 5Up) and the induction of egr-1 mRNA (Figure 6Up) by impulse flow-induced NO production, and downregulation of these transcription factors by sustained NO release (Figures 5Up and 6Up). The results we provided demonstrate for the first time that NO has two opposing (stimulatory and inhibitory) actions on the same genes, and NO exerts its dual actions on gene expression by coordinately regulating its transcription factor. This apparently conflicting action of NO may be related to the kinetics and signaling of NO production induced by different flow profiles. Previous work has demonstrated that temporal gradient in shear (impulse flow) produces a transient burst of NO, whereas steady shear (ramp flow) induces a sustained low rate of NO in EC; the former is dependent on and the latter is independent of calcium and G-protein.4 14 It needs to be further elucidated, however, how this bimodal NO production differently regulates gene expression.

Evidence provided here shows that NO plays a crucial role in temporal gradients in shear-induced stimulatory effects as well as steady shear-mediated inhibitory effects. It is not clear, however, why NOS inhibition enhanced expression of MCP-1 and PDGF-A by step flow (Figure 3Up). One speculative possibility regards the involvement of superoxide (O2-). Fluid shear can induce sustained production of reactive oxygen species,44 which induces the expression of ICAM-1 and c-fos.45 46 The breakdown of O2- occurs mainly by reaction with NO to form peroxynitrite (ONOO-),47 which has cytoprotective effects similar to those of NO at low concentration.48 Although eNOS can produce NO as well as O2-, NOS inhibitors may only block NO production,49 50 leading to excess O2-. Superoxide, in turn, can activate the transcription factors AP-1,51 NF{kappa}B,52 and egr-1,53 and therefore induce expression of MCP-1 and PDGF-A. Another source of O2- is activated Ras.54 Step flow has been shown to induce a transient and rapid activation of Ras in EC, leading to the induction of MCP-1 expression.55 NO has also been shown to inhibit Ras activation.56 With the inhibition of NO production by L-NAA, activation of Ras may be more sustained. Therefore, the Ras-O2- pathway may be another source for O2- involved in step flow-induced MCP-1 and PDGF-A expression when sustained NO production is inhibited.

In summary, we have shown that temporal gradients in shear stress lead to enhanced and sustained expression of MCP-1 and PDGF-A in HUVEC, whereas the presence of steady laminar shear stress reduces these levels. Both the stimulatory effects of temporal gradients in shear and the inhibitory effects of steady shear on gene expression have been found to be mediated by NO, probably by activation and inactivation of their transcription factors, NF{kappa}B and egr-1. Although additional investigation is necessary to further elucidate and refine the concepts presented in this paper, understanding the distinct contribution of each component in the complicated flow regimen may provide an important clue to the focal development of atherosclerosis and vascular disease in general.


*    Acknowledgments
 
This work was supported by National Heart, Lung, and Blood Institute grant HL-40696. X. Bao is the recipient of a National Research Service Award from the National Institute of Health. We thank T. McAllister for his excellent technical assistance, and C. Shilling and C.B. Clark for their helpful discussions. We also express our sincere appreciation to the nurses at the maternity wards of Sharp Memorial Hospital and Mercy Hospital of San Diego for supplying umbilical cords.

Received June 4, 1998; accepted September 30, 1998.


*    References
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