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Department of Medicine, Division of Cardiology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans.
vascular biology
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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VASCULAR ENDOTHELIAL DYSFUNCTION plays a major role in the pathogenesis of metabolic and cardiovascular diseases. Indirect measurement of reduced nitric oxide (NO) availability in the coronary or in the peripheral circulation is the most frequently assessed parameter of vascular endothelial dysfunction in patients with hypercholesterolemia, diabetes mellitus, hypertension, coronary artery disease, and chronic heart failure (CHF) (1, 16, 21, 22, 24, 29). However, the vascular endothelium mediates several other physiological and pathological processes besides NO-mediated control of the vasomotor tone. Inflammation, hemostasis, and angiogenesis are all modulated by the vascular endothelium through transitions between quiescent and activated states (30). These nonvasomotor functions of the vascular endothelium are not routinely characterized in patients, primarily because of limited access to the vascular endothelium.
Thus the aim of the present investigation was to develop a novel
approach to further characterize the vascular endothelial abnormalities
that accompany metabolic and cardiovascular diseases. A new minimally
invasive technique was designed to safely collect 200-1,000
endothelial cells from either a superficial forearm vein or from the
radial artery in human subjects. Measurements of protein expression
were performed using quantitative immunofluorescence analysis, an
innovative technique that requires only a small number of endothelial
cells to accurately quantify intracellular protein levels. Because of
our interest in the cellular mechanisms of endothelial dysfunction in
patients with CHF, we initially applied this novel approach to quantify
oxidative stress by nitrotyrosine formation, endothelial cell
activation by nuclear factor-
B (NF-B) nuclear translocation and
cyclooxygenase-2 (COX-2) expression, and NO synthesis by endothelial NO
synthase (eNOS) expression in the vascular endothelium of patients with
severe CHF.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Human subjects.
Five men (mean age of 68 ± 14 yr, mean body wt of 78 ± 22 kg) hospitalized for decompensated CHF and hypotension due to
low-output state without clinical evidence of sepsis were studied. Left
ventricular ejection fraction averaged 28 ± 8%. All patients
were receiving intravenous dobutamine at an infusion rate of 5 µg · kg
1 · min1 or
milrinone at an infusion rate of 0.3 µg · kg1 · min1 for <24
h, in addition to furosemide, digoxin, and angiotensin-converting enzyme inhibitors, when vascular endothelial biopsy was performed. Four
healthy age-matched men (mean age of 61 ± 10 yr) served as controls. All subjects signed an informed consent. The study protocol and the informed consent were approved by Albert Einstein College of
Medicine Committee of Clinical Investigation.
Vascular endothelial biopsy in patients with severe CHF and in
healthy subjects.
With the use of a 0.021-in.-diameter J-shaped wire (Daig, Minnetonka,
MN) or a 0.018-in.-diameter J-shaped wire (Arrow, Reading, PA) advanced
through an 18- or 20-gauge angiocath, venous endothelial cells were
scraped from the intimas of superficial forearm veins of five patients
with severe CHF and from those of four healthy subjects. The distal
portion of the wire was transferred to a 50-ml conical tube containing
dissociation buffer (0.5% bovine serum albumin, 2 mM EDTA, and 100 µg/ml heparin in PBS, pH 7.4) kept at 4°C. After 10 rinses with
dissociation buffer, the cells were recovered by centrifugation and
fixed with 3.7% formaldehyde in PBS for 10 min. The cells were washed
twice with PBS, transferred to eight poly-L-lysine-coated
slides (Sigma Chemical, St. Louis, MO), and air dried at 37°C. The
slides were stored at 
80°C until they were analyzed.
Cell culture. Rat capillary endothelial (RCE) cells (a gift of Dr. Robert D. Rosenberg, MIT; Ref. 7) were cultured in M199 media with 15% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B. Porcine aortic endothelial (PAE) cells (a gift of Dr. Bruce Terman, Cardiology Division, Albert Einstein College of Medicine; Ref. 27) were cultured in DMEM containing 10% newborn calf serum, 0.4 µg/ml puromycin, 10 U/ml penicillin, and 10 µg/ml streptomycin. Human umbilical vein endothelial (HUVE) cells were isolated and cultured as previously reported (2) until the second passage in M199 containing 20% newborn calf serum, 5% human serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate.
Quantitative immunofluorescence and immunoblot analysis of
protein expression in cultured endothelial cells.
Protein quantifications by immunofluorescence and by immunoblot
analysis for nitrotyrosine, NF-B nuclear translocation, COX-2, and
eNOS were compared in cultured endothelial cells. Nitrosylation of
protein tyrosine residues was measured in RCE cells exposed to 0, 0.16, 1, 2, and 4 mM peroxynitrate (Upstate Biotechnology, Lake Placid, NY)
for 5 min. NF-B nuclear translocation was assessed in RCE cells
after 0-, 3-, 6-, 12-, or 25-min exposure to 100 ng/ml tumor necrosis
factor-
(TNF-) (R&D Systems, Minneapolis, MN) (4).
COX-2 expression was measured in HUVE cells (grown in M199 containing
12% newborn calf serum for 24 h) after 0-, 0.75-, 1.5-, 3-, 4-, 5-, or 6-h exposure to 20 nM phorbol myristate acetate (Sigma
Chemical) (6). eNOS expression, which is modulated by KDR
(vascular endothelial growth factor receptor-2) activity (18), was measured in wild-type PAE cells and in PAE cells
stably transfected with either native KDR cDNA or one of three mutated constructs. In these constructs, portions of the receptor's
extracellular domain had been deleted, resulting in different levels of
constitutive activity in the tyrosine kinase domain of the receptor.
The first mutated construct [KDR(Ig1-3)] lacked Ig-like domains
4-7, the second construct [KDR(Ig1-6)] lacked Ig-like
domain 7, and the third construct [KDR(Ig1-3,7)] lacked Ig-like
domains 4-6 (27).
80°C. The cells on slides were rehydrated in PBS and rendered
permeable with 0.1% Triton X-100 for 15 min at room temperature.
Nonspecific sites were blocked with PBS-5% donkey serum, which was
also used for antibody dilution. The cells were incubated with one of
the following monoclonal antibodies against either nitrotyrosine
(Upstate Biotechnology), NF-B (Santa Cruz Biotechnologies, Santa
Cruz, CA), COX-2 (Cayman Chemicals, Ann Arbor, MI), or eNOS
(Transduction Laboratories, San Diego, CA) followed by Cy3-conjugated
donkey anti-mouse antibodies (Jackson ImmunoResearch Laboratories, West
Grove, PA). Negative control slides were generated by using preimmune
mouse IgG (Sigma Chemical) as primary antibodies. The nuclei were then
stained with diaminophenylindole (DAPI) (Molecular Probes, Eugene, OR). Immunofluorescence analysis was performed in a blinded fashion by
numerically coding each slide. Staining was visualized with ultraviolet
light under a fluorescent microscope (Nikon ECLIPSE E600, Melville,
NY). Cy3 staining (red) of nitrotyrosine, NF-B (Fig.
1B), COX-2, and eNOS in
endothelial cells was digitally captured by using a COHU charge-coupled
device camera (Danville, CA). Image processing was performed by using
commercially available software (Adobe Photoshop, San Jose, CA)
(26). With the use of the Adobe Photoshop "levels" and
"threshold" functions, the background was optimized (nonspecific
extracellular signal was reduced to a uniform black background). These
settings, used to optimize image quality, were then applied as
standards for the processing of all subsequent cell images. The
intensity of Cy3 staining was quantified by determining the number of
positive (bright) intracellular pixels (Fig. 1D). Slides
were systematically read left to right and top to bottom. Only cells
with both cellular and nuclear integrity were analyzed. Cellular and
nuclear integrity was assessed morphologically. Intact cells
were defined as those with continuous, unbroken cell membrane, as
analyzed by phase-contrast microscopy. Intact nuclei were defined as
well-circumscribed oval bodies as delineated by DAPI staining. The mean
intracellular pixel count between duplicate experiments was used for
statistical analysis. To assess NF-B nuclear translocation, DAPI and
NF-B images of the same cell were processed using Adobe Photoshop. The perimeter of the nucleus was traced (Fig. 1C) and
transferred with the use of the info-navigator function (coordinates
x,y), to exactly the same position within the
image representing NF-B intracellular distribution (Fig.
1D). Thus the intensity of NF-B staining could be
quantified independently for the nucleus and for the entire cell. Whole
cell and nuclear pixel counts were acquired. The ratio (nuclear
pixels/whole cell pixels) × 100 was considered representative of
nuclear translocation (%). Twenty-five to thirty-five consecutive
endothelial cells were analyzed from each slide.
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B
experiments, cytoplasmic and nuclear proteins were separated as
previously described (9). Twenty micrograms of nuclear
proteins and the corresponding volume of cytoplasmic proteins were
blotted onto a nitrocellulose membrane. Proteins were detected by
immunoblotting using specific monoclonal antibodies (see above) with a
peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) and a
chemiluminescent system (Amersham Pharmacia Biotech, Piscataway, NJ).
-Tubulin blotting (Sigma Chemical) was used to normalize for protein
loading and to verify adequate separation of cytoplasmic from nuclear
proteins. Protein quantification was performed by densitometry using a
molecular analyst software (Bio-Rad).
Quantitative immunofluorescence analysis of human arterial and
venous endothelial cells.
The cells on slides were fixed, rehydrated, and permeabilized as
described above. Nitrotyrosine, NF-B, COX-2, and eNOS were detected
with the use of the same monoclonal antibodies previously tested in
cultured endothelial cells (see above). The human endothelial cells
were identified by staining with polyclonal rabbit anti-human von
Willebrand factor antibodies (DAKO, Carpinteria, CA), followed by
secondary biotin-conjugated donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) preconjugated with streptavidin-oregon green (Molecular Probes). The nuclei were stained with DAPI. Slides were mounted, stored, and protected from light at 4°C until analyzed. Figure 2 shows typical nuclear (blue)
staining (Fig. 2A), punctate von Willebrand factor (green)
staining (Fig. 2B), and Cy3 (red) nitrotyrosine staining
(Fig. 2C) in a representative arterial endothelial cell.
Immunofluorescence analysis was performed as described above.
Twenty-five to thirty-five consecutive intact endothelial cells were
analyzed from each slide. The total number of endothelial cells on each
slide was manually counted.
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Statistical analysis. Arterial and venous pixel counts were compared by using the Student's t-test. Nonparametric bivariate correlation [Spearman rank correlation coefficient (rs)] was used to compare protein measurements by immunofluorescence and by immunoblot analysis in cultured endothelial cells. The reproducibility of duplicate immunofluorescence measurements of protein expression (pixel count or percent nuclear translocation) in cultured endothelial cells was assessed by both percent (mean percent difference between duplicate measurements and coefficient of variation) and absolute (measurement error and repeatability coefficient) indexes.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Yield of arterial and venous endothelial biopsy in human subjects. Arterial and venous endothelial cells were collected from a superficial forearm vein and from the radial artery in five patients with severe CHF and from a superficial forearm vein in four age-matched healthy subjects. With the use of fluorescent microscopy, endothelial cells were identified by von Willebrand factor staining. The arterial endothelial cell count per slide averaged 94 (range of 70-120, estimated total of 560-960 endothelial cells per patient) and the venous endothelial cell count per slide averaged 54 (range of 25-92, estimated total of 200-736 endothelial cells per patient) in the samples collected from the five patients with severe CHF. The venous endothelial cell count per slide averaged 56 (range of 33-77, estimated total of 264-616 endothelial cells per subject) in the samples collected from four age-matched healthy subjects. No complication (e.g., pain, phlebitis, infection, thrombosis) resulting from instrumentation of either veins or arteries was observed in subjects at the 1-wk clinical follow-up. More than 90% of the endothelial cells retained cellular and nuclear integrity.
Validation of quantitative immunofluorescence analysis in cultured
endothelial cells.
Quantitative immunofluorescence and immunoblot analysis of cellular
proteins were compared in cultured endothelial cells (Fig. 3). Incubation of RCE cells with
peroxynitrate for 5 min increased nitrosylation of cellular proteins in
a dose-dependent manner. The maximal effect was observed at a
concentration of 2 mM both by immunoblot and immunofluorescence
analyses. The correlation coefficient (rs) was
0.99 (P = 0.001) and reflected an excellent correlation
between these two methodologies (Fig. 3A). TNF- (100 ng/ml) induced NF-B nuclear translocation in a time-dependent manner
in RCE cells, as assessed by immunoblot analysis of cytoplasmatic and
nuclear proteins. Untreated RCE cells had minimal nuclear NF-B.
Treatment with TNF- for 6 min resulted in a twofold increase in
nuclear NF-B. Immunofluorescent mapping of NF-B distribution was
more sensitive than immunoblotting, with a twofold increase in nuclear
NF-B observed after only 3 min. However, the time to maximal
stimulation was the same (12 min) both by immunoblot and
immunofluorescence analyses. The correlation coefficient
(rs) for immunoblot and immunofluorescence
results was 0.93 (P = 0.002) (Fig. 3B).
Low-level COX-2 expression was detected in HUVE cells grown in 12%
newborn calf serum for 24 h. Phorbol myristate acetate (20 nM)
promoted a time-dependent increase in COX-2 expression over 6 h.
Correlation between immunoblot and immunofluorescence results was
excellent (rs = 0.93, P = 0.003) (Fig. 3C). Stable transfection of KDR mutants into
PAE cells resulted in different levels of eNOS expression. PAE cells
stably transfected with the constitutively active KDR(Ig1-3)
construct expressed more eNOS than did native PAE cells or those
transfected with native KDR in the absence of the ligand. Results of
immunoblot and immunofluorescence analyses for eNOS expression in PAE
cells were significantly correlated (rs = 0.99, P = 0.001) (Fig. 3D).
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Reproducibility of quantitative immunofluorescence analysis.
Immunofluorescence analysis of protein expression in cultured
endothelial cells was performed in duplicate and measurements were
compared. Reproducibility data for nitrotyrosine (5 duplicate measurements), COX-2 (7 duplicate measurements), eNOS (5 duplicate measurements), and NF-B nuclear translocation (5 duplicate
measurements) are summarized in Table 1.
The overall coefficient of variation and the mean measurement error for
nitrotyrosine, COX-2, and eNOS (17 duplicate measurements) were only
11% and 286 pixels, respectively. Duplicate measurements of NF-B
nuclear translocation showed a similar degree of reproducibility, with
a coefficient of variation of 6% and a measurement error of 2%
(%nuclear translocation). Immunofluorescence analysis was also
reliable in human endothelial cells harvested from patients. eNOS
expression was measured in duplicate slides with a mean difference
(range) of 7.5% (2.0-15.4), a measurement error of 895, a
repeatability coefficient of 2,478, and a coefficient of variation of
5.5%.
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Nitrotyrosine, NF-B, COX-2, and eNOS protein expression in the
arterial and the venous endothelium of patients with CHF and healthy
subjects.
Protein expression in vascular endothelial cells collected from
patients with CHF and from age-matched healthy subjects was assessed by
quantitative immunofluorescence analysis. Results are summarized in
Table 2. Levels of oxidative stress
(nitrotyrosine formation) and endothelial cell activation (NF-B
nuclear translocation and COX-2 expression) were similar in the
arterial and the venous endothelium of patients with CHF, whereas eNOS
expression was significantly greater in the arterial compartment. COX-2
expression was eightfold greater in venous endothelial cells collected
from patients with CHF, compared with venous endothelial cells from healthy subjects. A trend toward increased nitrotyrosine formation and
NF-B nuclear translocation in the venous endothelium of patients (P = 0.11 and P = 0.10, respectively)
was also observed.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Safe and minimally invasive collection of vascular endothelial
cells coupled to measurements of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of
the vascular endothelium in humans. Evaluation of vascular endothelial
dysfunction in patients with metabolic and cardiovascular diseases has,
so far, been mostly limited to the assessment of the NO-mediated
control of the vascular tone. Our novel approach will allow the
investigation of the cellular mechanisms by which the vascular
endothelium mediates other important physiological and pathological
processes, such as inflammation, hemostasis, and angiogenesis. In the
present study, levels of oxidative stress (nitrotyrosine formation),
endothelial cell activation (NF-B nuclear translocation, and COX-2
expression) and NO synthesis (eNOS expression) in the arterial and the
venous endothelium of patients with severe CHF and healthy age-matched
subjects were compared.
Vascular endothelial biopsy and measurement of protein expression
by quantitative immunofluorescence analysis.
Feng et al. (12) recently reported that a few hundred
endothelial cells could be rinsed from the J-shaped wire used during endovascular procedures. Smaller J-shaped wires, inserted through an
18- or 20-gauge angiocath into a peripheral forearm vein or the radial
artery, enabled us to collect 200-1,000 endothelial cells from
patients with severe CHF and from healthy subjects. This minimally
invasive procedure was performed without complications. Because
immunoblotting cannot quantify protein expression when only a
relatively small number of cells are available for analysis, we
assessed protein expression by quantitative immunofluorescence analysis. This new technique allows reliable measurements of
intracellular protein expression in a small number of endothelial
cells. The average intensity of the immunofluorescence signal in
25-35 endothelial cells directly correlated with protein
measurements by immunoblot analysis for nitrotyrosine formation and
NF-B nuclear translocation, COX-2, and eNOS expression in cultured
endothelial cells. Assessment of protein expression by
immunofluorescence analysis is reproducible: the overall coefficient of
variation between duplicate experiments was 11% for nitrotyrosine,
COX-2, and eNOS results and 6% for NF-B nuclear translocation.
Comparison of vascular endothelial protein expression in patients
with severe CHF and in healthy subjects.
Sustained low-grade systemic inflammation represents an important
feature of the clinical syndrome of CHF. In patients with severe CHF,
circulating levels of proinflammatory cytokines, prostanoids, and
oxidative stress metabolites are higher than those in healthy subjects
(10, 11, 17, 20, 28). TNF-, interleukin-1
, and
angiotensin II activate the NADPH oxidase system and enhance vascular
superoxide production (8, 13). Superoxide rapidly scavenges NO, generating peroxynitrate, a toxic metabolite that nitrosylates proteins on tyrosine residues forming nitrotyrosine, which
can be detected by immunofluorescence analysis. As previously noted in
patients undergoing coronary artery bypass surgery (14, 15), we found similar levels of oxidative stress (nitrotyrosine formation) in the arterial and the venous endothelium of patients with
severe CHF. Nitrotyrosine levels tended to be higher in the veins of
patients compared with healthy age-matched subjects (P = 0.11). Oxidative stress and cytokines activate cultured endothelial cells by inducing nuclear translocation of the transcription factor NF-B (5, 19). In turn, NF-B promotes the expression
of several proinflammatory genes, including COX-2 (3).
COX-2 induction is associated with increased release of proinflammatory
prostaglandins from cultured endothelial cells (23).
Venous COX-2 expression was eightfold greater in patients with CHF than
in healthy age-matched subjects. NF-B translocation also tended to
be greater in patients (P = 0.10). Finally, as
previously reported by Hamilton et al. (15), in patients
undergoing coronary artery bypass surgery, we observed that eNOS was
expressed at a significantly greater level in the arterial than in the
venous endothelium of patients with severe CHF.
Limitations.
Only five patients with severe CHF and four healthy age-matched
subjects were studied. It is likely that the differences in nitrotyrosine formation and NF-B nuclear translocation would have
reached statistical significance with a larger sample size. However,
the aim of the present investigation was to determine whether protein
expression could be reliably quantified in a small number of
endothelial cells collected by endovascular biopsy. Future studies
could address more conclusive remarks about the abnormalities in
vascular endothelial phenotype associated with CHF.
B-mediated transcription and
COX-2 expression are suppressed in vitro by elevated cAMP levels
(25), these drugs are not likely to be responsible for the
observed activation of the vascular endothelium.
In conclusion, in the present study we introduced and validated a novel
approach to accurately measure protein expression in human vascular
endothelial cells harvested from a superficial vein of the forearm and
the radial artery. Cell activation is present in the arterial and the
venous endothelium of patients with severe CHF. The possibility to
monitor over time the transition between quiescent and activated states
in the venous endothelium appears particularly attractive, since venous
rather than arterial sampling may allow serial measurements with
minimal hazards and discomfort to patients. This novel approach may
improve our understanding of the changes in vascular endothelial
phenotype associated with metabolic and cardiovascular disease states
such as hypercholesterolemia, diabetes mellitus, hypertension, coronary
artery disease, and CHF.
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ACKNOWLEDGEMENTS |
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We thank Dr. Bruce Terman for the helpful discussion and comments on the manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health (NIH) Grants HL-51043 and CA-86173 to J. A. Ware, by NIH Training Grant HL-07675 to P. C. Colombo, by American Heart Association Grant 95264104 to T. H. Le Jemtel, and by Fellowship Award 0020186T to A. W. Ashton.
Address for reprint requests and other correspondence: T. H. Le Jemtel, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461 (E-mail: lejemtel{at}aecom.yu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00680.2001
Received 2 July 2001; accepted in final form 6 November 2001.
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TOP
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METHODS
RESULTS
DISCUSSION
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