(Circulation. 1997;96:3610-3616.)
© 1997 American Heart Association, Inc.
Articles |
Pulsatile Stretch Stimulates Superoxide Production in Human Aortic Endothelial Cells
From Cardiology, University Hospital, Inselspital, Bern and Zurich, and Cardiovascular Research, Institute of Physiology, University Zurich, Switzerland.
Correspondence to Thomas F. Lüscher, MD, FACC, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland.
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Abstract |
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Background Free radicals such as superoxide and nitric oxide (NO) play a key role in the pathophysiology of atherosclerosis. Mechanical forces such as pulsatile stretch may be involved in free radical production. We studied superoxide production by pulsatile stretch in human endothelial cells.
Methods and Results Human cultured aortic endothelial cells were exposed to pulsatile stretch up to 24 hours, and superoxide production was examined. Short-term stretch for 1 hour (10% average elongation, 50 cycles per minute) increased superoxide production 2.2-fold. This effect was reduced by diphenyleneiodonium chloride, an NADPH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. Prolonged stretch up to 6 hours increased superoxide production, but it returned to near the control level after 24 hours of stretch. However, after blockade of NO production, 24 hours of stretch did increase superoxide production 2.4-fold compared with 24 hours of stretch alone. Moreover, 24-hour stretch doubled NO synthase (NOS) (III) protein and mRNA expression. The tetrahydrobiopterin synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine had no effect on unstretched cells but doubled superoxide production compared with 24-hour stretch alone; this increase was halved by cotreatment with 6-methyl-5,6,7,8-tetrahydropterine, a lipid-soluble form of tetrahydrobiopterine.
Conclusions Short-term stretch increased superoxide production from human aortic endothelial cells via NADPH oxidase and NOS (III), whereas prolonged stretch increased both superoxide and NO production. The increase in NOS (III) protein with prolonged stretch acts as a scavenger mechanism whereby NO inactivates superoxide. Tetrahydrobiopterin determines the balance of superoxide and NO production from NOS (III) after prolonged stretch in which NOS (III) level is upregulated.
Key Words: endothelium-derived factors • vasculature • arteriosclerosis • free radicals • tetrahydrobiopterin
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Introduction |
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Endothelial cells lie in a strategic anatomical position between the circulating blood and vascular smooth muscle. They are thus most exposed to physical forces such as shear stress,1 hydrostatic pressure,2 and pulsatile stretch, particularly in the arterial circulation. Changes in local flow acutely increase nitric oxide (NO) release in vitro1 and cause flow-dependent vasodilation in vivo.3 On the other hand, increases in hydrostatic pressure cause vasoconstriction,1 reduce NO production,4 and increase endothelin release.5 This may explain the greater endothelium-dependent relaxation in arteries than veins.6
Pulsatility and hence rhythmic stretching of vascular cells are equally important in the pathophysiology of atherosclerosis. In bovine endothelial cells, pulsatile stretch augments NO synthase (NOS) expression and NO production.7 The biological effects of NO, however, are determined not only by the amounts produced but also by the degree of inactivation by superoxide.8 9 Indeed, endothelial cells are a source of superoxide,10 11 and NOS is able to produce superoxide.12 Thus, to evaluate the biological effects of mechanical force, simultaneous characterization of both NO and superoxide production is essential to clarify the biological role of such stimuli in the cardiovascular system.
We investigated the effects and time course of pulsatile stretch on superoxide and NO production in human aortic endothelial cells in culture. Our results show for the first time that pulsatile stretch stimulates both superoxide and NO production from endothelial cells and that the concomitant expression of NOS and in turn NO release especially play an important role in regulating the balance of superoxide and NO.
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Methods |
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Materials
Cytochrome c, SOD, DPI, oxypurinol, indomethacin, L-NMMA, L-NAME, DAHP, and 6-methyl-5,6,7,8-tetrahydropterine were purchased from Sigma Chemical Co. Ionomycin was purchased from Calbiochem-Novabiochem Corp. Flexercell strain unit system and Flex I culture plates were purchased from Flexcell Co. EGM was purchased from Clonetics.
Cell Culture
HAECs were prepared from human aorta by enzyme
digestion.4 13 HAECs were identified by their typical
morphological and chemical characteristics, including growth of closely
opposed polygonal confluent monolayers, maintenance of
density-dependent inhibition after serial passage, and positive
immunofluorescence for factor VIII antigen. HAECs
were cultured on collagen type I–coated dishes or collagen type
I–coated plates for stretch experiments (Flex I) in EGM (Clonetech)
and 10% fetal calf serum (Hyclone). Cells between passages 2 and 6
were used for the experiments.
Stretch Device for Cultured Cells
Pulsatile stretch was given to HAECs as previously
reported.14 15 Briefly, HAECs were seeded onto Flex I
culture plates coated with type I collagen at an initial density of
105 cells/mL. Flex I culture plates were placed on a
computerized Flexercell Strain Unit gasketed base plate in the
incubator. The membranes were subjected to a vacuum of -5 and -20
kPa, respectively, at a frequency of 50 cycles/min for up to 24 hours.
A vacuum of -20 kPa causes a deformation pattern of the membrane
ranging from 0% at the center to 24% at the periphery (10%
average).14 15 16 A vacuum of -5 kPa evokes a membrane
deformation ranging from 0% at the center and 10% at the periphery
(6% average). In parallel, other Flex I culture plates not subjected
to stretch served as controls. The cell viability with and without
stretch, as assessed with trypan blue exclusion test, was >90%
throughout the experiments.
Measurement of O2- Production
O2- production was measured as
the SOD-inhibitable reduction of cytochrome
c.11 17 Briefly, HAECs were preincubated in
DMEM without phenolred for 30 minutes at 37°C, and then cytochrome
c (final concentration, 1 mg/mL) with or without SOD
(final concentration, 500 U/mL) was added in a CO2
incubator. At the indicated time points, the medium was removed from
the cells, and the absorbance was read at 550 nm against a distilled
water blank. Reduction of cytochrome c in the presence of
SOD was subtracted from the values without SOD. The portion of
O2--specific reduction of cytochrome
c was between 20% and 35% according to the experiments.
The optical density difference between comparable wells with or without
SOD was converted to equivalent O2-
production by use of the molar extinction coefficient for
cytochrome c [21.0x103
(mol/L)-1 ·
cm-1].17
Western Blot for NOS Type III
NOS type III (ie, endothelial NOS) protein was
analyzed by Western blot using an anti-human
endothelial NOS antibody (Transduction Laboratory). The
antibody was used at 250x dilution. This antibody is specific for
human NOS III and does not cross-react with NOS I and
II.18 After stretch experiments, HAECs were detached by
trypsin-EDTA, and the cell number was determined by Coulter counter
(Coulter Electronics). Then, 100 µL of lysis solution containing 10%
glycerol, 2.3% SDS, Tris-HCl, pH 6.8, 62.5 mmol/L, 0.01%
Bromophenol blue, and 5% mercaptoethanol was added to 105
cells. The lysate was then heated at 95°C to 100°C at 5 minutes.
Next, 30 µL of cell lysates containing 3.3x104 cells was
subjected to 7.5% single percentages gel (Ready Gel, BIO RAD). The
separated proteins were electrophoretically transferred to
Immunobilon-P membranes and then incubated with anti-human NOS III
antibody for 1 hour as previously described.18 The
membranes were finally visualized by the ECL kit (Amersham), and
densitometric analysis was done by NIH image 1.54.
Northern Blot for NOS Type III
Total RNA was isolated by Trizol reagent (GIBCO BRL) according
to manufacturer's instruction. Then, 20 µg of total RNA was
subjected to electrophoresis on 1% formaldehydeagarose gels and
transferred to a nylon membrane (Highland-N, Amersham International).
Blots were hybridized in QuickHyb (Strategene) with
32P-labeled cDNA probes prepared by random prime labeling.
A 5'-fragment (1.6-kb EcoRI/Bg lII) of human NOS
(III) cDNA clone19 (kindly provided by Dr T. Michel) was
used for a NOS (III) probe. Membranes were exposed to Kodak Bio Max
x-ray film at -70°C for 24 hours. Quantification of autoradiographs
was obtained using the image analysis program NIH Image.
Statistical Analysis
Data are presented as mean±SEM. Multiple comparisons
were evaluated by ANOVA followed by Fisher's protected least
significant difference test. Student's paired or unpaired t
tests were used for comparisons between two experiments. A value of
P<.05 was considered significant.
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Results |
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Time Course of O2- production in the Presence and Absence of Pulsatile Stretch
HAECs were incubated with reaction solution for the indicated time periods in the absence or presence of a physiological level of pulsatile stretch (10% average elongation, 50 cycles/min). Under both conditions, O2- production was increased in a time-dependent manner up to 60 minutes (Fig 1
). O2-
production stimulated by stretch was significantly increased
compared with that without stretch after 40 minutes (P<.05,
n=6). The curves began to level off after 80 minutes (data not shown).
Accordingly, a 60-minute interval was chosen for subsequent
experiments.
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) and presence
(
) of pulsatile stretch (10% average, 50 cycles/min) up to 60
minutes. Data are expressed mean±SEM (n=6). *P<.05 vs
control
Possible Pathway of O2- Production
Induced by Short-term Stretch
To clarify the mechanism of stretch-induced
O2- production, we studied the effects
of several enzyme inhibitors. DPI, an NADPH oxidase
inhibitor,23 significantly reduced
O2- production induced by short-term
stretch (Fig 2; P<.05, n=6).
On the other hand, OXY, a xanthine oxidase inhibitor, and
INDO, a cyclooxygenase inhibitor,
showed no effect on O2- production
induced by short-term stretch (Fig 2).
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Contribution of NOS in O2-
Production Induced by Short-term Stretch
Because NOS can act as an important source of
O2- production,12 we
further investigated the contribution of NOS by using NOS
inhibitors and agonists. L-NMMA occupies the substrate
binding site of NOS and thereby inhibits NO but not
O2- production by
NOS.12 20 In contrast to L-NMMA, L-NAME decreases electron
flux from NOS and hence inhibits both NO and
O2- production derived from the
enzyme.12 20 To clarify the contribution of NOS in
stretch-induced O2- production, we
compared the effects of L-NMMA and L-NAME. Because the incubation
medium is based on DMEM and contains 
400 µmol/L of
L-arginine, we choose a rather high concentration of both
inhibitors (1 mmol/L). As Fig 2
shows, L-NMMA
significantly increased O2- production
by short-term stretch (P<.05, n=6), whereas L-NAME had
no significant effect. IONO, an agonist that increases intracellular
calcium in endothelial cells, also showed no additional
effect on O2- production by short-term
stretch (Fig 2; P=NS).
Effect of Prolonged Pulsatile Stretch on
O2- Production
To examine the effect of prolonged stretch on
O2- production, the time course of
O2- production after exposure to
stretch was evaluated at the indicated time points. Up to 6 hours,
pulsatile stretch increased O2-
production. On the other hand, O2-
production gradually decreased after 12 hours of stretch. At 24
hours, there was no significant change compared with control conditions
(Fig 3; n=6).
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Possible Pathway of O2- Production
Induced by Prolonged Stretch
To clarify the pathway of O2-
production induced by prolonged stretch, we used the same
inhibitors as mentioned above. DPI significantly inhibited
O2- production induced by prolonged
stretch, but OXY or INDO showed no significant effect (Fig 4; n=6). On the other hand, L-NMMA and
L-NAME strikingly increased O2-
production induced by prolonged stretch (2.4- and 1.5-fold;
P<.05, n=6). After 24 hours of stretch, IONO significantly
reduced O2- production induced by
prolonged stretch (P<.05).
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Contribution of NADPH Oxidase and NOS in
O2- Production Induced by
Prolonged Stretch
In the presence of L-NMMA, we evaluated the net amount of
O2- production, including that which
is normally scavenged by NO. O2-
production by NADPH oxidase was calculated as the difference of
O2- production measured in the
presence of L-NMMA minus that measured in the presence of L-NMMA+DPI.
Superoxide production by NOS (III) was calculated as the
difference between that measured in the presence of L-NMMA minus
L-NMMA+L-NAME, because L-NAME in contrast to NMMA inhibits not only
NO production but also O2- from NOS
(III).12 As the Table shows,
short-term stretch increased net O2-
production via NADPH oxidase 3-fold and via NOS (III) 1.8-fold
compared with the basal production under static conditions
(P<.05, n=6). Surprisingly, prolonged stretch increased net
O2- production 6.5-fold via NADPH
oxidase and 3.6-fold from NOS (III) compared with basal
production (P<.05, n=6).
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Effect of Prolonged Stretch on NOS (III) Protein
Western blot with a specific antibody against human NOS
(III) was performed to clarify whether prolonged stretch increased NOS
protein in human aortic endothelia cells. Fig 5A shows that prolonged stretch increased
NOS (III) protein in a stretch-dependent manner. As indicated in Fig 5B, NOS (III) protein was also increased by prolonged stretch over 12
hours in a time-dependent manner. Moreover, we did Western blot using a
specific antibody against human NOS (II) (Santa Cruz Biotechnology),
but the all results were negative (data not shown).
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Effect of Prolonged Stretch on NOS (III) mRNA
To clarify the effect of stretch on NOS (III) mRNA, Northern
hybridization analysis was performed. Fig 6 shows that pulsatile stretch (10%
average, 50 cycles/min) increased mRNA of NOS (III) compared with
static control. Densitometric analysis clarified that 6, 12,
and 24 hours of stretch increased NOS (III) mRNA compared with GAPDH
2.8±0.2-, 2.5±0.1-, and 2.2±0.2-fold, respectively (n=4).
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Contribution of BH4 in O2-
Production From NOS (III)
To clarify the possible contribution of BH4 to the
regulation of O2- production from NOS,
we examined the effects of the inhibitor of GTP
cyclohydrolase I (DAHP)21 22 23 and the lipid-soluble analog
of BH4 (MEBH4).21 Without stretch,
neither DAHP nor MEBH4 exhibited a significant effect.
MEBH4 alone exhibited no effect (data not shown); DAHP,
however, significantly increased O2-
production after 24 hours of stretch (Fig 7; P<.05). Moreover, this
increase was significantly reduced by cotreatment with
MEBH4 (P<.05 versus DAHP alone).
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Discussion |
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The present experiments demonstrate for the first time in HAECs that (1) short-term and prolonged pulsatile stretch increases O2- production via NADPH oxidase and NOS (III), (2) prolonged pulsatile stretch increases both O2- and NOS (III) expression, and (3) NOS (III) upregulation by prolonged pulsatile stretch acts as a scavenger mechanism for O2-. Moreover, in contrast to static conditions, reduction of BH4 levels enhanced O2- production after prolonged pulsatile stretch.
DPI is a flavoprotein inhibitor, and it is also known to be a potent inhibitor of NADPH oxidase24 and NOS.25 In our experiments, both short-term and prolonged stretch–induced O2- production was significantly reduced by DPI. In bovine coronary endothelium26 and rat cultured aortic smooth muscle cell,27 NADH-dependent oxidase was reported to be a primary source of O2-. On the other hand and in line with our observations, NADPH oxidase was reported to be a major O2--generating system in rabbit aorta.28 This suggest that O2--generating systems differ among species and different kind of cells.
NOS is now considered to play a pivotal role not only in the production of NO but also as a source of O2-. Indeed, O2- production from purified NOS (I) has been reported.12 NO derived from NOS (III), on the other hand, can act as a scavenger that is able to capture O2- (6.7x109 mol/s) faster than SOD.29 Recently, NOS (III) was reported to be increased by stretch in bovine aortic endothelial cells.7 Our hypothesis, therefore, was that if NOS (III) can be upregulated by stretch in HAECs similar to bovine cells, NOS (III) upregulation may play a pivotal role in determining the balance between O2- and NO production by the endothelium.
Of particular interest was the time course of stretch-induced O2- production. Indeed, in the absence of L-NMMA, prolonged stretch exhibited weaker effects on O2- production than short-term stretch. Stretch has been reported to activate protein kinase C,30 and protein kinase C is known to activate NADPH oxidase to produce O2-.31 Therefore, it may be possible that prolonged stretch downregulates protein kinase C, and this in turn might reduce O2- production, but we considered another possibility. In this article, O2- production was measured with cytochrome c reduction.17 It would also be possible to evaluate O2- production with NBT reduction by histochemistry, but we choose cytochrome c reduction to obtain quantitative information. This method is quite specific for the O2- anion,32 but it detects only SOD-inhibitable or active O2-. Obviously, therefore, O2- that has been inactivated by NO is not capable of reducing of cytochrome c and hence is not detected by this assay. This, in turn, means that to obtain a measure of true O2- production, measurements must be performed in the presence of L-NMMA to inhibit NO. Our results using NMMA clearly showed that prolonged stretch increased net O2- production from NADPH oxidase by 6.5-fold and from NOS (III) by 3.6-fold. Moreover, prolonged stretch increased NOS (III) protein and NO production in HAECs. Thus, the biphasic time-production course of O2- production can be explained by the increased NO production with prolonged stretch. These results therefore suggest that the upregulation of NOS (III) by prolonged stretch plays a physiological role as a scavenger mechanism of O2-.
To get further insights into the regulation of the balance of O2- and NO production from NOS (III), we examined the role of BH4, an essential cofactor for NOS to produce NO.22 33 34 35 36 37 BH4 is produced via GTP cyclohydrolase I in endothelial cells,38 and we used DAHP as an inhibitor of this enzyme.38 39 In unstretched HAECs, DAHP had no effect on O2- production. However, in stretched (24 hours) HAECs, O2- production was markedly increased by DAHP. This increase in O2- production induced by DAHP in stretched HAECs could be partially reversed by 6-methyltetrahydropterine, a lipid-soluble analog of BH4.23 Hence, with prolonged stretch, the requirements of HAECs for BH4 are increased, and depletion of BH4 leads to an increased O2- rather than NO production by NOS (III). The increased requirement for BH4 under conditions of prolonged stretch can be explained by the increase in NOS (III) protein by stretch.
Upregulation of NOS (I) and (III) was reported by shear stress,40 estrogen,18 41 42 and stretch.7 This increase in NOS, at a glance, seems to play vasculoprotective and compensatory roles. Upregulated NOS may produce larger amounts of NO, but it may also produce more O2- to scavenge NO. Although 6-methyltetrahydropterine alone showed no significant effect on O2- production from static and prolonged stretched HAECs without DAHP treatment (data not shown), the reduction in O2- production by this analog of BH4 during DAHP treatment suggest a therapeutic potential of such compounds under the condition of BH4 depletion or increased demands for the cofactor.
In conclusion, our results demonstrate the importance of mechanical forces as determinants of the balance of O2- and NO in human endothelial cells. These mechanisms are likely to play a key role in the pathophysiology of hypertensive vascular disease43 and atherosclerosis,32 disease states in which both NO and O2- production in endothelial cells are upregulated.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Swiss National Research Foundation (grant 32-32541.91/2) and a grant-in aid by Mobiliar Insurance, Bern, Switzerland, and the Karl Mayer Foundation, Liechtenstein.
Received May 6, 1997; revision received June 17, 1997; accepted June 26, 1997.
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 C. A. Ruiz-Feria Concurrent supplementation of arginine, vitamin E, and vitamin C improve cardiopulmonary performance in broilers chickens Poult. Sci., March 1, 2009; 88(3): 526 - 535. [Abstract] [Full Text] [PDF]
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 T. Wu, W. C. Willett, N. Rifai, and E. B. Rimm Plasma Fluorescent Oxidation Products as Potential Markers of Oxidative Stress for Epidemiologic Studies Am. J. Epidemiol., September 1, 2007; 166(5): 552 - 560. [Abstract] [Full Text] [PDF]
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I. Suzuma, T. Murakami, K. Suzuma, H. Kaneto, D. Watanabe, T. Ojima, Y. Honda, H. Takagi, and N. Yoshimura Cyclic Stretch-Induced Reactive Oxygen Species Generation Enhances Apoptosis in Retinal Pericytes Through c-Jun NH2-Terminal Kinase Activation Hypertension, February 1, 2007; 49(2): 347 - 354. [Abstract] [Full Text] [PDF]
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E. Takimoto and D. A. Kass Role of Oxidative Stress in Cardiac Hypertrophy and Remodeling Hypertension, February 1, 2007; 49(2): 241 - 248. [Full Text] [PDF]
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A. G. Lorenzoni and C. A. Ruiz-Feria Effects of Vitamin E and L-Arginine on Cardiopulmonary Function and Ascites Parameters in Broiler Chickens Reared Under Subnormal Temperatures Poult. Sci., December 1, 2006; 85(12): 2241 - 2250. [Abstract] [Full Text] [PDF]
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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]
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C. A. Lemarie, P.-L. Tharaux, B. Esposito, A. Tedgui, and S. Lehoux Transforming Growth Factor-{alpha} Mediates Nuclear Factor {kappa}B Activation in Strained Arteries Circ. Res., August 18, 2006; 99(4): 434 - 441. [Abstract] [Full Text] [PDF]
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S. Lehoux Redox signalling in vascular responses to shear and stretch Cardiovasc Res, July 15, 2006; 71(2): 269 - 279. [Abstract] [Full Text] [PDF]
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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]
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H. Huang, J. Sylvan, M. Jonas, R. Barresi, P. T. C. So, K. P. Campbell, and R. T. Lee Cell stiffness and receptors: evidence for cytoskeletal subnetworks Am J Physiol Cell Physiol, January 1, 2005; 288(1): C72 - C80. [Abstract] [Full Text] [PDF]
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G.-X. Zhang, S. Kimura, A. Nishiyama, T. Shokoji, M. Rahman, and Y. Abe ROS During the Acute Phase of Ang II Hypertension Participates in Cardiovascular MAPK Activation But Not Vasoconstriction Hypertension, January 1, 2004; 43(1): 117 - 124. [Abstract] [Full Text] [PDF]
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S. Kinugawa, H. Post, P. M. Kaminski, X. Zhang, X. Xu, H. Huang, F. A. Recchia, M. Ochoa, M. S. Wolin, G. Kaley, et al. Coronary Microvascular Endothelial Stunning After Acute Pressure Overload in the Conscious Dog Is Caused by Oxidant Processes: The Role of Angiotensin II Type 1 Receptor and NAD(P)H Oxidase Circulation, December 9, 2003; 108(23): 2934 - 2940. [Abstract] [Full Text] [PDF]
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P. R. Standley, A. Camaratta, B. P. Nolan, C. T. Purgason, and M. A. Stanley Cyclic stretch induces vascular smooth muscle cell alignment via NO signaling Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1907 - H1914. [Abstract] [Full Text] [PDF]
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D. F. Kahn, S. J. Duffy, D. Tomasian, M. Holbrook, L. Rescorl, J. Russell, N. Gokce, J. Loscalzo, and J. A. Vita Effects of Black Race on Forearm Resistance Vessel Function Hypertension, August 1, 2002; 40(2): 195 - 201. [Abstract] [Full Text] [PDF]
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T. Aizawa, N. Ishizaka, S.-I. Usui, N. Ohashi, M. Ohno, and R. Nagai Angiotensin II and Catecholamines Increase Plasma Levels of 8-Epi-Prostaglandin F2{alpha} With Different Pressor Dependencies in Rats Hypertension, January 1, 2002; 39(1): 149 - 154. [Abstract] [Full Text] [PDF]
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B.S. Wung, J.J. Cheng, S.-K. Shyue, and D.L. Wang NO Modulates Monocyte Chemotactic Protein-1 Expression in Endothelial Cells Under Cyclic Strain Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1941 - 1947. [Abstract] [Full Text] [PDF]
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R. A. Beswick, A. M. Dorrance, R. Leite, and R. C. Webb NADH/NADPH Oxidase and Enhanced Superoxide Production in the Mineralocorticoid Hypertensive Rat Hypertension, November 1, 2001; 38(5): 1107 - 1111. [Abstract] [Full Text] [PDF]
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I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
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S. J. Duffy, N. Gokce, M. Holbrook, L. M. Hunter, E. S. Biegelsen, A. Huang, J. F. Keaney Jr., and J. A. Vita Effect of ascorbic acid treatment on conduit vessel endothelial dysfunction in patients with hypertension Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H528 - H534. [Abstract] [Full Text] [PDF]
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Q. Hu and R. C. Ziegelstein Hypoxia/Reoxygenation Stimulates Intracellular Calcium Oscillations in Human Aortic Endothelial Cells Circulation, November 14, 2000; 102(20): 2541 - 2547. [Abstract] [Full Text] [PDF]
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J. A. Leopold and J. Loscalzo Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2477 - H2485. [Abstract] [Full Text] [PDF]
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U. Bayraktutan, L. Blayney, and A. M. Shah Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91-phox and p22-phox in Endothelial Cells Arterioscler Thromb Vasc Biol, August 1, 2000; 20(8): 1903 - 1911. [Abstract] [Full Text] [PDF]
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M. J. Somers, K. Mavromatis, Z. S. Galis, and D. G. Harrison Vascular Superoxide Production and Vasomotor Function in Hypertension Induced by Deoxycorticosterone Acetate-Salt Circulation, April 11, 2000; 101(14): 1722 - 1728. [Abstract] [Full Text] [PDF]
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D. L. Sherman, J. F. Keaney Jr, E. S. Biegelsen, S. J. Duffy, J. D. Coffman, and J. A. Vita Pharmacological Concentrations of Ascorbic Acid Are Required for the Beneficial Effect on Endothelial Vasomotor Function in Hypertension Hypertension, April 1, 2000; 35(4): 936 - 941. [Abstract] [Full Text] [PDF]
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J. Yang, K. Fukuo, S. Morimoto, T. Niinobu, T. Suhara, and T. Ogihara Pranidipine Enhances the Action of Nitric Oxide Released From Endothelial Cells Hypertension, January 1, 2000; 35(1): 82 - 85. [Abstract] [Full Text] [PDF]
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K. Hishikawa, B. S. Oemar, F. C. Tanner, T. Nakaki, T. Fujii, and T. F. Luscher Overexpression of Connective Tissue Growth Factor Gene Induces Apoptosis in Human Aortic Smooth Muscle Cells Circulation, November 16, 1999; 100(20): 2108 - 2112. [Abstract] [Full Text] [PDF]
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G. Kojda and D. Harrison Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure Cardiovasc Res, August 15, 1999; 43(3): 652 - 671. [Full Text] [PDF]
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K. Yamamoto, Q. N. Dang, S. P. Kennedy, R. Osathanondh, R. A. Kelly, and R. T. Lee Induction of Tenascin-C in Cardiac Myocytes by Mechanical Deformation. ROLE OF REACTIVE OXYGEN SPECIES J. Biol. Chem., July 30, 1999; 274(31): 21840 - 21846. [Abstract] [Full Text] [PDF]
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K. Hishikawa and T. F. Luscher Felodipine Inhibits Free-Radical Production by Cytokines and Glucose in Human Smooth Muscle Cells Hypertension, December 1, 1998; 32(6): 1011 - 1015. [Abstract] [Full Text] [PDF]
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T. Marumo, V. B. Schini-Kerth, R. P. Brandes, and R. Busse Glucocorticoids Inhibit Superoxide Anion Production and p22 Phox mRNA Expression in Human Aortic Smooth Muscle Cells Hypertension, December 1, 1998; 32(6): 1083 - 1088. [Abstract] [Full Text] [PDF]
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K. Yamamoto, Q. N. Dang, R. A. Kelly, and R. T. Lee Mechanical Strain Suppresses Inducible Nitric-oxide Synthase in Cardiac Myocytes J. Biol. Chem., May 8, 1998; 273(19): 11862 - 11866. [Abstract] [Full Text] [PDF]
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Q. Hu, G. Zheng, J. L. Zweier, S. Deshpande, K. Irani, and R. C. Ziegelstein NADPH Oxidase Activation Increases the Sensitivity of Intracellular Ca2+ Stores to Inositol 1,4,5-Trisphosphate in Human Endothelial Cells J. Biol. Chem., May 19, 2000; 275(21): 15749 - 15757. [Abstract] [Full Text] [PDF]
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K. Hishikawa, B. S. Oemar, and T. Nakaki Static Pressure Regulates Connective Tissue Growth Factor Expression in Human Mesangial Cells J. Biol. Chem., May 11, 2001; 276(20): 16797 - 16803. [Abstract] [Full Text] [PDF]
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