Oscillatory and Steady Laminar Shear Stress Differentially Affect Human Endothelial Redox State

Role of a Superoxide-Producing NADH Oxidase

From the Division of Cardiology, Emory University School of Medicine (G.W. De K., D.C.C., N.I., R.W.A., K.K.G.), and Georgia Institute of Technology, School of Mechanical Engineering (R.M.N.), Atlanta, Ga.

Correspondence to Kathy K. Griendling, PhD, Emory University, Division of Cardiology, 1639 Pierce Dr, 319 WMB, Atlanta, GA 30322. E-mail kgriend{at}emory.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O₂·^-)–producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O₂·^- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (±5 dyne/cm², 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174±16% (P<0.05) and 505±111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm²) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O₂·^-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.

Key Words: shear stress • endothelium • NADH/NADPH oxidase • oxygen-derived free radical • heme oxygenase-1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Since the pathologists Rokitansky¹ and Virchow² described the focal nature of atherosclerotic plaque formation in the vascular tree, involvement of hemodynamic forces in the pathogenesis of atherosclerosis has been postulated. In the early 1980s, comparisons between the pattern of blood flow and the localization of atherosclerotic lesions showed that low rather than high mean shear was associated with atherogenesis.³ In addition, marked oscillations of flow (between -7 and +4 dyne/cm²), but not turbulent flow, were observed at sites of atherosclerotic lesions.⁴ From these observations, it was postulated that oscillatory shear stress exerted a stimulus to the vascular wall that induced lesion formation and that high unidirectional shear stress might be protective.

Meanwhile, crucial discoveries in vascular physiology, morphology, and molecular biology have demonstrated that vascular endothelial integrity is obligatory for normal cardiovascular functioning and that endothelial dysfunction is a key initial event during atherogenesis. Endothelial dysfunction is responsible for impaired vessel relaxation and vasospasm and participates in endothelial-monocyte adhesion, oxidation of LDL, and increased susceptibility to thrombosis (for review, see Reference 5⁵ ). Numerous studies have shown that shear stress both activates and inactivates many cellular processes, including the synthesis and release of vasoactive and coagulation factors (for review, see Reference 6⁶ ) and the expression of inflammatory molecules, such as adhesion molecules.^{7 8 9} Recently, it was shown that steady unidirectional shear stress decreases the expression of VCAM-1^{8 10} and almost completely prevents postshear cytokine-induced expression of VCAM-1 in human endothelial cells.¹¹ Prolonged oscillatory shear, in contrast, robustly upregulates VCAM-1 expression.¹² These observations provide initial evidence that human endothelial cells can distinguish between different types of shear stress and suggest that steady laminar shear might induce protective anti-inflammatory responses.

As a first approach to clarify these observations, we examined the effect of oscillatory and unidirectional shear stress on endothelial oxidative mechanisms. Cellular redox state is an important functional parameter that modulates gene expression,¹³ activity of signaling pathways¹⁴ and paracrine factors,¹⁵ apoptosis,¹⁶ and cell growth.¹⁷ Cellular redox state reflects a balance between processes that promote either oxidative or reductive pathways in the cell. Thus, we examined the effect of steady laminar and oscillatory shear stress on the function of the superoxide radical (O₂·^-)–producing NADH oxidase (a major source of reactive oxygen species in vascular cells^18,19) and on the expression of the cytosolic O₂·^-–scavenging Cu/Zn SOD (an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress). In addition, the influence of oscillatory and steady laminar shear on the expression of the redox-sensitive gene HO-1 was assessed. The results of the present study suggest that steady and oscillatory shear stress differentially affect the endothelial redox state.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Cryopreserved primary HUVECs were obtained from Clonetics Corp. HUVECs were grown in medium 199 supplemented with 20% FCS, 16 U/mL heparin, 50 µg/mL endothelial growth supplement, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin and cultured at 37°C in dishes coated with 0.1% (wt/vol) gelatin to assist cell adhesion. For experiments, cells between passages 2 and 6 were seeded onto gelatin-coated plastic plates, fed every other day, and used at confluence. Cells for use as statically maintained samples were plated similarly. None of these conditions had any effect on cell viability.

Flow System
The steady laminar flow system has been described in detail elsewhere.²⁰ Briefly, the cell culture slide containing the endothelial monolayer was inserted into a parallel-plate flow chamber, which was installed between upper and lower reservoirs containing culture medium. The chamber had a length of 11 cm, width of 6 cm, and height of 0.025 cm. After passage through the flow chamber, medium collects in the lower reservoir and is recirculated to the upper reservoir by means of a peristaltic pump. The height between the 2 reservoirs determines the steady flow rate. The mean shear stress (_w) to which the cells were exposed was calculated using the following formula: _w=(6 µ/h²b)Q, where µ refers to the dynamic viscosity, b to the flow chamber width, h to the flow chamber height, and Q to the flow rate.

The oscillatory flow system has also been previously documented.²¹ A motor-driven syringe pump was inserted sidearm into the laminar flow system so that longitudinal oscillatory motion could be generated. This oscillatory component allowed pulsatility to be superimposed on the steady flow via a displacement of the medium back and forth above the endothelial cell surface. In order to dampen the motion so as to ensure a sinusoidal wave form, a small reservoir was placed between the syringe and the flow chamber. The frequency and amplitude of the oscillatory flow were controlled by adjusting the speed or the amplitude of the syringe motion, respectively. The pulsatile flow rate was monitored using an electromagnetic flowmeter probe that was calibrated with an in-line flowmeter. All shear stress experiments were conducted in a warm (37°C) room, and the flow medium was exposed to a 95% air/5% CO₂ mixture to maintain a pH of 7.2.

The monolayers were subjected either to a steady laminar shear stress of 5 dyne/cm² or to a very low mean shear stress with an instantaneous oscillatory shear stress between +5 and -5 dyne/cm² at a frequency of 1 Hz, in an attempt to approximate physiological conditions. These experimental conditions were imposed for a range of time periods. For some experiments, the monolayers were incubated with the antioxidant NAC (20 mmol/L) either for 30 minutes, followed by shear stress in the presence of the antioxidant, or, in the case of the unsheared control samples, for the duration of the experiment.

Because of the requirements for securing the plate within the chamber, installation of the monolayer into the flow chamber necessarily destroys cells at the edges of the plate. In order to discount any effect of substances released by dead cells in the experimental homogenates, all control monolayers were secured within flow chambers for the length of time used in the shear experiments.

NADH Oxidase Assay
NADH oxidase activity was measured using a lucigenin assay as previously described.²² Briefly, control cultures and cultures that had been exposed to shear were washed and lysed in a buffer containing protease inhibitors (20 mmol/L monobasic potassium phosphate [pH 7.0], 1 mmol/L EGTA, 10 µmol/mL aprotinin, 0.5 µg/mL leupeptin, 0.7 µg/mL pepstatin, and 0.5 mmol/L phenylmethylsulfonyl fluoride). The cell suspension was then dounce-homogenized 100 times on ice, and the homogenate was stored on ice until use. Protein content was measured in an aliquot of the homogenate by the method of Lowry et al.²³ NADH oxidase activity was measured using lucigenin, which is specific for O₂·^-. The assay was performed in a 50 mmol/L phosphate buffer (pH 7.0) containing 1 mmol/L EGTA, 150 mmol/L sucrose, 500 µmol/L lucigenin as the electron acceptor, and NADH as the substrate (final volume, 0.9 mL). NADH was used at a final concentration of 100 µmol/L. The reaction was started by the addition of 100 µL of homogenate (50 to 150 µg protein). Photon emission was measured every 15 seconds for 10 minutes in a luminometer. No activity could be measured in the absence of NADH. A buffer blank containing lucigenin and NADH was subtracted (<5% of the cell signal) from each reading before transformation of the data by comparison with a standard curve generated with xanthine/xanthine oxidase. The chemiluminescence signal observed during the present study is unlikely to be related to lucigenin-induced O₂·^- formation in the cell homogenate,²⁴ because no chemiluminescence signal was detected when lucigenin was assayed with cell lysate in the absence of NADH, and the validity of the assay was confirmed in separate experiments using cytochrome c as the electron acceptor.²⁵

In some experiments, inhibitors (diphenylene iodonium, KCN, 4,5-dihydroxy-1,3-benzene disulfonic acid [Tiron], allopurinol, and L-NAME) were added to samples 10 minutes before readings. As previously described, NADH oxidase activity was insensitive to KCN (100 µmol/L), L-NAME (100 µmol/L), and allopurinol (100 µmol/L), indicating that O₂·^- production was not derived from mitochondrial electron transport, nitric oxide synthase, or xanthine oxidase. NADH oxidase activity was, however, reduced by the flavin protein inhibitor diphenylene iodonium and by 10 mmol/L Tiron, verifying that the reactive oxygen species is O₂·^- and confirming that the endothelial oxidase is a flavin-containing enzyme.

Measurement of Intracellular O₂·^- in Intact Cells
To measure intracellular O₂·^- in intact HUVECs, we used dihydroethidium, a cell-permeant dye that is oxidized by O₂·^- to yield the fluorescent ethidium cation.²⁶ This dye has been shown to faithfully measure O₂·^- in endothelial cells.²⁷ Shear-preconditioned and static monolayers were rinsed 3 times with ice-cold PBS, treated with versene (0.2 g/L EDTA in PBS), and scraped from the plate. Cell suspensions were centrifuged at 500g for 10 minutes at 4°C, and the supernatant was aspirated. Pellets were resuspended in 25 µmol/L dihydroethidium in serum-free culture medium and incubated in a light-protected environment for 30 minutes at 37°C. After centrifugation (500g for 10 minutes at 4°C), cells were washed and fixed with paraformaldehyde in PBS at a final concentration of 1% (wt/vol). The relative fluorescence intensities were quantified using flow cytometry in a fluorescence-activated cell sorter (FACScan IV, Becton Dickinson) with absorption set at 518 nm and the detector set at 605 nm. Untreated fixed HUVECs were used as a reference sample.

Northern Blot Analysis
Total RNA was isolated using TRI reagent. RNA (12 µg) was separated on 1% denaturing formaldehyde agarose gels, transferred to Nytran membranes (Schleicher & Schuell) by overnight upward capillary blotting with 10x SSC, and immobilized by UV cross-linking as described previously.²⁸ Consistency of total RNA loading between samples was controlled by densitometric analysis of 28S RNA ultraviolet fluorescence in the presence of ethidium bromide. Double-stranded full-length human Cu/Zn SOD cDNA and the full-length human HO-1 cDNA were labeled using a random primer labeling kit (Prime It II) and [³²P]dCTP. Blots were prehybridized at least 2 hours and hybridized overnight at 42°C in the following solution: 1 mol/L NaCl, 50 mmol/L Tris HCl (pH 7.4), 5x Denhardt's solution, 50% formamide, 0.5% SDS, and 100 µg/mL sheared and denatured salmon sperm DNA. Denhardt's solution was omitted during hybridization. After hybridization, the blots were washed 3 times in 1x SSC and 1% SDS at 50°C. The blots were autoradiographed using Hyperfilm-MP at -80°C, and the relative density of each band was determined using laser densitometry. Staining of the 28S band by ethidium bromide after transfer to the membrane was used for normalization.

Western Blot Analysis
Cells were lysed at 1 mL per dish with lysis buffer (mmol/L: HEPES 50, EDTA 5, NaCl 50, and n-octylglucoside 60, along with 1% Triton X-100, [pH 7.5]) containing protease inhibitors (10 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin) and phosphatase inhibitors (50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, and 10 mmol/L sodium pyrophosphate). Extracted protein was quantified by the Lowry assay.²³ Equal amounts of proteins were separated on 12% polyacrylamide gels using SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with PBS containing 5% nonfat dry milk, 2% BSA, and 0.2% Tween 20. Antibodies against HO-1 and Cu/Zn-SOD were used at 1:500 and 1:200 dilutions, respectively. The enhanced chemiluminescence Western blotting system was used for detection.

Calculations and Statistical Analysis
Comparison between groups was performed by unpaired t test. NADH oxidase enzyme activity was quantified by calculating the mean amount O₂·^- produced per milligram protein per minute over a 10-minute period. Enzyme activity was linear over a wide range of protein content (<10 to 200 µg) within one sample.

Materials
All chemicals were of analytical grade or better. BSA and phenylmethylsulfonyl fluoride were from Boehringer Mannheim. Soybean trypsin inhibitor, glutamine, penicillin, streptomycin, calf serum, and trypsin/EDTA were purchased from GIBCO. FCS was purchased from Hyclone Laboratories. Endothelial growth supplement was purchased from Biomedical Technology. Common buffer salts and medium 199 were obtained from Fisher. Dihydroethidium was from Molecular Probes. Diphenylene iodonium was purchased from Toronto Research Chemicals. [³²P]dCTP was purchased from DuPont NEN. Nytran membranes were purchased from Schleicher & Schuell, and Biospin columns were from Bio-Rad. Prime-It II probe labeling kits were purchased from Amersham Life Sciences. Cu/Zn SOD antibody was from Biodesign, Intl, and HO-1 antibody was purchased from StressGen Biotechnologies. All other chemicals and reagents were from Sigma Chemical Co. The full-length Cu/Zn SOD cDNA was a kind gift from Dr David G. Harrison (Emory University, Atlanta, Ga), and the human HO-1 cDNA was a kind gift from Dr Shigeki Shibaharu (Tohoku University, Japan).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Oscillatory and Steady Laminar Shear Stress on NADH Oxidase Activity and O₂·^- Levels
The effect of oscillatory shear stress (±5 dyne/cm², 1 Hz) on NADH oxidase activity is shown in Figure 1. NADH oxidase was time-dependently activated by exposure to oscillatory shear. Activation was observed only when exposure was >1 hour and progressively increased in magnitude up to 24 hours after the onset of oscillatory shear (from 2342±333 nmol O₂·^-/mg protein per minute in static cells to 3940±505 nmol O₂·^-/mg protein per minute in cells after 24-hour shear, P<0.05).

View larger version (29K): [in this window] [in a new window]   Figure 1. Effect of oscillatory shear on NADH-driven superoxide anion production in HUVECs. A, Representative experiment shows the effect of 5 hours of exposure to oscillatory shear on NADH-mediated O2·- production in HUVEC homogenates. Cells were maintained in static conditions () or exposed to oscillatory shear () for 5 hours (±5 dyne/cm2, 1 Hz). Cells were washed, homogenized, and assayed for NADH (100 µmol/L)–driven O2·- production as described in "Materials and Methods." B, HUVECs were maintained in static conditions (open bars) or exposed to oscillatory shear (±5 dyne/cm2, 1 Hz, shaded bars) and harvested at the indicated time points. Cells were washed, homogenized, and assayed for NADH (100 µmol/L)–driven O2·- production as described in "Materials and Methods." Values are calculated as the mean±SEM of O2·- production/min per milligram protein calculated over 10 minutes for 5 to 7 experiments (*P<0.05).

Initiation of steady, but transient, laminar shear at 5 dyne/cm² also resulted in a significant time-dependent increase in oxidase activity (Figure 2). Enzyme activity was increased as early as 1 hour after exposure to steady laminar shear. However, when steady laminar shear stress was prolonged for 24 hours, oxidase activity in sheared cells was not different from activity in static cells (1568±194 versus 1972±295 nmol O₂·^-/mg protein per minute in sheared cells, P=NS).

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of steady laminar shear on NADH-driven superoxide anion production in HUVECs. HUVECs were maintained in static conditions (open bars) or exposed to steady laminar shear (5 dyne/cm2, shaded bars) and harvested at the indicated time points. Cells were washed, homogenized, and assayed for NADH (100 µmol/L)–driven O2·- production as described in "Materials and Methods." Values are calculated as the mean±SEM of O2·- production/min per milligram protein calculated over 10 minutes for 6 to 8 experiments (*P<0.05).

Increased NADH oxidase activity most likely resulted from activation of the existing enzyme, since neither type of shear stress altered the expression of p22phox, the only cloned subunit of the vascular NADH oxidase (data not shown).

This difference in oxidase activity between cells exposed to oscillatory and laminar shear stress for 24 hours was reflected in the O₂·^- levels in intact cells. As measured by ethidium fluorescence, O₂·^- levels in cells exposed to oscillatory shear were significantly higher (mean fluorescence intensity, 357±29; n=2) than those in HUVECs exposed to laminar shear (mean fluorescence intensity, 102±14; n=2) (Figure 3). Both were lower than in static cells (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 3. Effect of oscillatory and steady laminar shear on O2·- levels in HUVECs. HUVECs were exposed to oscillatory (±5 dyne/cm2, 1 Hz) or steady laminar (5 dyne/cm2) shear stress for 24 hours, incubated with dihydroethidium, and assayed by flow cytometry to measure ethidium fluorescence as an indicator of intracellular O2·-. Top, Ethidium fluorescence in cells exposed to oscillatory shear for 24 hours. Bottom, Fluorescence in HUVECs exposed to steady laminar shear for 24 hours. This experiment is representative of 2 identical experiments.

Effect of Oscillatory and Steady Laminar Shear Stress on Cu/Zn SOD Expression
The discrepancy in intracellular O₂·^- levels between HUVECs exposed to oscillatory and laminar shear suggests a possible differential regulation of scavenging systems. Application of 24 hours of steady laminar shear (5 dyne/cm²) increased the abundance of Cu/Zn SOD mRNA (138±5% versus static conditions, n=3, P<0.05) and protein (142±18%, n=3) (Figure 4). In contrast, when cells were exposed to 24 hours of oscillatory shear (±5 dyne/cm², 1 Hz), Cu/Zn SOD mRNA and protein levels were not altered (99.6±14% [n=5] for mRNA and 110±8% for protein [n=3] versus static conditions, P=NS) (Figure 4). Identical results were obtained after 5 hours of oscillatory shear stress (data not shown). Thus, oscillatory shear apparently causes a sustained increase in pro-oxidative mechanisms, whereas steady laminar shear induces only a transient oxidative insult.

View larger version (58K): [in this window] [in a new window]   Figure 4. Effect of oscillatory and laminar shear stress on Cu/Zn SOD expression. Top, Northern analysis shows the effect of oscillatory (±5 dyne/cm2, 1 Hz) and laminar (5 dyne/cm2) shear stress on Cu/Zn SOD mRNA expression in HUVECs. Cells were maintained in static conditions or exposed to shear for 24 hours. RNA was extracted and prepared for Northern analysis as described in "Materials and Methods." Middle, 28S rRNA is shown as an indication of equal loading. Bottom, Western analysis of Cu/Zn SOD protein expression is shown in cells treated as described above. These results are representative of results obtained in 2 or 3 independent experiments.

Effect of Oscillatory and Steady Laminar Shear Stress on HO-1 Expression
HO-1 has been shown to be redox-sensitive in other nonendothelial cell types.^{29 30} In experiments anticipating studies on shear stress, we investigated the effects of oxidant stress on HO-1 mRNA expression in endothelial cells. As shown in Figure 5, extracellular reactive oxygen species (generated by xanthine oxidase [20 mU/mL] in the presence of xanthine [0.4 mmol/L] or by hydrogen peroxide [10 to 100 µmol/L]) markedly increased HO-1 mRNA expression. Under both conditions, this increase could be prevented, or at least attenuated, by previous administration of Tiron (an O₂·^- scavenger) or catalase (an enzymatic hydrogen peroxide scavenger), respectively. These data confirm that HO-1 mRNA expression is sensitive to changes in the intracellular redox state in endothelial cells.

View larger version (39K): [in this window] [in a new window]   Figure 5. Effect of extracellular reactive oxygen species on HO-1 expression. Northern analysis shows the effect of extracellular reactive oxygen species on HO-1 mRNA expression in HUVECs. A, HUVECs were cultured in 100-mm dishes until confluence and exposed for 5 minutes to xanthine (X, 400 µmol/L) and xanthine oxidase (XO, 20 mU/mL) in either the presence of absence of Tiron (10 mmol/L). After 5 minutes, cells were washed and harvested 2 hours later. RNA was extracted and prepared for Northern analysis as described in "Materials and Methods." B, HUVECs were cultured in 100-mm dishes until confluence and exposed for 2 hours to indicated concentrations of hydrogen peroxide (H2O2) in either the presence or absence of catalase (900 U/mL). RNA was extracted and prepared for Northern analysis as described in "Materials and Methods."

Thus, we used HO-1 mRNA expression as an additional readout of net oxidative state of HUVECs exposed to shear stress. Figure 6 shows the time-dependent effect of oscillatory and steady laminar shear stress on HO-1 mRNA expression. When cells were exposed to oscillatory shear stress, a sustained increase in HO-1 mRNA was observed. HO-1 mRNA levels were increased by 4.5-fold above baseline (n=3, P<0.05) after 5 hours and remained upregulated by 5.1-fold above baseline (n=3, P<0.05) after 24 hours. This increase was reflected in protein levels, which were increased by 1.3-fold at 5 hours and by 2.0-fold at 24 hours (n=2–4). In contrast, but consistent with its effects on oxidase activity, steady laminar shear stress induced an initial large increase in HO-1 mRNA expression that began to attenuate by 24 hours. Levels of HO-1 mRNA increased by 8.6-fold above baseline (n=3, P<0.05) after 5 hours but subsequently decreased to 3.5-fold above baseline (n=3, P<0.05) after 24 hours. HO-1 protein levels were elevated after 5 hours (1.4-fold) and remained elevated for 24 hours (2.7-fold), perhaps because of the long half-life of HO-1 protein³¹ or to changes in stability (n=2 or 3). To demonstrate that HO-1 mRNA upregulation was redox-sensitive, in some experiments HUVECs were preincubated with the antioxidant thiol compound NAC (20 mmol/L) for 30 minutes before the application of oscillatory or steady laminar shear stress. As shown in Figure 7, for both types of shear, induction of HO-1 mRNA expression was completely inhibited. Incubation with NAC also decreased HO-1 protein expression for both types of shear (79% inhibition).

View larger version (43K): [in this window] [in a new window]   Figure 6. Time course of oscillatory and steady laminar shear–induced HO-1 expression. Top, Representative Northern blots showing the effect of oscillatory (±5 dyne/cm2, 1 Hz) and steady laminar (5 dyne/cm2) shear on HO-1 mRNA expression at different time points. Cells from the same split were maintained in static conditions or exposed to shear for 5 or 24 hours. Cells were harvested the same day to avoid interference of differences in confluence. RNA was extracted and prepared for Northern analysis as described in "Materials and Methods." Bottom, Summary of results of Northern analysis obtained from 3 experiments.

View larger version (66K): [in this window] [in a new window]   Figure 7. Effect of oscillatory and laminar shear stress on HO-1 mRNA and effect of the thiol-containing antioxidant NAC. Representative Northern blots show the effect of oscillatory (±5 dyne/cm2, 1 Hz) and laminar (5 dyne/cm2) shear on HO-1 mRNA expression. Cells were maintained in static conditions or exposed to 5 hours of shear in the absence or presence of NAC (20 mmol/L, 30-minute pretreatment). RNA was extracted and prepared for Northern analysis as described in "Materials and Methods."

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study is focused on the differential effects of oscillatory and steady laminar shear stress on endothelial oxidative mechanisms. The data show that oscillatory shear, used at an amplitude modeled in vivo at vascular bifurcations (±5 dyne/cm²),⁴ progressively enhanced the activity of the O₂·^--producing NADH oxidase and the expression of the redox-sensitive gene HO-1, whereas levels of the O₂·^--scavenger enzyme Cu/Zn SOD remained unchanged. Steady laminar shear, used at 5 dyne/cm² to match mean peak levels of shear, initially increased NADH oxidase activity and HO-1 mRNA expression, but these responses were transient and returned toward baseline after 24 hours. Moreover, Cu/Zn SOD mRNA and protein expression were increased after 24 hours of steady laminar shear, suggesting that in cells exposed to this type of shear stress, mechanisms are induced to compensate for the oxidative stress. This was confirmed by measurement of intracellular O₂·^-, which showed that the overall O₂·^- levels in cells exposed to oscillatory shear for 24 hours were 3-fold higher than levels in cells exposed to laminar shear. Together, these data indicate that fluid shear forces may have important effects on endothelial oxidative mechanisms and that different types of shear stress may have different effects. Since fluid shear forces and endothelial redox state have been proposed to be involved in the pathogenesis of atherosclerosis, these observations may have mechanistic importance.

Growing experimental evidence suggests that the generation of reactive oxygen species participates in cellular activation and intracellular signal transduction. For example, both O₂·^- and H₂O₂ have been implicated in the activation of phospholipase D, p42/p44 mitogen-activated protein kinases, and p38 mitogen-activated protein kinase.^{32 33 34} Laurindo et al³⁵ demonstrated that shear stress induced by increased flow through intact isolated rabbit aortas was accompanied by endothelium-dependent production of free radicals. Our data suggest that increased production of reactive oxygen species induces HO-1 mRNA expression. The biochemical pathways responsible for generation of reactive oxygen species during cell activation, however, remain unclear. Recently, several reports have shown that vascular smooth muscle and endothelial cells express an NADH/NADPH-dependent oxidase that is considered to be one of the most potent sources of O₂· ^- in the vascular cell.^{18 22 36 37} We recently reported that enzyme activity of the vascular p22phox-based NADH oxidase is induced during cell activation by the cytokine tumor necrosis factor- in vitro and by angiotensin II in vitro and in vivo.^{18 22 38 39} After induction with both stimuli, enzyme activity remained elevated for prolonged periods, as long as the stimulus was present (experiments were performed up to 24 hours). The major role of this oxidase may be as an inducible enzyme, since baseline NADH oxidase activity is unaffected by expression of antisense p22phox.⁴⁰ This might explain why NADH oxidase activity levels are induced by shear stimuli, even though basal O₂·^- levels are high in endothelial cells.

In the present study, oscillatory shear–induced oxidase activity was sustained, similar to that observed after angiotensin II or tumor necrosis factor-.^{22 39} The transient activation by steady laminar shear, however, is somewhat unusual. Numerous other cellular processes have indeed been demonstrated to be only transiently increased by acute in vitro application of steady laminar shear stress, such as ion channel activity, surface adenine nucleotide concentrations, and intracellular calcium (for review, see Reference 6⁶ ). This phenomenon, known as feedback inhibition of mechanotransduction, is explained by adaptation or signal filtering.⁶ Feedback inhibition of mechanotransduction has been postulated to be necessary to prevent overstimulation by the normal mechanical environment. In the case of shear-induced activation of O₂·^- production, feedback inhibition seems crucial to avoid overproduction and adverse effects of reactive oxygen species. The inability of endothelial cells to adapt in the oscillatory environment may be a key feature leading to continuous activation of pro-oxidant processes under these conditions, whereas the lack of sustained NADH oxidase activity in cells exposed to steady laminar shear stress may contribute to the antiatherogenic effects of this type of shear stress.

The biological action of reactive oxygen species is counterbalanced by nonenzymatic and enzymatic cellular scavenger systems. One of the major determinants of intracellular O₂·^- is the cytosolic copper/zinc-containing enzyme Cu/Zn SOD, which accelerates the conversion of O₂·^- to H₂O₂. In the present study, we observed that application of steady laminar shear during 24 hours increases Cu/Zn SOD mRNA and protein expression in HUVECs. This observation is consistent with the observations by Inoue et al⁴¹ in human arterial endothelial cells. Interesting is the somewhat smaller increase observed at 5 dyne/cm² in the present study (140% versus 200% by Inoue et al), a discrepancy that may point to differences between venous and arterial endothelial cells. The present finding that levels of Cu/Zn SOD mRNA were unchanged after exposure to oscillatory shear stress, however, is novel and intriguing. Differences in gene expression between various types of shear stress are only beginning to be observed. Although the 5' flanking promoter region of the Cu/Zn SOD gene contains 3 copies of the shear-responsive nucleotide sequence GAGACC⁴² and 2 copies of the activator protein-1 binding site,⁴³ it is not known whether steady laminar shear–induced Cu/Zn SOD mRNA expression is modulated through these sequences. Whether oscillatory and steady laminar shear differentially affect Cu/Zn SOD mRNA expression by a differential effect on the activation of these promoter regions requires further investigation.

HO-1 is a member of the heat shock protein family, which catalyzes the initial rate-limiting step of heme degradation to CO and biliverdin. The physiological function of HO-1 is not yet fully understood, but CO-mediated changes in intracellular cGMP may be involved in the regulation of vascular tone and in the expression of endothelial mitogens.^{44 45} In addition, HO-1 may contribute to the control of oxidative stress in the vessel wall, since it decreases the cellular pro-oxidant heme and indirectly (via the action of biliverdin reductase on biliverdin) increases the antioxidant bilirubin.⁴⁶ Induction of HO-1 is also accompanied by increased expression of ferritin, a chelator of free iron.⁴⁷ Previous experiments in fibroblasts and vascular smooth muscle cells have demonstrated that HO-1 is exquisitely sensitive to oxidative stress, including UV irradiation, O₂·^-, and H₂O₂.^{29 30 48} Promoter studies of the human heme oxygenase gene have identified binding sites for nuclear factor-B and activator protein-1 (both redox-sensitive transcription factors)⁴⁹ and have shown that elements within 121 bp immediately upstream from the mRNA cap site respond to various forms of oxidative stress.⁵⁰ Although we did not directly assess the involvement of these redox-sensitive DNA binding sites in shear-induced upregulation of HO-1, the ability of NAC to block HO-1 expression supports this hypothesis, especially since there is no evidence for the shear-responsive element GAGACC sequence in the HO-1 promoter. Whatever the mechanism, the potential importance of increased HO-1 expression in shear stress, which has also recently been reported in vascular smooth muscle cells,⁵¹ resides in endothelial cells as far as its potent antioxidant properties are concerned.⁴⁷ Indeed, the antioxidant effects of HO-1 may be partly responsible for the lower intracellular O₂·^- in sheared cells compared with static cells.

In the present study, after confirming the redox sensitivity of HO-1 in HUVECs, HO-1 mRNA expression was used as a parameter of functional intracellular oxidative stress in order to better appreciate the overall balance between the observed increase in NADH oxidase activity and the expression of Cu/Zn SOD mRNA. Strikingly, results for HO-1 mRNA expression were consistent with results for NADH oxidase, Cu/Zn SOD expression, and intracellular O₂·^- levels. First, as a novel finding, both oscillatory and steady laminar shear upregulated HO-1 mRNA expression in endothelial cells. Second, experiments with NAC confirmed that redox pathways are involved in shear-induced HO-1 mRNA expression. Third, the time dependence of shear-induced HO-1 mRNA expression was consistent with the data observed for the NADH oxidase activation. Our attempts to further analyze the specific contribution of NADH oxidase activity in inducing HO-1 mRNA expression were restricted by a marked toxicity (at concentrations as low as 2 µmol/L) of the flavin enzyme inhibitor diphenylene iodonium on HUVECs. Therefore, although the vascular NADH oxidase is among the most powerful O₂·^--regulating systems in the endothelial cell,³⁶ we cannot completely exclude the possibility that other oxidases participate in the induction of HO-1 mRNA. Finally, the sustained induction of HO-1 may contribute to the antioxidant defenses summoned by the cell to combat the oxidative stress resulting from NADH oxidase activation.

The distribution of hemodynamic forces is thought to contribute to the focal nature of atherosclerosis. Comparisons between the pattern of blood flow and the localization of atherosclerotic lesions showed that low rather than high mean shear was associated with atherogenesis.⁴ Moreover, marked intimal thickening was most extensive when instantaneous shear oscillated between -7 and +4 dyne/cm². Elevated steady laminar shear stress tends to protect against intimal thickening. The present study, showing different effects of oscillatory and steady laminar shear on endothelial oxidative mechanisms, may help to explain these observations. The endothelial redox state may contribute to the initiation and progression of atherosclerosis by influencing the oxidative state of LDL, modulating the half-life of nitric oxide, and regulating inflammatory gene expression and cell growth. Thus, although both types of shear stress are initially pro-oxidant, the induction of SOD and the lowering of intracellular O₂·^- (ie, the net antioxidant effect) once laminar shear stress has been established may be protective against atherogenic stimuli, whereas oscillatory shear, by virtue of its inability to increase SOD expression and its higher intracellular O₂·^- levels, does not exert that protection and, thus, may contribute to atherogenesis.

In summary, the present study shows experimental evidence that continuous exposure of endothelial cells to oscillatory and steady laminar shear stress differentially affects endothelial oxidative mechanisms. This difference includes at least (1) the absence of feedback inhibition of oscillatory shear compared with steady laminar shear on the activity of the O₂·^--producing enzyme, NADH oxidase, and (2) the differential regulation of the mRNA expression of the antioxidant enzymes Cu/Zn SOD and HO-1. Further studies are necessary to understand the molecular signaling pathways that allow endothelial cells to discriminate between various types of shear stress.

	Selected Abbreviations and Acronyms

 

HO-1 = heme oxygenase-1 HUVEC = human umbilical vein endothelial cell L-NAME = NG-nitro-L-arginine methyl ester NAC = N-acetylcysteine SOD = superoxide dismutase VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-38206 and HL-58863 and by a fellowship from the Belgian American Educational Foundation (to Dr De Keulenaer). The authors are grateful to Drs Bob Taylor and Stanislas Sys for helpful discussions.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received January 26, 1998; accepted March 16, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Rokitanski C. The Pathological Anatomy of the Organs of Respiration and Circulation: A Manual of Pathological Anatomy. London, UK: Sydenham Society; 1952.

2. Virchow R. Cellular Pathology as Based Upon Physiological and Pathological Histology. London, UK: Churchill; 1860.

3. Zarins CK, Giddens DP, Bharadvaj BK, Sottiuarai VS, Mabon RF, Glagov S. Carotid bifurcation atherosclerosis: quantitative correlation of plaque localization with flow velocity profiles and wall shear stress. Circ Res. 1983;53:502–514.[Abstract/Free Full Text]

4. Ku D, Giddens D, Zarins C, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low and oscillating shear stress. Arteriosclerosis. 1985;5:293–302.[Abstract/Free Full Text]

5. De Meyer GR, Herman AG. Vascular endothelial dysfunction. Prog Cardiovasc Dis. 1997;39:325–342.[Medline] [Order article via Infotrieve]

6. Davies PF, Tripathi SC. Mechanical stress mechanisms and the cell: an endothelial paradigm. Circ Res. 1993;72:239–245.[Abstract/Free Full Text]

7. Walpola PL, Gotlieb AI, Cybylsky MI, Langille BL. Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. Arterioscler Thromb Vasc Biol. 1995;15:2–10.[Abstract/Free Full Text]

8. Medford R, Erickson S, Chappell D, Offermann M, Nerem R, Alexander R. Laminar shear stress and redox sensitive regulation of human vascular endothelial cell VCAM-1 gene expression [abstract]. Circulation. 1994;909(suppl I):I-83.

9. Nagel T, Resnick N, Atkinson W, Dewey CF Jr, Gimbrone MA Jr. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest. 1994;94:885–891.

10. Ando J, Tsuboi H, Korenaga R, Takada Y, Toyama-Sorimach N, Miyasaka M, Kamiya A. Down-regulation of vascular adhesion molecule-1 by fluid shear stress in cultured mouse endothelial cells. Ann N Y Acad Sci. 1996;748:148–157.

11. Tsao PS, Buitrago R, Chan JR, Cooke JP. Fluid flow inhibits endothelial adhesiveness, nitric oxide and transcriptional regulation of VCAM-1. Circulation. 1996;94:1682–1689.[Abstract/Free Full Text]

12. Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res. 1998;82:532–539.[Abstract/Free Full Text]

13. Marui N, Offerman M, Swerlick R, Kunsch C, Roxen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell-adhesion molecule-1 (VCAM-1) gene-transcription and expression are regulated through an antioxidant sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866–1874.

14. Rao GN. Hydrogen peroxide induces complex formation of SHC-Grb2-SOS with receptor tyrosine kinase and activates ras and extracellular signal-regulated protein kinases group of mitogen activated protein kinases. Oncogene. 1996;13:713–719.[Medline] [Order article via Infotrieve]

15. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature. 1986;320:454–456.[Medline] [Order article via Infotrieve]

16. Stoian I, Oros A, Moldoveanu E. Apoptosis and free radicals. Biochem Mol Med. 1996;59:93–97.[Medline] [Order article via Infotrieve]

17. Rao GN, Berk BC. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res. 1992;70:593–599.[Abstract/Free Full Text]

18. Rajagopalan S, Kurz S, Münzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996;97:1916–1923.[Medline] [Order article via Infotrieve]

19. Mohazzab-H KM, Wolin MS. Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol. 1994;267:L815–L822.[Abstract/Free Full Text]

20. Levesque MJ, Nerem RM. The elongation and orientation of cultured endothelial cells in response to shear stress. J Biomech Eng. 1985;107:341–347.[Medline] [Order article via Infotrieve]

21. Thoumine O, Nerem RM, Girard PR. Oscillatory shear and hydrostatic pressure modulate cell-matrix attachment proteins in cultured endothelial cells. In Vitro Cell Dev Biol Anim. 1995;31:45–54.[Medline] [Order article via Infotrieve]

22. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994;74:1141–1148.[Abstract/Free Full Text]

23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]

24. Vasquez-Vivar J, Hogg N, Kirkwood AP, Martasek P, Kalyanaraman B. Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study. FEBS Lett. 1997;403:127–130.[Medline] [Order article via Infotrieve]

25. Bellavite P, Corso F, Dusi S, Grzeskowiak M, Della-Bianca V, Rossi F. Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid. J Biol Chem. 1988;263:8210–8214.[Abstract/Free Full Text]

26. Budd SL, Castilho RF, Nicholls DG. Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. FEBS Lett. 1997;415:21–24.[Medline] [Order article via Infotrieve]

27. Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol. 1994;55:253–258.[Abstract]

28. Lassègue B, Alexander RW, Nickenig G, Clark M, Murphy TJ, Griendling KK. Angiotensin II down-regulates the vascular AT₁ receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation. Mol Pharmacol. 1995;48:601–609.[Abstract]

29. Keyse SM, Tyrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide and sodium arsenite. Proc Natl Acad Sci U S A.. 1989;86:99–103.[Abstract/Free Full Text]

30. Applegate LA, Luscher P, Tyrrell RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 1990;51:974–978.[Abstract/Free Full Text]

31. Srivastava KK, Cable EE, Donohue SE, Bonkovsky HL. Molecular basis for heme-dependent induction of heme oxygenase in primary cultures of chick embryo hepatocytes: demonstration of acquired refractoriness to heme. Eur J Biochem. 1993;213:909–917.[Medline] [Order article via Infotrieve]

32. Huot J, Houle F, Marceau F, Landry J. Oxidative stress–induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res. 1997;80:383–392.[Abstract/Free Full Text]

33. Baas AS, Berk BC. Differential activation of mitogen-activated protein kinases by H₂O₂ and O₂^- in vascular smooth muscle cells. Circ Res. 1995;77:29–36.[Abstract/Free Full Text]

34. Natarajan V, Taher MM, Roehm B, Parinandi NL, Schmid HHO, Kiss Z, Garcia JGN. Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide. J Biol Chem. 1993;268:930–937.[Abstract/Free Full Text]

35. Laurindo FRM, Pedro M de A, Barbeiro HV, Pileggi F, Carvalho MHC, Augusto O, da Luz PL. Vascular free radical release: ex vivo and in vivo evidence for a flow-dependent endothelial mechanism. Circ Res. 1994;74:700–709.[Abstract/Free Full Text]

36. Mohazzab KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol. 1994;266:H2568–H2572.[Abstract/Free Full Text]

37. Pagano PJ, Ito Y, Tornheim K, Gallop PM, Tauber AI, Cohen RA. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol. 1995;268:H2274–H2280.[Abstract/Free Full Text]

38. Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Q IV, Taylor WR, Harrison DG, de Leon H, Wilcox JN, Griendling KK. p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res. 1997;80:45–51.[Abstract/Free Full Text]

39. De Keulenaer GW, Alexander RW, Ushio-Fukai M, Ishizaka N, Griendling KK. Tumour necrosis factor- activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochem J. 1998;329:653–657.

40. Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem. 1996;271:23317–23321.[Abstract/Free Full Text]

41. Inoue N, Ramaswamy S, Fukai R, Nerem RM, Harrison DG. Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells. Circ Res. 1996;79:32–37.[Abstract/Free Full Text]

42. Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF Jr, Gimbrone MA Jr. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. Proc Nat Acad Sci U S A.. 1993;90:4591–4595.[Abstract/Free Full Text]

43. Kim HT, Kim YH, Nam JW, Lee HJ, Rho HM, Jung G. Study of 5'-flanking region of human Cu/Zn superoxide dismutase. Biochem Biophys Res Commun. 1994;201:1526–1533.[Medline] [Order article via Infotrieve]

44. Johnson RA, Lavesa M, Askari B, Abraham NG, Nasjletti A. A heme oxygenase product, presumably carbon monoxide, mediates a vasopressor function in rats. Hypertension. 1995;25:166–169.[Abstract/Free Full Text]

45. Morita T, Kourembanas S. Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. J Clin Invest. 1995;96:2676–2682.

46. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 1987;235:1043–1046.[Abstract/Free Full Text]

47. Vile GF, Tyrell RM. Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase of ferritin. J Biol Chem. 1993;268:14678–14681.[Abstract/Free Full Text]

48. Ishizaka N, Griendling KK. Heme oxygenase-1 is regulated by angiotensin II in rat vascular smooth muscle cells. Hypertension. 1997;29:790–795.[Abstract/Free Full Text]

49. Lavrovsky Y, Schwartzman ML, Levere RD, Kappas A, Abraham NG. Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promotor region of the human heme oxygenase 1 gene. Proc Natl Acad Sci U S A. 1994;91:5987–5991.[Abstract/Free Full Text]

50. Tyrrell RM, Applegate LA, Tromvoukis Y. The proximal promotor region of the human heme oxygenase contains elements involved in stimulation of transcriptional activity by a variety of agents including oxidants. Carcinogenesis. 1993;14:761–765.[Abstract/Free Full Text]

51. Wagner CT, Durante W, Christodoulides N, Hellums JD, Schafer AI. Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells. J Clin Invest. 1997;100:589–596.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				A. H. Wagner, O. Kautz, K. Fricke, M. Zerr-Fouineau, E. Demicheva, B. Guldenzoph, J. L. Bermejo, T. Korff, and M. Hecker Upregulation of Glutathione Peroxidase Offsets Stretch-Induced Proatherogenic Gene Expression in Human Endothelial Cells Arterioscler Thromb Vasc Biol, November 1, 2009; 29(11): 1894 - 1901. [Abstract] [Full Text] [PDF]

				J. R. Durrant, D. R. Seals, M. L. Connell, M. J. Russell, B. R. Lawson, B. J. Folian, A. J. Donato, and L. A. Lesniewski Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down-regulation of NADPH oxidase J. Physiol., July 1, 2009; 587(13): 3271 - 3285. [Abstract] [Full Text] [PDF]

				K. Kruger, S. Frost, E. Most, K. Volker, J. Pallauf, and F. C. Mooren Exercise affects tissue lymphocyte apoptosis via redox-sensitive and Fas-dependent signaling pathways Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2009; 296(5): R1518 - R1527. [Abstract] [Full Text] [PDF]

				J.-L. Balligand, O. Feron, and C. Dessy eNOS Activation by Physical Forces: From Short-Term Regulation of Contraction to Chronic Remodeling of Cardiovascular Tissues Physiol Rev, April 1, 2009; 89(2): 481 - 534. [Abstract] [Full Text] [PDF]

				Z. Han, S. Varadharaj, R. J. Giedt, J. L. Zweier, H. H. Szeto, and B. R. Alevriadou Mitochondria-Derived Reactive Oxygen Species Mediate Heme Oxygenase-1 Expression in Sheared Endothelial Cells J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 94 - 101. [Abstract] [Full Text] [PDF]

				W. G. Mayhan, D. M. Arrick, G. M. Sharpe, and H. Sun Nitric oxide synthase-dependent responses of the basilar artery during acute infusion of nicotine Nicotine Tob Res, March 1, 2009; 11(3): 270 - 277. [Abstract] [Full Text] [PDF]

				R. Zhang, Y.-G. Bai, L.-J. Lin, J.-X. Bao, Y.-Y. Zhang, H. Tang, J.-H. Cheng, G.-L. Jia, X.-L. Ren, and J. Ma Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats J Appl Physiol, January 1, 2009; 106(1): 251 - 258. [Abstract] [Full Text] [PDF]

				Y.-C. Liao, H.-F. Lin, T. Rundek, R. Cheng, Y.-C. Guo, R. L. Sacco, and S.-H. H. Juo Segment-Specific Genetic Effects on Carotid Intima-Media Thickness: The Northern Manhattan Study Stroke, December 1, 2008; 39(12): 3159 - 3165. [Abstract] [Full Text] [PDF]

				M. Zakkar, H. Chaudhury, G. Sandvik, K. Enesa, L. A. Luong, S. Cuhlmann, J. C. Mason, R. Krams, A. R. Clark, D. O. Haskard, et al. Increased Endothelial Mitogen-Activated Protein Kinase Phosphatase-1 Expression Suppresses Proinflammatory Activation at Sites That Are Resistant to Atherosclerosis Circ. Res., September 26, 2008; 103(7): 726 - 732. [Abstract] [Full Text] [PDF]

				A. W. Orr, C. Hahn, B. R. Blackman, and M. A. Schwartz p21-Activated Kinase Signaling Regulates Oxidant-Dependent NF-{kappa}B Activation by Flow Circ. Res., September 12, 2008; 103(6): 671 - 679. [Abstract] [Full Text] [PDF]

				X. Zhou, H. G. Bohlen, S. J. Miller, and J. L. Unthank NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1008 - H1016. [Abstract] [Full Text] [PDF]

				J. O. Fledderus, R. A. Boon, O. L. Volger, H. Hurttila, S. Yla-Herttuala, H. Pannekoek, A.-L. Levonen, and A. J.G. Horrevoets KLF2 Primes the Antioxidant Transcription Factor Nrf2 for Activation in Endothelial Cells Arterioscler Thromb Vasc Biol, July 1, 2008; 28(7): 1339 - 1346. [Abstract] [Full Text] [PDF]

				L. Ai, M. Rouhanizadeh, J. C. Wu, W. Takabe, H. Yu, M. Alavi, R. Li, Y. Chu, J. Miller, D. D. Heistad, et al. Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1576 - C1585. [Abstract] [Full Text] [PDF]

				M. H. Laughlin, S. C. Newcomer, and S. B. Bender Importance of hemodynamic forces as signals for exercise-induced changes in endothelial cell phenotype J Appl Physiol, March 1, 2008; 104(3): 588 - 600. [Abstract] [Full Text] [PDF]

				J. L. Garvin and N. J. Hong Cellular Stretch Increases Superoxide Production in the Thick Ascending Limb Hypertension, February 1, 2008; 51(2): 488 - 493. [Abstract] [Full Text] [PDF]

				A. L. Mowbray, D.-H. Kang, S. G. Rhee, S. W. Kang, and H. Jo Laminar Shear Stress Up-regulates Peroxiredoxins (PRX) in Endothelial Cells: PRX 1 AS A MECHANOSENSITIVE ANTIOXIDANT J. Biol. Chem., January 18, 2008; 283(3): 1622 - 1627. [Abstract] [Full Text] [PDF]

				Md. R. Abid, K. C. Spokes, S.-C. Shih, and W. C. Aird NADPH Oxidase Activity Selectively Modulates Vascular Endothelial Growth Factor Signaling Pathways J. Biol. Chem., November 30, 2007; 282(48): 35373 - 35385. [Abstract] [Full Text] [PDF]

				V. R. Kalapatapu, L. Satterfield, A. T. Brown, Hongjiang Chen, N. Ercal, T. O. Price, Jie Gao, K. Ibrahim, and M. M. Moursi The Effects of Toradol on Postoperative Intimal Hyperplasia in a Rat Carotid Endarterectomy Model: Laboratory Research Vascular and Endovascular Surgery, November 1, 2007; 41(5): 402 - 408. [Abstract] [PDF]

				J. D. Widder, W. Chen, L. Li, S. Dikalov, B. Thony, K. Hatakeyama, and D. G. Harrison Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress Circ. Res., October 12, 2007; 101(8): 830 - 838. [Abstract] [Full Text] [PDF]

				G. Dai, S. Vaughn, Y. Zhang, E. T. Wang, G. Garcia-Cardena, and M. A. Gimbrone Jr Biomechanical Forces in Atherosclerosis-Resistant Vascular Regions Regulate Endothelial Redox Balance via Phosphoinositol 3-Kinase/Akt-Dependent Activation of Nrf2 Circ. Res., September 28, 2007; 101(7): 723 - 733. [Abstract] [Full Text] [PDF]

				M. H. Pennestri, J. Montplaisir, R. Colombo, G. Lavigne, and P. A. Lanfranchi Nocturnal blood pressure changes in patients with restless legs syndrome Neurology, April 10, 2007; 68(15): 1213 - 1218. [Abstract] [Full Text] [PDF]

				M. L. McCormick, D. Gavrila, and N. L. Weintraub Role of Oxidative Stress in the Pathogenesis of Abdominal Aortic Aneurysms Arterioscler Thromb Vasc Biol, March 1, 2007; 27(3): 461 - 469. [Abstract] [Full Text] [PDF]

				Z. Han, Y.-R. Chen, C. I. Jones III, G. Meenakshisundaram, J. L. Zweier, and B. R. Alevriadou Shear-induced reactive nitrogen species inhibit mitochondrial respiratory complex activities in cultured vascular endothelial cells Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1103 - C1112. [Abstract] [Full Text] [PDF]

				N. J. Hong and J. L. Garvin Flow increases superoxide production by NADPH oxidase via activation of Na-K-2Cl cotransport and mechanical stress in thick ascending limbs Am J Physiol Renal Physiol, March 1, 2007; 292(3): F993 - F998. [Abstract] [Full Text] [PDF]

				A. W. Orr, R. Stockton, M. B. Simmers, J. M. Sanders, I. J. Sarembock, B. R. Blackman, and M. A. Schwartz Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis J. Cell Biol., February 26, 2007; 176(5): 719 - 727. [Abstract] [Full Text] [PDF]

				J. Suo, D. E. Ferrara, D. Sorescu, R. E. Guldberg, W. R. Taylor, and D. P. Giddens Hemodynamic Shear Stresses in Mouse Aortas: Implications for Atherogenesis Arterioscler Thromb Vasc Biol, February 1, 2007; 27(2): 346 - 351. [Abstract] [Full Text] [PDF]

				A. W. Orr, M. H. Ginsberg, S. J. Shattil, H. Deckmyn, and M. A. Schwartz Matrix-specific Suppression of Integrin Activation in Shear Stress Signaling Mol. Biol. Cell, November 1, 2006; 17(11): 4686 - 4697. [Abstract] [Full Text] [PDF]

				N. Duerrschmidt, C. Stielow, G. Muller, P. J. Pagano, and H. Morawietz NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells J. Physiol., October 15, 2006; 576(2): 557 - 567. [Abstract] [Full Text] [PDF]

				J.-X. Chen, H. Zeng, M. L Lawrence, T. S. Blackwell, and B. Meyrick Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1563 - H1572. [Abstract] [Full Text] [PDF]

				F. M. Kouri and O. Eickelberg Transforming Growth Factor-{alpha}, a Novel Mediator of Strain-Induced Vascular Remodeling Circ. Res., August 18, 2006; 99(4): 348 - 350. [Full Text] [PDF]

				Z. Li, X. Hyseni, J. D. Carter, J. M. Soukup, L. A. Dailey, and Y.-C. T. Huang Pollutant particles enhanced H2O2 production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells Am J Physiol Cell Physiol, August 1, 2006; 291(2): C357 - C365. [Abstract] [Full Text] [PDF]

				T. M. Paravicini and R. M. Touyz Redox signaling in hypertension Cardiovasc Res, July 15, 2006; 71(2): 247 - 258. [Abstract] [Full Text] [PDF]

				S. Lehoux Redox signalling in vascular responses to shear and stretch Cardiovasc Res, July 15, 2006; 71(2): 269 - 279. [Abstract] [Full Text] [PDF]

				M. Schmelter, B. Ateghang, S. Helmig, M. Wartenberg, and H. Sauer Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation FASEB J, June 1, 2006; 20(8): 1182 - 1184. [Abstract] [Full Text] [PDF]

				J. H. Traverse, Y. E. Nesmelov, M. Crampton, P. Lindstrom, D. D. Thomas, and R. J. Bache Measurement of myocardial free radical production during exercise using EPR spectroscopy Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2453 - H2458. [Abstract] [Full Text] [PDF]

				M. P. Merker, S. H. Audi, R. D. Bongard, B. J. Lindemer, and G. S. Krenz Influence of pulmonary arterial endothelial cells on quinone redox status: effect of hyperoxia-induced NAD(P)H:quinone oxidoreductase 1 Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L607 - L619. [Abstract] [Full Text] [PDF]

				H. Cai A new mechanism for flow-mediated vasoprotection? Focus on "Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species" Am J Physiol Cell Physiol, January 1, 2006; 290(1): C35 - C36. [Full Text] [PDF]

				A. Cave, D. Grieve, S. Johar, M. Zhang, and A. M Shah NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology Phil Trans R Soc B, December 29, 2005; 360(1464): 2327 - 2334. [Abstract] [Full Text] [PDF]

				A. A. Miller, G. R. Drummond, H. H.H.W. Schmidt, and C. G. Sobey NADPH Oxidase Activity and Function Are Profoundly Greater in Cerebral Versus Systemic Arteries Circ. Res., November 11, 2005; 97(10): 1055 - 1062. [Abstract] [Full Text] [PDF]

				K. E. Chapman, S. E. Sinclair, D. Zhuang, A. Hassid, L. P. Desai, and C. M. Waters Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L834 - L841. [Abstract] [Full Text] [PDF]

				C. F.H. Mueller, J. D. Widder, J. S. McNally, L. McCann, D. P. Jones, and D. G. Harrison The Role of the Multidrug Resistance Protein-1 in Modulation of Endothelial Cell Oxidative Stress Circ. Res., September 30, 2005; 97(7): 637 - 644. [Abstract] [Full Text] [PDF]

				M. Li, K.-R. Chiou, A. Bugayenko, K. Irani, and D. A. Kass Reduced Wall Compliance Suppresses Akt-Dependent Apoptosis Protection Stimulated by Pulse Perfusion Circ. Res., September 16, 2005; 97(6): 587 - 595. [Abstract] [Full Text] [PDF]

				Y. Castier, R. P. Brandes, G. Leseche, A. Tedgui, and S. Lehoux p47phox-Dependent NADPH Oxidase Regulates Flow-Induced Vascular Remodeling Circ. Res., September 16, 2005; 97(6): 533 - 540. [Abstract] [Full Text] [PDF]

				I. Fleming, B. Fisslthaler, M. Dixit, and R. Busse Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells J. Cell Sci., September 15, 2005; 118(18): 4103 - 4111. [Abstract] [Full Text] [PDF]

				A. Pines, L. Perrone, N. Bivi, M. Romanello, G. Damante, M. Gulisano, M. R. Kelley, F. Quadrifoglio, and G. Tell Activation of APE1/Ref-1 is dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP Nucleic Acids Res., August 2, 2005; 33(14): 4379 - 4394. [Abstract] [Full Text] [PDF]

				G. Kojda and R. Hambrecht Molecular mechanisms of vascular adaptations to exercise. Physical activity as an effective antioxidant therapy? Cardiovasc Res, August 1, 2005; 67(2): 187 - 197. [Abstract] [Full Text] [PDF]

				T. Hosoya, A. Maruyama, M.-I. Kang, Y. Kawatani, T. Shibata, K. Uchida, K. Itoh, and M. Yamamoto Differential Responses of the Nrf2-Keap1 System to Laminar and Oscillatory Shear Stresses in Endothelial Cells J. Biol. Chem., July 22, 2005; 280(29): 27244 - 27250. [Abstract] [Full Text] [PDF]

				J. Amar, J.-B. Ruidavets, J.-C. Peyrieux, J.-M. Mallion, J. Ferrieres, M. E. Safar, and B. Chamontin C-Reactive Protein Elevation Predicts Pulse Pressure Reduction in Hypertensive Subjects Hypertension, July 1, 2005; 46(1): 151 - 155. [Abstract] [Full Text] [PDF]

				A. W. Orr, J. M. Sanders, M. Bevard, E. Coleman, I. J. Sarembock, and M. A. Schwartz The subendothelial extracellular matrix modulates NF-{kappa}B activation by flow: a potential role in atherosclerosis J. Cell Biol., April 11, 2005; 169(1): 191 - 202. [Abstract] [Full Text] [PDF]

				J. M. Cook-Mills and T. L. Deem Active participation of endothelial cells in inflammation J. Leukoc. Biol., April 1, 2005; 77(4): 487 - 495. [Abstract] [Full Text] [PDF]

				D. D. Gutterman, H. Miura, and Y. Liu Redox Modulation of Vascular Tone: Focus of Potassium Channel Mechanisms of Dilation Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 671 - 678. [Abstract] [Full Text] [PDF]

				B. Braam, R. de Roos, H. Bluyssen, P. Kemmeren, F. Holstege, J. A. Joles, and H. Koomans Nitric Oxide-Dependent and Nitric Oxide-Independent Transcriptional Responses to High Shear Stress in Endothelial Cells Hypertension, April 1, 2005; 45(4): 672 - 680. [Abstract] [Full Text] [PDF]

				N. A. Zakopoulos, G. Tsivgoulis, G. Barlas, C. Papamichael, K. Spengos, E. Manios, I. Ikonomidis, V. Kotsis, I. Spiliopoulou, K. Vemmos, et al. Time Rate of Blood Pressure Variation Is Associated With Increased Common Carotid Artery Intima-Media Thickness Hypertension, April 1, 2005; 45(4): 505 - 512. [Abstract] [Full Text] [PDF]

				R. P. Brandes and J. Kreuzer Vascular NADPH oxidases: molecular mechanisms of activation Cardiovasc Res, January 1, 2005; 65(1): 16 - 27. [Abstract] [Full Text] [PDF]

				N. Lauer, T. Suvorava, U. Ruther, R. Jacob, W. Meyer, D. G. Harrison, and G. Kojda Critical involvement of hydrogen peroxide in exercise-induced up-regulation of endothelial NO synthase Cardiovasc Res, January 1, 2005; 65(1): 254 - 262. [Abstract] [Full Text] [PDF]

				J. Herrmann, S. T. Higano, R. J. Lenon, C. S. Rihal, and A. Lerman Myocardial bridging is associated with alteration in coronary vasoreactivity Eur. Heart J., December 1, 2004; 25(23): 2134 - 2142. [Abstract] [Full Text] [PDF]

				X Lu and G. S Kassab Nitric oxide is significantly reduced in ex vivo porcine arteries during reverse flow because of increased superoxide production J. Physiol., December 1, 2004; 561(2): 575 - 582. [Abstract] [Full Text] [PDF]

				J. Hendrickx, K. Doggen, E. O. Weinberg, P. Van Tongelen, P. Fransen, and G. W. De Keulenaer Molecular diversity of cardiac endothelial cells in vitro and in vivo Physiol Genomics, October 4, 2004; 19(2): 198 - 206. [Abstract] [Full Text] [PDF]

				L. L. Schott, R. P. Wildman, S. Brockwell, L. R. Simkin-Silverman, L. H. Kuller, and K. Sutton-Tyrrell Segment-Specific Effects of Cardiovascular Risk Factors on Carotid Artery Intima-Medial Thickness in Women at Midlife Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1951 - 1956. [Abstract] [Full Text] [PDF]

				E. Sho, M. Sho, H. Nanjo, K. Kawamura, H. Masuda, and R. L. Dalman Hemodynamic Regulation of CD34+ Cell Localization and Differentiation in Experimental Aneurysms Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1916 - 1921. [Abstract] [Full Text] [PDF]

				X.-L. Chen, J. Y. Grey, S. Thomas, F.-H. Qiu, R. M. Medford, M. A. Wasserman, and C. Kunsch Sphingosine kinase-1 mediates TNF-{alpha}-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1452 - H1458. [Abstract] [Full Text] [PDF]

				T. Suvorava, N. Lauer, and G. Kojda Physical inactivity causes endothelial dysfunction in healthy young mice J. Am. Coll. Cardiol., September 15, 2004; 44(6): 1320 - 1327. [Abstract] [Full Text] [PDF]

				C. M. Waters Reactive oxygen species in mechanotransduction Am J Physiol Lung Cell Mol Physiol, September 1, 2004; 287(3): L484 - L485. [Full Text] [PDF]

				C.-W. Ni, H.-J. Hsieh, Y.-J. Chao, and D. L. Wang Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow Am J Physiol Cell Physiol, September 1, 2004; 287(3): C771 - C780. [Abstract] [Full Text] [PDF]

				D. M. Browe and C. M. Baumgarten Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl- Current Elicited by {beta}1 Integrin Stretch in Rabbit Ventricular Myocytes J. Gen. Physiol., August 30, 2004; 124(3): 273 - 287. [Abstract] [Full Text] [PDF]

				J. L. Unthank, K. M. Sheridan, and M. C. Dalsing Collateral Growth in the Peripheral Circulation: A Review Vascular and Endovascular Surgery, July 1, 2004; 38(4): 291 - 313. [Abstract] [PDF]

				D. Sun, A. Huang, E. H. Yan, Z. Wu, C. Yan, P. M. Kaminski, T. D. Oury, M. S. Wolin, and G. Kaley Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2249 - H2256. [Abstract] [Full Text] [PDF]

				T. J. Rabelink, H. C. de Boer, E. J.P. de Koning, and A.-J. van Zonneveld Endothelial Progenitor Cells: More Than an Inflammatory Response? Arterioscler Thromb Vasc Biol, May 1, 2004; 24(5): 834 - 838. [Abstract] [Full Text]

				P. J. Kuhlencordt, E. Rosel, R. E. Gerszten, M. Morales-Ruiz, D. Dombkowski, W. J. Atkinson, F. Han, F. Preffer, A. Rosenzweig, W. C. Sessa, et al. Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1195 - C1202. [Abstract] [Full Text] [PDF]

				A. G. Passerini, D. C. Polacek, C. Shi, N. M. Francesco, E. Manduchi, G. R. Grant, W. F. Pritchard, S. Powell, G. Y. Chang, C. J. Stoeckert Jr., et al. Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta PNAS, February 24, 2004; 101(8): 2482 - 2487. [Abstract] [Full Text] [PDF]

				S. M Wasserman and J. N Topper Adaptation of the endothelium to fluid flow: in vitro analyses of gene expression and in vivo implications Vascular Medicine, February 1, 2004; 9(1): 35 - 45. [Abstract] [PDF]

				P. A. VanderLaan, C. A. Reardon, and G. S. Getz Site Specificity of Atherosclerosis: Site-Selective Responses to Atherosclerotic Modulators Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 12 - 22. [Abstract] [Full Text] [PDF]

				J. A. Vita and G. F. Mitchell Effects of shear stress and flow pulsatility on endothelial function: Insights gleaned from external counterpulsation therapy J. Am. Coll. Cardiol., December 17, 2003; 42(12): 2096 - 2098. [Full Text] [PDF]

				J. Hwang, M. H. Ing, A. Salazar, B. Lassegue, K. Griendling, M. Navab, A. Sevanian, and T. K. Hsiai Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit Expression: Implication for Native LDL Oxidation Circ. Res., December 12, 2003; 93(12): 1225 - 1232. [Abstract] [Full Text] [PDF]

				J. S. McNally, M. E. Davis, D. P. Giddens, A. Saha, J. Hwang, S. Dikalov, H. Jo, and D. G. Harrison Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2290 - H2297. [Abstract] [Full Text] [PDF]

				Y. Taniyama and K. K. Griendling Reactive Oxygen Species in the Vasculature: Molecular and Cellular Mechanisms Hypertension, December 1, 2003; 42(6): 1075 - 1081. [Abstract] [Full Text] [PDF]

				J. Hwang, A. Saha, Y. C. Boo, G. P. Sorescu, J. S. McNally, S. M. Holland, S. Dikalov, D. P. Giddens, K. K. Griendling, D. G. Harrison, et al. Oscillatory Shear Stress Stimulates Endothelial Production of O2- from p47phox-dependent NAD(P)H Oxidases, Leading to Monocyte Adhesion J. Biol. Chem., November 21, 2003; 278(47): 47291 - 47298. [Abstract] [Full Text] [PDF]

				M. Kitada, D. Koya, T. Sugimoto, M. Isono, S.-i. Araki, A. Kashiwagi, and M. Haneda Translocation of Glomerular p47phox and p67phox by Protein Kinase C-{beta} Activation Is Required for Oxidative Stress in Diabetic Nephropathy Diabetes, October 1, 2003; 52(10): 2603 - 2614. [Abstract] [Full Text] [PDF]

				Y. Liu, H. Zhao, H. Li, B. Kalyanaraman, A. C. Nicolosi, and D. D. Gutterman Mitochondrial Sources of H2O2 Generation Play a Key Role in Flow-Mediated Dilation in Human Coronary Resistance Arteries Circ. Res., September 19, 2003; 93(6): 573 - 580. [Abstract] [Full Text] [PDF]

				T. K. HSIAI, S. K. CHO, P. K. WONG, M. ING, A. SALAZAR, A. SEVANIAN, M. NAVAB, L. L. DEMER, and C.-M. HO Monocyte recruitment to endothelial cells in response to oscillatory shear stress FASEB J, September 1, 2003; 17(12): 1648 - 1657. [Abstract] [Full Text] [PDF]

				G. Mancia, G. Parati, P. Castiglioni, R. Tordi, E. Tortorici, F. Glavina, and M. Di Rienzo Daily Life Blood Pressure Changes Are Steeper in Hypertensive Than in Normotensive Subjects Hypertension, September 1, 2003; 42(3): 277 - 282. [Abstract] [Full Text] [PDF]

				R. Magid, T. J. Murphy, and Z. S. Galis Expression of Matrix Metalloproteinase-9 in Endothelial Cells Is Differentially Regulated by Shear Stress: ROLE OF c-Myc J. Biol. Chem., August 29, 2003; 278(35): 32994 - 32999. [Abstract] [Full Text] [PDF]

				B. Lassegue and R. E. Clempus Vascular NAD(P)H oxidases: specific features, expression, and regulation Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2003; 285(2): R277 - R297. [Abstract] [Full Text] [PDF]

				O. Sorop, J. A.E. Spaan, T. E. Sweeney, and E. VanBavel Effect of Steady Versus Oscillating Flow on Porcine Coronary Arterioles: Involvement of NO and Superoxide Anion Circ. Res., June 27, 2003; 92(12): 1344 - 1351. [Abstract] [Full Text] [PDF]

				D. G Harrison, Hua Cai, U. Landmesser, and K. K Griendling The Pickering Lecture British Hypertension Society, 10th September 2002: Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease Journal of Renin-Angiotensin-Aldosterone System, June 1, 2003; 4(2): 51 - 61. [Abstract] [PDF]

				B.A. Kelly, B.C. Bond, and L. Poston Gestational profile of matrix metalloproteinases in rat uterine artery Mol. Hum. Reprod., June 1, 2003; 9(6): 351 - 358. [Abstract] [Full Text] [PDF]

				C.-W. Ni, H.-J. Hsieh, Y.-J. Chao, and D. L. Wang Shear Flow Attenuates Serum-induced STAT3 Activation in Endothelial Cells J. Biol. Chem., May 23, 2003; 278(22): 19702 - 19708. [Abstract] [Full Text] [PDF]

				R. Ceravolo, R. Maio, A. Pujia, A. Sciacqua, G. Ventura, M. C. Costa, G. Sesti, and F. Perticone Pulse pressure and endothelial dysfunction in never-treated hypertensive patients J. Am. Coll. Cardiol., May 21, 2003; 41(10): 1753 - 1758. [Abstract] [Full Text] [PDF]

				A. Lerman and J. Herrmann Endothelial function under pressure J. Am. Coll. Cardiol., May 21, 2003; 41(10): 1759 - 1760. [Full Text] [PDF]

				E. R. Gross, J. F. LaDisa Jr., D. Weihrauch, L. E. Olson, T. T. Kress, D. A. Hettrick, P. S. Pagel, D. C. Warltier, and J. R. Kersten Reactive oxygen species modulate coronary wall shear stress and endothelial function during hyperglycemia Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1552 - H1559. [Abstract] [Full Text] [PDF]

				L. Belhassen, G. Pelle, J.-L. Dubois-Rande, and S. Adnot Improved endothelial function by the thromboxane a2 receptor antagonist s 18886 in patients with coronary artery disease treated with aspirin J. Am. Coll. Cardiol., April 2, 2003; 41(7): 1198 - 1204. [Abstract] [Full Text] [PDF]

				J. W. E. Rush, J. R. Turk, and M. H. Laughlin Exercise training regulates SOD-1 and oxidative stress in porcine aortic endothelium Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1378 - H1387. [Abstract] [Full Text] [PDF]

				H. Miura, J. J. Bosnjak, G. Ning, T. Saito, M. Miura, and D. D. Gutterman Role for Hydrogen Peroxide in Flow-Induced Dilation of Human Coronary Arterioles Circ. Res., February 7, 2003; 92 (2): e31 - e40. [Abstract] [Full Text] [PDF]

				D. X. Zhang, A.-P. Zou, and P.-L. Li Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H605 - H612. [Abstract] [Full Text] [PDF]

				X. Peng, S. Haldar, S. Deshpande, K. Irani, and D. A. Kass Wall Stiffness Suppresses Akt/eNOS and Cytoprotection in Pulse-Perfused Endothelium Hypertension, February 1, 2003; 41(2): 378 - 381. [Abstract] [Full Text] [PDF]

				X.-L. Chen, S. E. Varner, A. S. Rao, J. Y. Grey, S. Thomas, C. K. Cook, M. A. Wasserman, R. M. Medford, A. K. Jaiswal, and C. Kunsch Laminar Flow Induction of Antioxidant Response Element-mediated Genes in Endothelial Cells. A NOVEL ANTI-INFLAMMATORY MECHANISM J. Biol. Chem., January 3, 2003; 278(2): 703 - 711. [Abstract] [Full Text] [PDF]

				S. M. Wasserman, F. Mehraban, L. G. Komuves, R.-B. Yang, J. E. Tomlinson, Y. Zhang, F. Spriggs, and J. N. Topper Gene expression profile of human endothelial cells exposed to sustained fluid shear stress Physiol Genomics, December 26, 2002; 12(1): 13 - 23. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by De Keulenaer, G. W. Articles by Griendling, K. K. Search for Related Content PubMed PubMed Citation Articles by De Keulenaer, G. W. Articles by Griendling, K. K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH