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Am J Physiol Cell Physiol 293: C87-C94, 2007. First published February 21, 2007; doi:10.1152/ajpcell.00585.2006
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VASCULAR BIOLOGY

Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types

Hak-Joon Sung, Andrew Yee, Suzanne G. Eskin, and Larry V. McIntire

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia

Submitted 21 November 2006 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H2O2 production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H2O2 levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC.

phenotype; reactive oxygen species; inflammation; shear stress

MECHANICAL FORCES associated with blood flow and pressure have long been recognized as potential instigators of cardiovascular diseases (40). Locations of atherosclerotic plaques in the cardiovascular system have been correlated with regions of disturbed blood flow (43). Venous bypass grafts are more prone to accelerated atherosclerosis than arterial grafts (3, 30). Recently, transcriptional studies of endothelial cell (EC) diversity reveal that ECs from different vascular beds have distinct phenotypes that may contribute to the site specificity of vascular pathogenesis (8, 11).

Injury to the vascular endothelium from oxidative stress, imposed exogenously or generated endogenously, leads to vascular disease (29). Oxidative stress is induced by overproduction of reactive oxygen species (ROS) or insufficient metabolism of ROS. However, low levels of ROS are essential participants in cell signaling (16). ROS are generated by oxidative processes in the cell, principally by NAD(P)H oxidase, endothelial nitric oxide synthase (eNOS), xanthine oxidase, and mitochondrial electron transport (34). ROS are then metabolized to hydrogen peroxide (H2O2) by dismutation of superoxide (O2bullet–) by superoxide dismutase (SOD) and converted to water principally by glutathione peroxidase (GPx) and catalase, although GPx also catalyzes reactions that turn organic peroxides into the corresponding stable alcohols, using glutathione as a source of reducing equivalents.

Cyclic strain studies of cultured EC have been shown to increase ROS production (1, 20, 21, 31, 45). Microarray studies of EC subjected to cyclic strain showed that most differentially expressed genes are associated with oxidative stress (14). In addition, inclusion of a motion control condition indicates that cyclic strain, per se, may not be as potent as fluid agitation in mediating endothelial gene expression (14). To ask these questions more directly, we subjected arterial (human aortic endothelial cells; HAEC) and venous (human umbilical vein endothelial cells; HUVEC) ECs to cyclic strain and motion control and measured indicators of oxidative stress. In vivo, arterial ECs are exposed to higher levels of cyclic strain than venous ECs and thus can be considered adapted to physiological levels (10%) of cyclic strain (12). HUVEC may be particularly sensitive to oxidative damage, in light of findings that 50–100 µM exogenous H2O2 is apoptotic for HUVEC, but 200 µM exogenous H2O2 produces no significant cell death in arterial EC (4).

In the present study, we show that two types of cultured EC, HAEC and HUVEC, respond surprisingly differently to cyclic strain and motion control in their ROS production, oxidative stress-related enzyme activity, and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 expression. We hypothesize that venous ECs are more susceptible to oxidative damage when exposed to cyclic strain, whereas arterial ECs are more susceptible to damage from oscillatory fluid agitation generated in the motion control, in which the protective properties of cyclic strain are absent.


   METHODS
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 METHODS
RESULTS
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Cell culture. HUVEC and HAEC purchased from Cambrex (Walkersville, MD) were cultured in EGM-2 medium, according to the supplier's instructions. HAEC were derived from three individual donors; HUVEC were derived from three independently pooled lots. Cells (passages 4–6) were seeded at ~100,000 cells/cm2 onto silicone membranes (Specialty Manufacturing, Saginaw, MI) coated with 1% gelatin cross-linked with 0.5% glutaraldehyde (38). Twenty-four hours after seeding, the cells were given fresh medium and used in experiments.

Cyclic strain experiments. Cells were subjected to cyclic strain (1 Hz, 10% or 20% strain) for 24 h, as previously described (14). The stretcher device consisted of a silicone membrane suspended slightly above and parallel to the bottom of a polycarbonate box between two sets of stainless-steel plates. Each set contained two plates that clamped the ends of the membrane. For cyclic strain, one set of plates was fixed to the end of the box, while the second set was attached to a cam-driven movable piston at the opposite end of the box, allowing for cyclic, unidirectional deformation of the membrane. For motion control, one end was fixed by a set of immobile plates (similarly to cyclic strain), while the other end was fixed around a stainless-steel template that prevented the membrane from stretching. The same cam as that driving the strain membrane drove a piston-plate assembly at the template end, resulting in a similar oscillatory fluid motion without stretching the cells. The maximal shear stress oscillations were approximated from the velocity profile of fluid flow near an oscillating flat plate (37) for cyclic strain (0 ± 0.15 and 0 ± 0.24 dyn/cm2 for 10% and 20% strain, respectively) and from the velocity profile of the shear wave layer of oscillatory fluid flow over a plate with high characteristic frequency fluctuations (19) for motion control (0 ± 0.21 and 0 ± 0.42 dyn/cm2 for 10% and 20% deformation, respectively). For the static condition, the assembly was identical to that for the motion control and strain conditions, but the piston was not moved. For each condition (cyclic strain, motion control, and static), three independent experiments were performed.

Immunofluorescence. Immediately after cyclic strain or control experiments, the membranes were removed from the strain apparatus, and the cells were washed with PBS and fixed with 10% formalin. F-actin was stained with Oregon Green 488 phalloidin (Molecular Probes, Eugene, OR), and nuclei were counterstained with Hoechst 33258 (Sigma, St. Louis, MO). Cells were imaged by confocal microscopy (Zeiss, Thornwood, NY). The experiments for actin visualization were separate from those in which ROS production and metabolism were measured.

ROS production. Levels of intracellular ROS were determined by incubation of EC monolayers with the H2O2-sensitive fluorophore dichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes) or the O2bullet–-sensitive fluorophore dihydroethidium (DHE; Molecular Probes) (2, 32). After the membranes were removed from the apparatus, the cells were washed in ice-cold PBS. Cells were incubated with 5 µM DCFDA in PBS for 20 min or with 5 µM of DHE in PBS for 15 min and imaged by confocal microscopy. Fluorescence intensity (4 image fields per condition) was measured with ImageJ (National Institutes of Health). Background-subtracted values were normalized to cell number.

Enzyme activity. Immediately after experiments, the membranes were cut from the apparatus and washed in PBS, and the cells were lysed in 10 mM sodium phosphate, pH 7.2, containing 150 mM NaCl (PBS), 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 0.2% sodium azide (15). Catalase activity was assessed with the Catalase-520 kit, GPx activity was measured with the GPx-340 kit, and SOD activity was measured with the SOD-525 kit (all kits from OXIS International, Portland, OR), according to the manufacturer's instructions. Total protein concentration was quantitated with the Bradford protein assay kit (Bio-Rad, Hercules, CA), and activity was normalized to total protein content.

Western blot analysis. The cells were lysed as above for enzyme assays (15), and protein content was measured. Ten micrograms of protein from the cell lysate was mixed with Laemmli buffer, boiled for 10 min, separated by SDS-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Biosciences, Piscataway, NJ). For VCAM-1, the membranes were incubated with mouse anti-human VCAM-1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Primary antibody binding was detected with horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Biosciences). For ICAM-1, the membranes were incubated with rabbit anti-human ICAM-1 polyclonal antibody (Santa Cruz Biotechnology). Bound primary antibodies were detected with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Biosciences). Signals were visualized by chemiluminescent detection according to the manufacturer's protocol (ECL plus Western blotting detection system, Amersham Biosciences). The membranes were stripped and reblotted with mouse anti-human beta-actin monoclonal antibody (Sigma) to verify equal protein loading and to normalize band intensity. Band intensities were analyzed by densitometry (Kodak ID image analysis, Kodak, Rochester, NY).

To investigate the effect of ROS inhibition on VCAM-1 and ICAM-1 protein expression, cells were mechanically stimulated in the presence or absence of 4 µM diphenyleneiodonium chloride (DPI; Sigma D2926). The effect of DPI on cell viability was assessed with the Live/Dead Viability/Cytotoxicity Kit (L-3224, Molecular Probes). To stimulate VCAM-1 and ICAM-1 production, cells were treated with 100 pg/ml of interleukin (IL)-1beta under static conditions for 24 h.

Statistical analysis. All comparisons were performed with Student's t-test. A P value <0.05 was considered significant. Results are means ± SE.


   RESULTS
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 RESULTS
DISCUSSION
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Baseline values. Examination of baseline ROS indicators in HUVEC and HAEC in static culture (Table 1) showed differences in levels of H2O2, O2bullet–, and GPx and SOD activity. HUVEC produced 2.5 ± 0.5-fold more H2O2 and 1.23 ± 0.12 fold more O2bullet– than HAEC. Also, SOD activity in HAEC was 2.7 ± 0.9-fold higher and GPx activity was 1.4 ± 0.1-fold higher than in HUVEC. Catalase activity and VCAM-1 and ICAM-1 expression were similar in both cell types.


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  		Table 1. Baseline values in static culture

 
Morphology. Actin filaments aligned perpendicular to the axis of stretch after 24 h of 10% cyclic strain but not in the matched motion control and static cultures, in which the cells appeared randomly oriented. Cell adhesion was not obviously compromised after 24 h of either cyclic strain or motion control in either HUVEC or HAEC (Fig. 1).


Figure 1
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  		Fig. 1. Actin microfilaments in human umbilical vein endothelial cells (HUVEC; A–C) and human aortic endothelial cells (HAEC; D–F) after 24-h exposure to 10% cyclic strain (CS; A, D), motion control (MC; B, E), and static conditions (SC; C, F) Green, Oregon green phalloidin; blue, Hoechst nuclear stain; yellow arrow, direction of strain. Cell alignment perpendicular to the principal axis of stretch is consistent with earlier reports (23). Scale bar = 50 µm.

 
ROS production. The greatest increase in production of intracellular H2O2 (33.0 ± 3.5-fold over static levels) was in HAEC exposed to 20% motion control, compared with a 13.4 ± 0.7-fold increase produced by 20% cyclic strain (Fig. 2A; P < 0.05). Also, significantly more H2O2 was produced by 10% motion control than by 10% cyclic strain in HAEC (9.8 ± 1.2-fold vs. 5.0 ± 0.6-fold, respectively; P < 0.05). Conversely, we observed more H2O2 produced in HUVEC under cyclic strain (7.4 ± 0.2-fold at 10% and 7.4 ± 0.3-fold at 20%) than under motion control (1.9 ± 0.2-fold at 10% and 3.9 ± 0.2-fold at 20%; P < 0.05 for cyclic strain vs. motion control at both 10% and 20% deformation). It is noteworthy that H2O2 responses in HAEC appeared more sensitive to an increase in deformation than HUVEC (Fig. 2A, compare 10% to 20%). In Fig. 2B, both HUVEC and HAEC showed significant increases in O2bullet– production in response to increasing substrate deformation from 10% to 20% under both cyclic strain and motion control conditions. Significance (P < 0.05) was detected only in HAEC under 20% motion control (4.25 ± 0.27-fold over static) vs. 20% cyclic strain (3.5 ± 0.23-fold over static).


Figure 2
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  		Fig. 2. Production of H2O2 (A) and O2bullet– (B) was measured in HUVEC and HAEC subjected to CS or MC and normalized to static control levels. Measurements were taken from confocal microscope images of 4 fields per condition, 3 independent experiments and normalized to total cell number. Results are means ± SE. *P < 0.05 for CS vs. MC; {dagger}P < 0.05 for 10% vs. 20% strain.

 
SOD, GPx, and catalase activity. Since HUVEC and HAEC showed opposite responses in intracellular H2O2 content to cyclic strain vs. motion control and since the trend in O2bullet– responses was consistent with the H2O2 results, we examined the activity of three different enzymes that regulate the equilibrium of intracellular H2O2. SOD converts O2bullet– to H2O2, which is then converted to water and O2 by catalase and GPx. SOD activity in HUVEC increased 11.7 ± 0.9-fold under 10% cyclic strain and 7.8 ± 0.5-fold under 10% motion control (P < 0.05). At 20% deformation, SOD activity increased 14.4 ± 1.8-fold under cyclic strain and 10.5 ± 1.9-fold under motion control (Fig. 3A). In HAEC, 10% cyclic strain and 10% motion control increased SOD activity 4.7 ± 0.4-fold and 7.3 ± 0.04-fold, respectively. SOD activity increased 7.6 ± 0.6-fold under 20% cyclic strain relative to 11.2 ± 0.7-fold under 20% motion control.


Figure 3
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  		Fig. 3. Enzyme activities of superoxide dismutase (SOD; A), glutathione peroxidase (GPx; B), and catalase (C) measured from 3 independent experiments and normalized to milligrams of protein. Values for CS and MC have been normalized to static controls. Results are means ± SE. *P < 0.05 for CS vs. MC; {dagger}P < 0.05 for 10% vs. 20% strain.

 
GPx (Fig. 3B) was significantly more active (P < 0.05) in HUVEC under 10% cyclic strain (17.3 ± 1.4-fold) than under 10% motion control (5.5 ± 0.6-fold). At 20% deformation, increase in GPx activity due to cyclic strain (15.8 ± 1.7-fold) was not significantly different from increase due to motion control (12.2 ± 1.7-fold). In HAEC, 10% cyclic strain increased GPx activity 2.8 ± 0.4-fold, whereas 10% motion control further activated GPx 6.6 ± 0.6-fold (P < 0.05). Under 20% cyclic strain and 20% motion control, GPx activity increased 10.6 ± 0.9-fold and 14.4 ± 1.7-fold, respectively, but no significant differences were found. Significant (P < 0.05) responses to an increase in deformation were found in HUVEC and HAEC under motion control and in HAEC under cyclic strain.

Catalase activity (Fig. 3C) in HUVEC responded similarly to GPx. At 10% deformation, cyclic strain increased catalase activity more than motion control (6.6 ± 0.4-fold vs. 3.9 ± 0.4-fold, P < 0.05). At 20% deformation, increases in catalase activity were not statistically different between cyclic strain and motion control (7.3 ± 0.7-fold vs. 6.3 ± 0.7-fold). Conversely, in HAEC, catalase activity was increased more (P < 0.05) under 10% motion control (6.1 ± 0.3-fold) than under 10% cyclic strain (2.9 ± 0.5-fold). This trend in HAEC catalase activity remained significant at 20% deformation (6.7 ± 0.8-fold for motion control vs. 3.7 ± 0.5-fold for cyclic strain; P < 0.05).

VCAM and ICAM expression. To examine functional responses to the elevated ROS levels in HUVEC and HAEC, we studied protein expression of the inflammatory markers VCAM-1 and ICAM-1 (Figs. 4 and 5). The effects of cyclic strain and motion control on VCAM-1 protein expression were consistent with our findings on ROS production and metabolism. In HUVEC, 10% cyclic strain had a greater stimulatory effect than 10% motion control (3.8 ± 0.8-fold vs. 1.9 ± 0.6-fold; P < 0.05). However, in HAEC, 10% motion control elicited a greater response in VCAM-1 expression than 10% cyclic strain (3.9 ± 0.1-fold vs. 2.8 ± 0.25-fold; P < 0.05). At 20% deformation, the fold changes in VCAM-1 expression were not significantly different between cyclic strain and motion control for either cell type. To inhibit ROS, we subjected the cells to cyclic strain or motion control in the presence of 4 µM DPI. The optimum DPI concentration was determined based on balancing cell viability and maximizing inhibitory effect on ROS production. Treatment of HUVEC with 4 µM DPI during cyclic strain or motion control reduced production of O2bullet– to 27.5 ± 8.0% of static control values with no loss of cell viability. Inhibition of ROS production with DPI decreased VCAM-1 expression to the values in stationary cultures, regardless of mechanical stimulus (Fig. 4). As a positive control, to induce endogenous ROS, we treated the cells with 100 pg/ml IL-1beta in static culture for 24 h, which increased VCAM-1 protein expression in HUVEC 3.94 ± 1.25-fold and in HAEC 5.38 ± 0.96-fold.


Figure 4
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  		Fig. 4. A: representative Western blots for vascular cell adhesion molecule (VCAM)-1 and beta-actin expression. DPI, diphenyleneiodonium. B: densitometry results averaged from 3 independent experiments and normalized to beta-actin. Values for CS and MC have been normalized to static controls. CS-DPI and MC-DPI, CS and MC with DPI inhibition. Results are means ± SE. *P < 0.05 for CS vs. MC; {dagger}P < 0.05 for 10% vs. 20% strain; {ddagger}P < 0.05 for with vs. without DPI.

 

Figure 5
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  		Fig. 5. A: representative Western blot for intercellular adhesion molecule (ICAM)-1 and beta-actin expression. B: densitometry results averaged from 3 independent experiments and normalized to beta-actin. Values for CS and MC have been normalized to static controls. Results are means ± SE. *P < 0.05 for CS vs. MC; {dagger}P < 0.05 for 10% vs. 20% strain; {ddagger}P < 0.05 for with vs. without DPI.

 
The effects on ICAM-1 expression were consistent with our findings on VCAM-1. In HUVEC, 10% cyclic strain increased ICAM-1 protein expression (2.3 ± 0.4-fold) more than 10% motion control (1.4 ± 0.3-fold; P < 0.05). ICAM-1 upregulation was not statistically different at 20% deformation (3.3 ± 0.3-fold for cyclic strain vs. 3.4 ± 0.7-fold for motion control). In HAEC, 10% and 20% cyclic strain attenuated ICAM-1 upregulation (1.6 ± 0.2-fold and 3.3 ± 0.2-fold, respectively) relative to 10% and 20% motion control (2.6 ± 0.3-fold and 4.5 ± 0.4-fold, respectively), but significance was found only at 20% deformation. DPI treatment inhibited ICAM-1 expression induced by motion control and cyclic strain in both HUVEC and HAEC; significance (P < 0.05) was found for all deformations except for 10% motion control in HUVEC and 10% cyclic strain in HAEC. ICAM-1 protein expression was increased 1.95 ± 0.19-fold in HUVEC and 2.84 ± 0.15-fold in HAEC in response to IL-1beta.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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GRANTS
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In HUVEC, we found greater increased production and metabolism of ROS under cyclic strain than under motion control, consistent with our previous microarray study (14). In HAEC, the reverse was true, with motion control providing a greater stimulus than cyclic strain. Bovine aortic ECs behaved similarly to HAEC in that motion control responses increased over cyclic strain (data not shown), which provides evidence that the differences are not due to variables in culture conditions (e.g., passage number, age of donor at cell harvest, pooling of donor cells). The differences in ROS production were even more pronounced between HUVEC and HAEC than is evident in Fig. 2, because the static control values against which the fold changes were calculated differ. Intracellular levels of H2O2 and O2bullet– were greater in HUVEC than in HAEC. HAEC had higher GPx and SOD activity levels in static culture than HUVEC (Table 1). These findings are consistent with each other; in HUVEC, more ROS would result from less ROS catabolism. They are also consistent with our hypothesis that HUVEC may be more susceptible to oxidative damage under cyclic strain.

There is precedent for global differences between venous and arterial ECs. Gene expression profiling of ECs from different parts of the vasculature reveals differences that persisted for generations in vitro. Chi et al. (8) showed many differences among EC types in microarray studies of five arterial EC types (including HAEC) and two vein EC types (including HUVEC), as well as microvascular ECs from many tissues. Deng et al. (11) suggested that antioxidative genes are more highly expressed in cultured saphenous vein EC than in coronary artery EC.

The sources of ROS in ECs are controversial (18). NAD(P)H oxidase activity, xanthine oxidase, nitric oxide synthases, myeloperoxidases, cytochrome P-450, and mitochondrial electron transport contribute to ROS generation, and the major source is a function of the mechanical stimulus and EC type (28). The effect of shear stress on ROS generation is clearer because of comparisons between steady-shear stress and oscillatory shear stress, whereas the ROS response of ECs to cyclic strain is less well characterized (28). Howard et al. (21) showed that porcine aortic ECs released H2O2 in response to cyclic strain (20%, 1 Hz) for 6 or 24 h. They postulated that NAD(P)H oxidase was the source of the H2O2 and showed that NAD(P)H oxidase activity increased in response to 2 h of cyclic strain. However, after 24-h cyclic strain, NAD(P)H levels returned to baseline, suggesting that cyclic strain activated another source(s) of O2bullet–. Our finding that differences in O2bullet– levels between cyclic strain and motion control (Fig. 2B) were not as dramatic as differences in H2O2 levels (Fig. 2A) in both HAEC and HUVEC suggests that cyclic strain regulates EC oxidative state through H2O2-generating and -reducing enzymes.

The activities of SOD, GPx, and catalase were strongly upregulated by cyclic strain and motion control in HUVEC and HAEC (Fig. 3). Similar to our findings on H2O2 production (Fig. 2A), motion control had a greater effect in HAEC than cyclic strain, while cyclic strain was a more potent stimulus than motion control in HUVEC. In HUVEC, we observed significant increases in H2O2 content by cyclic strain relative to motion control at both at 10% and 20% deformation (Fig. 2A). Although cyclic strain significantly increased SOD, GPx, and catalase activities at 10% deformation relative to motion control, we observed no such increases in any of the enzyme activities at 20% deformation in HUVEC (Fig. 3). This observation implies that in HUVEC the significant increase in H2O2 content by 20% cyclic strain (relative to 20% motion control; Fig. 2A) may have resulted from other H2O2-contributing enzymes (e.g., myeloperoxidase, xanthine oxidase, eNOS) that are sensitive to cyclic strain. Furthermore, H2O2 content and the activities of GPx and catalase were dose responsive to motion control but not to cyclic strain (Figs. 2A and 3), suggesting that the stimulus is saturated by 10% cyclic strain and that augmented H2O2 production precedes changes in the activities of H2O2-consuming enzymes in HUVEC.

In HAEC, cyclic strain attenuated increases in H2O2 levels (Fig. 2A) and SOD, GPx, and catalase activities (Fig. 3) that were induced by motion control. SOD activity in HAEC under all mechanical conditions was consistent with the intracellular H2O2 profile. However, the activities of GPx and catalase were not as consistent, despite showing the same trend as SOD in cyclic strain-attenuated activity. There was insufficient evidence that 20% cyclic strain, relative to 20% motion control, significantly attenuated GPx activity in HAEC. Also, catalase activity was not dose responsive to either cyclic strain or motion control. More importantly, the differing responses to mechanical stimulation of GPx and catalase in HAEC, but comparable responses of these enzymes in HUVEC, may be a demonstration that ROS-metabolizing enzymes behave differently from each other to maintain oxidative equilibrium in a mechanically active environment in HAEC but not in HUVEC.

Previous studies reported that expression of proinflammatory molecules in EC was mediated by production of ROS and contributed to vascular remodeling (6, 17, 44). In the present study, VCAM-1 and ICAM-1 protein levels followed the differences we observed with ROS production (Fig. 2) and ROS metabolizing enzymes (Fig. 3) in both HAEC and HUVEC. Motion control and cyclic strain induced a proinflammatory state as indicated by increased VCAM-1 (Fig. 4) and ICAM-1 (Fig. 5) expression. DPI reduced VCAM-1 and ICAM-1 expression in both cell types under motion control and cyclic strain, indicating that ROS generation was responsible for the increases. Supporting our findings, Yun et al. (47) observed a threefold increase in monocyte adhesion to cyclically strained HUVEC (25% strain, 0.5 Hz); antibodies against ICAM or VCAM decreased the monocyte adhesion (46). Ali et al. (1) found that cyclic strain (~25%, 0.25 Hz, 6 h) induced VCAM-1 and ICAM-1 expression 5.5-fold and 2.2-fold, respectively, in HUVEC, which was attenuated by DPI. The opposite H2O2 profiles, enzyme activities, and cell adhesion molecule expressions between HAEC and HUVEC in response to cyclic strain and motion control provided evidence that ECs discriminate different mechanical stimuli and that the resulting phenotypes are specific to EC type. Treating different EC types with oxidized LDL resulted in a transcriptome that yielded a significant overrepresentation of "oxidoreductase activity" and "regulation of inflammatory response" in venous ECs but not arterial ECs (11).

The promoter sequence for VCAM-1 contains AP-1, GATA, and NF-{kappa}B sites (9, 35), whereas the ICAM-1 promoter contains AP-2/3, TPA response element (TRE), and NF-{kappa}B sites (42) and is insensitive to GATA inhibition (22). Du et al. (13) showed that cyclically strained HUVEC and HAEC (10% deformation average, 1 Hz) both induced AP-1 and NF-{kappa}B in a monophasic manner, consistent with our findings that 10% cyclic strain upregulated VCAM-1 in both cell types. Interestingly, as noted by Dagia et al. (9), redox-sensitive GATA is involved in VCAM-1 expression in tumor necrosis factor (TNF)-{alpha}-stimulated HUVEC (41) but not TNF-{alpha}-stimulated HAEC.

All devices that apply cyclic strain deformation to cells also produce some oscillatory fluid motion due to the "nonslip" boundary conditions at the wall. In the present study, the fluid agitation generated by cyclic strain and motion control produced a complex, reversing, low-magnitude shear stress (5), possibly resulting in concentration gradients of medium components. The response of HAEC to low-magnitude shear stress has been shown to increase NF-{kappa}B activity and consequent VCAM-1 expression and monocyte adhesion (33). Adhesion of leukocytes occurs at regions of the vasculature characterized by low shear stresses and recirculatory flow patterns (24, 27). Thus it is conceivable that motion control and cyclic strain (which generates both oscillatory fluid shear stress and cellular deformation) have significantly different effects on the EC.

While complex, reversing flow predisposes both arterial and venous ECs to oxidative stress and inflammation (7, 10, 36, 39), our findings suggest that cyclic strain, per se, ameliorates ROS-mediated inflammation in arterial ECs but contributes to venous EC dysfunction. Interestingly, higher levels of H2O2 in aortic ECs have been correlated with hypertension (a parallel to increasing cyclic strain) (26); the internal circumference of healthy large arteries (including the aorta) distend 2–18% with each cardiac cycle (12), supporting the hypothesis that arterial ECs are adapted to cyclic strain in vivo. This would be consistent with observations that vein-to-artery autografts were more prone to develop accelerated atherosclerosis and stenoses than artery-to-artery grafts (3, 30).

The demonstration that oscillating fluid motion provides a greater stimulus than cyclic strain to HAEC and not to HUVEC has not been previously demonstrated, to our knowledge, and has implications regarding in vitro modeling of the disease processes in which ECs participate in vivo. The differences in mechanosensing and mechanotransduction pathways between arterial and venous EC may stem from their location in the vascular tree.


   GRANTS
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 GRANTS
REFERENCES
 
Funding for these studies was provided through National Heart, Lung, and Blood Institute Grants R21-HL-072039 and RO1-HL-18672.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical help of Yumiko Sakurai.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. V. McIntire, Dept. of Biomedical Engineering, Georgia Inst. of Technology, 313 Ferst Dr., Suite 2116, Atlanta, GA 30332-0535 (e-mail: larry.mcintire{at}bme.gatech.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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