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Expanded Methods
Male SHR and WKY rats aged 12 weeks were obtained from Charles River Breeding
Laboratories. The experimental procedures, which were approved by Genentech's
Institutional Animal Care and Use Committee, conform to the guiding principles of the
American Physiological Society.
Measurements of Arterial Pressure and Heart Rate
MAP and HR were measured by catheterization as previously described (20). After
anesthesia with intraperitoneal injection of ketamine 80 mg/kg (Aveco Co., Inc., Fort
Dodge, Iowa) and xylazine 10 mg/kg (Rugby Laboratories, Inc., Rockville Center, NY),
catheters (PE-10 fused with PE-50) filled with heparin-saline (50 U/ml) were implanted
into the abdominal aorta via the right femoral artery for measurement of MAP and HR,
and into the right femoral vein for VEGF administration.
MAP and HR were measured in conscious, unrestrained SHR (n=53) and WKY rats
(n=51) one day after catheterization by connecting the catheters to a Model P23XL
pressure transducer (Viggo-Spectramed, Oxnard, CA, USA) coupled to a Grass Model 7
polygraph (Grass Instruments, W. Warwick, RI, USA). After a 45-min stabilization
period, VEGF (recombinant human VEGF165) (Genentech, Inc. South San Francisco, CA)
was infused intravenously at 0.05, 0.3, 1.5, or 4.5 g/kg/min in normal saline (5 l/min)
for 2 hours. Intravenous infusion (5 l/min for 2 hours) of saline vehicle alone did not
affect MAP and HR. Each animal received only a single administration of VEGF to
avoid possible tachyphylaxis with repeated administration.
Plasma NO Assessment
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To avoid influence of blood loss on hemodynamics, blood collections were performed
in separate groups of rats. Blood (0.2-0.25 ml) was collected from the arterial catheter
into blood collection tubes (Vacutainer, Becton Dickinson) pre-rinsed with sterile water
to eliminate any potential nitrite and nitrate contaminants before and 2 hours after
intravenous infusion of VEGF (1.5 g/kg/min) in SHR and WKY rats (n=9 in each
group). Blood samples were placed on ice, centrifuged and serum was separated and
frozen at -70C until analysis of nitrates by capillary electrophoresis (23). Briefly serum
samples were diluted 1:10 in MilliQ+ water (Millipore, Bedford, MA) in pre-rinsed
polypropylene vials, ultrafiltered at 5000g through Ultrafree-MC filters (Millipore; 5 kD
nominal molecular weight cut-off) and were analyzed using an HP 3D Capillary
Electrophoresis system (Hewlett-Packard information here), using 72 cm fused silica
capillaries (75 m ID). The electrolyte buffer consisted of 25 mM sodium sulphate
containing 5% NICE-Pak OFM Anion-BT (Water sproprietary osmotic flow modifier) in
MilliQ+ water. Samples were injected by electromigration for 20 seconds at –6 kV and
analyzed at an applied negative potential of 300 V/cm. The capillary was purged with
electrolyte for 1 minute between runs. Data was acquired at a response time of 0.1
seconds, with 0.01 peak width selection at 214 nm onto an HP 3D CE Chem Station data
system. The instrument was calibrated by nitrates standards of various concentrations.
Measurement linearity and assay precision were verified by using serum samples pooled
from untreated animals to which nitrates were added at various concentrations.
Pharmacokinetic Study
Blood collection. In a parallel experiment, VEGF was administered by IV via the
femoral vein to male WKY (n=6) and SHR rats (n=6) at a dose of 1.5 g/kg/min for 120
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min. In each rat, serial blood samples were taken. Blood (300 l) was collected on
EDTA via the femoral artery at 0, 30, 45, 60, 90, 120, 130, 140, 150, 180, 190, 210 and
240 min. Blood samples were centrifuged at 10,000 x g for 5 min, separated and stored
at –70°C until ready for analysis.
VEGF Assay. Dose solutions and rat plasma samples were analyzed for measurement
of rhVEGF using a dual monoclonal antibody-based Enzyme Linked Immunosorbent
Assay (ELISA). This assay utilizes a capture antibody 3.5 F8 (Genentech, Inc. South San
Francisco, CA) that binds to the VEGF heparin binding domain (amino acid #111-165)
and a detecting antibody, MAb A 4.6.1 (Genentech, Inc. South San Francisco, CA), that
binds to the KDR binding region (amino acid #82, 84, 86). It uses a fluorometric
detection method and has an assay sensitivity range of 1-128 pg/mL in rat plasma.
Pharmacokinetic Analysis. A non-compartmental method (WinNonLin®, Pharsight
Corporation, Mountain View, CA) was used to calculate VEGF PK parameters following
the 120 min IV infusion. Parameters (AUC = pg/mL*min, Clearance = mL/min/kg,
Cmax = pg/mL, Volume of distribution = mL/kg) were estimated from plasma
concentration versus time data using truncated area under the curve analysis from 0 to
240 min.
Plasma PGI2 Assay
The levels of 6-keto Prostaglandin F1, a non-enzymatically hydrated product from
PGI2, in the plasma samples were measured by using an EIA kit from Cayman
Chemicals (Ann Arbor, MI). This assay is based on the competition between 6-keto
PGF1 and a 6-keto PGF1-acetylcholinesterase (AChE) conjugate (6-keto PGF1
tracer) for a limited number of 6-keto PGF-1-specific rabbit antiserum binding sites.
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Because the concentration of 6-keto PGF1 tracer is held constant while the
concentration of 6-keto PGF1 varies, the amount of 6-keto PGF1 tracer that is able to
bind to the rabbit antiserum will be inversely proportional to the concentration of 6-keto
PGF1 in the well. Briefly, plasma was diluted with EIA buffer. The assay was done
step by step according to the Manufacture's Instructions. Plate was developed with
Ellman's Reagent and read at a single wavelength of 420 nm. The amount of 6-keto
Prostaglandin F1 (pg/ml) was calculated based on a standard curve.
Chronic Administration of VEGF
An Alzet pump (model 2001, Alza, Palo Alto, CA) was implanted with a catheter that
was inserted into the right jugular vein for chronic iv infusion of saline vehicle or VEGF
at the dose of 0.16, 0.32, or 0.42 g/kg/min for 7 days in 28 SHR and 28 WKY rats.
Then, 6 days after the continuous infusion, the arterial catheter was implanted as
described above. One day later, MAP and HR were measured in conscious rats.
Microarterial preparation and mounting in vitro
Fresh mesentery arteries of rats were placed in a container filled with cold (4˚C) Krebs
solution and immediately transferred to the laboratory. The mesentery arteries with a
diameter of 270 - 490 m at pressure of 100 mmHg were carefully dissected out. The
vessel was cut into cylindrical rings of 2 mm long under a microscope. A pair of a stainless
steel wires (40 m in diameter) was guided through the lumen of each ring. One wire was
fixed tightly on a jaw in a 2-channel myograph (Model 500A, J P Trading, Aarhus,
Denmark), and another wire was passed lightly through the vascular lumen and was
anchored to another jaw of the same chamber. These two wires were attached to a force
transducer or a micrometer, respectively. An adjustable micrometer can pull the jaws apart
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and stretch the artery between the 2 parallel wires. A calibrated force transducer (model
FT03, Grass Instruments, Quincy, MA) was used to measure the force, with the output
shown on a computer screen and printed in a printer. Measurements were made on 2 rings
simultaneously using two organ chambers.
The Krebs solution had the following composition (in mmol/L): Na+ 144, K+ 5.9, Ca2+
2.5, Mg2+ 1.2, Cl- 128.7, HCO3- 25, SO42- 1.2, H2PO4- 1.2, and glucose 11. During the
experiment, the solution at 37˚C was aerated with a gas mixture of 95 % O2 + 5 % CO2
(PO2 >200 mm Hg). Oxygen partial pressure of oxygen (PO2) was measured by an Oxygen
Meter (Model 781, Strathkelvin Instrument, U.K.).
All rings were equilibrated for 45 minutes before and after normalization as described
previously (23). In myograph (Model 500 A), the normalization procedure was performed
automatically.
Statistical Analysis
Results are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was
performed to assess differences in parameters under the same condition between groups
and to compare changes over time within each group. Two-way ANOVA was used to
compare parameters in response to chronic VEGF infusion. Significant differences were
then subjected to posthoc analyses using the Newman-Keuls method.
P<0.05 was
considered to be statistically significant.
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