Original Study

The association between vascular endothelial

growthfactor levelsandclinicallyevident peripheral
edema in dogs with systemic inflammatory
response syndrome
Deborah C. Silverstein, DVM, DACVECC; Catalina Montealegre, VMD; Frances S. Shofer, PhD and
Cynthia M. Otto, DVM, PhD, DACVECC
Abstract
Objective To determine the relationship between plasma vascular endothelial growth factor (VEGF) levels,
severity of illness, and edema formation in critically ill dogs.
Design Prospective, observational, descriptive, clinical study.
Setting University Teaching Hospital.
Animals Twenty-eight dogs.
Interventions None.
Measurements and Main Results Physical examination and multifrequency bioimpedance (MFBIA)
measurements were performed daily on 28 critically ill dogs with evidence of severe inammatory disease
and compared with the corresponding plasma VEGF levels. The change in VEGF values and the relationship
between MFBIA measurements and clinical evidence of edema were also examined. Eighteen dogs had a
positive VEGF level, 12 dogs had clinical evidence of edema, and 7 dogs had both a positive VEGF level and
clinical evidence of edema. There was no statistically signicant correlation between VEGF levels and the
presence of edema on physical examination (P50.2). VEGF values were also evaluated with respect to WBC
count, survival prediction index, presence of known sepsis, change in extracellular water, and outcome. No
statistical relationship could be identied between VEGF levels in the blood of dogs with inammatory
disease and their survival prediction index (P50.1), the WBC count (P50.2), or presence of sepsis (P50.2).
Dogs with a VEGF level 470 pg/mL (n53) were less likely to survive (P50.04). Because of high variability
within and between animals, conclusions regarding changes in MFBIA could not be made, suggesting that this
technology requires further renement and investigation in critically ill dogs.
Conclusions A relationship between VEGF and clinically evident increased vascular permeability was not
found in this study. Dogs with markedly elevated VEGF levels may be more likely to die, but further studies
are needed to determine the diagnostic and prognostic value of VEGF in critically ill dogs.
(J Vet Emerg Crit Care 2009; 19(5): 459466) doi: 10.1111/j.1476-4431.2009.00457.x
Keywords: body water changes, canine, vascular leak, vascular permeability, SIRS
Introduction
Critically ill animals commonly suffer from diseases
that result in complex cellular processes that involve
the balanced interaction between pro-inammatory and
anti-inammatory mediators. Many inammatory
mediators contribute to dysfunction of the vascular
endothelium during various stages of inammation.
Leukotrienes (LTs) (LTC4, LTD4, LTE4), cyclooxgenase
products, platelet activating factor, neurogenic peptides
(neurokinins and atrial natriuretic peptide), nitric ox-
ide, peroxynitrite, metalloproteases, tumor necrosis
factor-alpha (TNF-a), interleukins (IL), and interferon
are associated with the development of increases in
vascular permeability.
110
Additionally, decreased va-
somotor tone, vasopressor hyporesponsiveness, and
hypercoagulability commonly occur. Treatment of
patients with endothelial dysfunction is especially
Gift support: Crazy Kim Foundation.
The authors declare no conicts of interest.
Address correspondence and reprint requests to
Dr. Deborah Silverstein, Department of Clinical Studies, University of
Pennsylvania Matthew J. Ryan Veterinary Hospital, 3900 Delancey St,
Philadelphia, PA 19104-6010, USA.
Email: dcsilver@vet.upenn.edu
From the Department of Clinical Studies, University of Pennsylvania
Matthew J. Ryan Veterinary Hospital, Philadelphia, PA 19104-6010.
Journal of Veterinary Emergency and Critical Care 19(5) 2009, pp 459466
doi:10.1111/j.1476-4431.2009.00457.x
& Veterinary Emergency and Critical Care Society 2009 459
difcult because the combination of increased hydro-
static pressure (due to uid resuscitation), decreased
colloid osmotic pressure (COP) (due to decreased al-
bumin production, increased losses, or both), and in-
creased vascular permeability typically results in the
development of interstitial tissue edema.
The changes in vascular permeability caused by the
pro-inammatory cytokines mentioned above most
commonly occur secondary to an increase in the num-
ber of intracellular fenestrations (small pores), caveolae
(small plasmalemmal invaginations that allow vesicu-
lar transport of small proteins through the cytoplasm of
a single endothelial cell), or vesiculo-vacuolar organ-
elles (fused vesicles that form a channel through the
endothelial cytoplasm).
11,12
These changes result in an
increased ux of water, solutes, and protein from the
vascular space. The resultant increase in interstitial
uid lengthens the diffusion distance across which O
2
and CO
2
must traverse in order to enter, or diffuse out
of, the adjacent cells.
11,12
Subsequently, the cells become
hypoxic, sodium transport out of the cells is compro-
mised, and cellular swelling and death ensues. Edema
typically forms rst in the potential spaces of the peri-
toneal and subcutaneous spaces, thus delaying edema
formation in other organs such as the kidney, heart,
brain, and muscles, preserving oxygen diffusion to
these cells.
Hyperpermeability is associated with normal physi-
ologic conditions such as fetal development and wound
healing,
11
in addition to many pathologic conditions
such as diabetic retinopathy, neoplasia, protein-losing
nephropathy, atherosclerosis, systemic inammatory re-
sponse syndrome (SIRS), sepsis, and acute respiratory
distress syndrome (ARDS).
13
Increased vascular perme-
ability can also cause enlargement of cerebral infarcts.
14
Clinically, it is difcult to measure the severity and pro-
gression of tissue edema. However, total body water
(TBW), intracellular water (ICW), and extracellular wa-
ter (ECW) has been measured using multifrequency
bioelectrical impedance analysis (MFBIA) in humans.
15
MFBIA is immediate, noninvasive, inexpensive, and has
been validated for use in dogs.
16
Animals receiving iso-
tonic uid therapy develop an increase in their TBW
and ECW as measured using MFBIA.
16
Because the
changes are all isotonic, the ICW typically remains the
same unless the cells become severely hypoxic, at which
point the cellular membrane pumps may fail, leading to
the accumulation of sodium and thus water in the in-
tracellular space.
Although the development of edema is a multifacto-
rial process, one cytokine that may play a signicant
role in increasing vascular permeability during severe
inammation is vascular endothelial growth factor
(VEGF). VEGF is produced by many cells and tissues
in the body, including peripheral blood monocytes,
neutrophils, platelets, vascular smooth muscle cells,
cardiac myocytes, osteoblasts, neurons, myobroblasts,
and lung epithelium.
17,18
It exerts its effects by inter-
acting primarily with receptors on endothelial cells and
bone marrow cells.
19
VEGF was originally discovered
as a permeability-enhancing protein secreted by tu-
mors.
20
It has because been shown to be a potent per-
meability-enhancing agent in vitro,
21,22
in situ,
23
and in
vivo.
10,24
VEGF is reported to be 50,000 times more po-
tent than histamine for inducing vascular permeabil-
ity.
25
The method by which VEGF increases endothelial
permeability to macromolecules is thought to be by in-
creasing the number of fenestrations, caveolae, or ve-
siculo-vacuolar organelles, or by widening tight
junctions.
22,2628
VEGF also induces vasodilation
29
and is essential for
the de novo formation of new blood vessels (vasculo-
genesis) and the sprouting growth of capillaries (an-
giogenesis).
30
Other roles for VEGF include enhancing
endothelial cell-induced coagulation, stimulating -
brinolysis, preventing endothelial cell apoptosis, and
causing endothelial cells to promote adhesion and ac-
tivation of resting platelets.
31
Stimuli for increased cellular VEGF expression in-
clude hypoxia,
32,33
inammatory cytokines,
19,31,34
and
high ambient glucose concentrations (both in vitro in
several cell types as well as in the glomeruli of diabetic
rats).
35
Plasma levels of VEGF have also been found to
be increased in some inammatory diseases such as
SIRS,
36
sepsis,
37
and ARDS.
38
This increase in vascular
permeability can cause interstitial edema, cellular hy-
poxia, and transcellular uid leakage.
12
Identication of
mediators that contribute to the cascade of progressive
edema may provide new prognostic biomarkers as
well as targets for prevention and treatment of this
complication.
Because VEGF is produced by inammatory cells
and may play a role in the development of vascular
leak syndromes and multiorgan dysfunction, we hy-
pothesized that animals with the elevated VEGF levels
would also have increased WBC counts, decreased sur-
vival prediction index (SPI2) (lower value means more
likely to die), be more likely to have an inammatory
disease such as sepsis, have increased interstitial edema
development, greater increases in ECW, and lower
survival rates.
Materials and Methods
This was a prospective observational, descriptive study.
The subjects for this study were 28 client-owned dogs
presented for treatment at a veterinary referral teaching
hospital that t the following inclusion criteria: an
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00457.x 460
D.C. Silverstein et al.
inammatory disease process causing an increase or
decrease in the WBC count (420 10
9
/L or o5 10
9
/
L) or 45% bands, body weight 45 kg, critical illness
requiring at least 24 hours in the intensive care unit
(ICU), animals requiring uid therapy, and client con-
sent. The ICU at the facility only accepts animals for
admission if they are unstable and require intensive
cardiovascular or respiratory support. Dogs were ex-
cluded from the study if they had any evidence of
neoplasia, severe hypoxemia (PaO
2
o60 mm Hg or
SpO
2
o90%), or clinical evidence of edema on presen-
tation (chemosis, increased skin turgor, pitting intersti-
tial edema).
Age, sex, and breed were recorded for each patient.
Upon admission to the ICU, physical examination was
performed (including rectal temperature, heart rate, re-
spiratory rate). Mean arterial blood pressure (MAP)
and MFBIA were measured. Blood was collected for
VEGF immunoreactivity and COP measurements.
Arterial blood gas analysis or pulse oximetry was
performed. The SPI2 was determined from admission
biochemistry analysis and MAP measurement. Except
for the SPI2, all measurements were repeated every
24 hours.
In order to determine the relationship between VEGF
and clinically evident edema formation in critically ill
dogs, we evaluated edema formation in critically ill
dogs via physical examination and MFBIA and com-
pared these ndings to the highest measured level of
plasma VEGF. VEGF values were also evaluated with
respect to WBC count, SPI2, presence of known sepsis
via positive bacteriologic culture results, change in
ECW, and outcome. The relationship between MFBIA
measurements and clinically evident edema were also
examined.
SPI
The SPI2 was determined by following the modied
equation as described by King et al.
39
Values obtained
for the SPI2 were taken within 24 hours of admission to
the ICU. Based on a recent report showing that serial
SPI2 calculations do not improve the sensitivity of
the model, only admission SPI2 was included.
40
The
equation for SPI2 is dened as Logit P50.32731
(0.0108 MAP) (0.0102 respiratory rate) (0.2183
creatinine)1(0.0164 PCV)1(0.3553 albumin)
(0.1184 age) (0.8069 medical versus surgical status).
Physical examination
The presence or absence of clinically evident edema
was subjectively determined daily during physical ex-
amination of each patient by 1 of 2 veterinarians with at
least 5 years of clinical experience and recorded as
edema or no edema. The veterinarian was not aware of
the MFBIA or VEGF measurements during the assess-
ment. In addition, rectal temperature, heart rate, and
respiratory rate abnormalities were recorded.
MFBIA
MFBIA was measured daily using a bioimpedance
spectroscopy analyzer
a
using the previously published
method.
16
The dogs were placed in sternal or lateral
recumbency while 2 electrodes were inserted subcuta-
neously immediately caudal to the occipital protuber-
ance, 2.5 cm (1 in) apart, and another 2 electrodes were
placed subcutaneously at the tail base, 2.5 cm apart. The
path length from the occipital protuberance to the tail
base was measured in centimeters. All values for ECW,
ICW, and TBW were obtained using the instruments
software for water volume analysis.
VEGF ELISA
Venous blood was obtained daily for the VEGF ELISA
via an IV catheter and was placed into vacuum
b
col-
lection tubes containing ethylene diamine triacetic
acid as an anticoagulant. The samples were then cen-
trifuged at 1000 g for 3 minutes. The plasma was as-
pirated from the tube after centrifugation and
transferred to a separate plastic tube. This tube was
then frozen at 801C until batch analysis using a quan-
titative sandwich enzyme immunoassay kit
c
and fol-
lowing the manufacturers instructions. One hundred
microliters of diluent was added to each well followed
by 100 mL of plasma sample, control, or standard.
Plasma samples, controls, and standards were run in
duplicate. To correct for optical imperfections in the
plate, the plate was simultaneously read at 540 nm and
these readings were subtracted from the primary read-
ings taken at 450 nm. This VEGF ELISA measures the
amount of circulating VEGF
165
and VEGF
121
and has
been used for plasma VEGF analysis in dogs.
41,42
Values that were read as 0 pg/mL were all consid-
ered negative for circulating VEGF.
COP
In order to determine the contribution of COP to
clinically evident edema formation, venous blood was
obtained via an indwelling venous catheter and anti-
coagulated with lithium heparin for measurement of
COP daily using a colloid osmometer.
d
Arterial PaO
2
/pulse oximetry
An arterial blood sample was obtained via an indwell-
ing arterial catheter for measurement of the PaO
2
using
a point-of-care analyzer.
e
If an arterial blood sample
was not obtained then a pulse oximeter
f
was used to
measure arterial oxyhemoglobin saturation using light
absorption. This information was used to ensure that
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00457.x 461
VEGF levels and impedance in critically ill dogs
the dogs were not hypoxemic while participating in the
study.
Survival
Survival was dened as those animals that were alive at
the time of discharge from the hospital. All survivors
were sent home when the disease state was stable and
the animal no longer required intensive monitoring or
therapeutic support.
Data analysis
Summary data are presented as frequencies and per-
centages for categorical variables (sepsis, outcome, clin-
ical evidence of edema) and medians with interquartile
ranges (IQR) for continuous variables (WBC count,
SPI2). For purposes of analysis, dogs were grouped as
VEGF1 (VEGF40 pg/mL) or VEGF (VEGF 0 pg/
mL). For comparisons between the VEGF groups, the w
2
or Fisher exact test and the Mann-Whitney U-test for
categorical or continuous data, respectively, was used.
In addition, to compare VEGF levels between dogs with
and without clinical evidence of edema, a Mann-Whit-
ney U-test was used. All data were analyzed using sta-
tistical software.
g
A P-value o0.05 was considered
statistically signicant.
Animal care committee approval
The protocol was approved by the Institutional Animal
Care and Use Committee. All clients provided in-
formed consent before enrollment of their pets in the
study.
Results
Twenty-eight dogs were included in the study. The eti-
ology of critical illness varied between dogs and is pre-
sented in Table 1. The overall survival rate was 64%
(18/28 dogs were discharged). All of the dogs had ev-
idence of SIRS as described by Hauptman et al,
43
and 14
of the dogs had a known septic focus with a positive
bacteriologic culture (the other 14 had nonseptic SIRS).
Twelve dogs had clinical evidence of edema via phys-
ical examination on at least 1 day while in the ICU and
18 dogs had at least 1 positive serum VEGF level. Seven
dogs had both a positive VEGF level and clinical ev-
idence of edema. All animals had a PaO
2
80 mm Hg
(n 525) or a SpO
2
95% (n 53) at all measurements
during inclusion in the study.
The median SPI2 for all dogs was 0.702 (IQR: 0.620
0.770). Dogs that died or were euthanized had a median
SPI2 of 0.650 (IQR: 0.4960.751) compared with 0.732
(IQR: 0.6360.777) for those dogs that survived to dis-
charge (P50.2). The SPI2 in animals that developed
clinical evidence of edema was 0.657 (IQR: 0.5680.803)
versus 0.711 (IQR: 0.6400.764) in those that did not
develop edema (P50.55). Animals that had a positive
VEGF value at any time during the hospital stay had a
median SPI2 of 0.682 (IQR: 0.6140.758) and animals
with a VEGF value of 470 pg/mL (n 53) had a median
SPI2 of 0.751 (IQR: 0.1780.803). Dogs that were
VEGF throughout their hospitalization had a median
SPI2 of 0.738 (IQR: 0.6360.882).
The COP in clinically edematous animals was
415.5 mm Hg at all times. The animals with clinical
evidence of edema did not have a higher VEGF level
than those without clinical evidence of edema (animals
with edema 543 88 pg/mL, animals without edema
531 59 pg/mL, P50.4). There were 9 animals with a
positive change in VEGF levels (delta VEGF) during the
course of hospitalization, but only 1 of these dogs had
clinical evidence of edema.
The presence of circulating VEGF was not signi-
cantly associated with the WBC count (P50.4). The
median WBC count in the animals with circulating
VEGF was 23 10
9
/L (IQR: 1432.2 10
9
/L), while
the median WBC count in animals with no circulating
VEGF was 18.8 10
9
/L (IQR: 7.522.7 10
9
/L).
Three dogs had a VEGF levels 470 pg/mL (median
121 pg/mL; IQR 72186 pg/mL) and 25 dogs had VEGF
levels o70 pg/mL (median 33 pg/mL; IQR 066 pg/
mL). Dogs with a VEGF level 470 pg/mL were less
likely to survive compared with dogs with VEGF
level o70 pg/mL (0% versus 72% survival; P50.04).
In comparison, there was no difference in survival
between VEGF and VEGF1 dogs (66% versus 60%
survived; P51.0).
Table1: Signalment and diseases of dogs enrolled in study
Age in years (mean SD) 6.7 3.5
Sex (n) Female intact (3)
Male intact (4)
Male castrated (10)
Female spayed (11)
Breed (n) Mix (5)
Labrador (6)
Other (17)
Disease (n) Gastric dilatation volvulus (1)
Endocarditis/septic polyarthropathy (1)
Bite wounds (2)
Immune-mediated hemolytic anemia (2)
Prostatic abscess (2)
Pneumonia (2)
Gastroenteritis (2)
Pyometra (2)
Hepatopathy (2)
Postoperative complication (2)
Gastrointestinal perforation (4)
Hit by car (4)
Pancreatitis (5)
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00457.x 462
D.C. Silverstein et al.
The values obtained using MFBIA were highly vari-
able between days, with both repeated measures in in-
dividual dogs as well as between dogs. Statistical
analyses of values over time were therefore not per-
formed. The clinical signs of edema did not correlate
with MFBIA measurements of ECW, ICW, or TBW (eg,
several dogs developed clinical signs of edema despite
a decrease in TBWand ECWor vice versa). In addition,
the VEGF level was not predictive of clinical edema
formation, regardless of the method of detection (phys-
ical examination [P50.55] or MFBIA).
The variability of MFBIA values within patients was
unacceptably high. The values obtained did not appear
to correlate with the clinical signs of the patient and the
MFBIA predicted uid shifts were often not physically
possible. MFBIA, while holding promise, failed to pro-
vide sufciently valid information, therefore, values
obtained are not reported.
Discussion
Forty-three percent of the critically ill dogs in this study
developed clinical evidence of interstitial edema, de-
spite the presence of a COP 415.5 mm Hg, a value that,
by itself, does not typically lead to interstitial edema.
Despite previous studies suggesting VEGF was associ-
ated with SIRS and the known function of VEGF as a
vascular permeability factor, the present study was un-
able to demonstrate a relationship between VEGF levels
and clinical evidence of interstitial edema or WBC
counts. Similarly, animals with a positive delta VEGF
were not more likely to develop clinical evidence of
edema than those with a zero or negative delta VEGF.
However, animals with a VEGF level 470 pg/mL were
more likely to die or be euthanized, although only 3
animals had VEGF levels in this range. Because SIRS
represents a progressive inammatory disease, a change
in biomarker levels may be more informative than a
single value.
Similar to VEGF levels, SPI2 values were not pre-
dictive of survival. Typically, the closer the SPI2 value
is to 0, the greater the severity of disease. The SPI2 is
generally more valuable as a predictor of survival in a
population or group of animals rather than individual
patients. However, the SPI2 was not predictive of
survival, the development of clinical edema, or the
presence of a positive VEGF serum level. The median
SPI2 for all animals in the study was 0.666 and
this was associated with an overall survival rate to
discharge of 64%. Further studies examining the role
of SPI2 and vascular leak syndromes may be infor-
mative.
Previous studies have examined VEGF levels in
septic human patients. Children with meningococcal
infections were found to have higher VEGF plasma
concentrations if presented with shock than those
without shock. Additionally, the VEGF level at the
time of admission correlated with both the severity of
disease and the amount of uid administered within
the rst 24 hours.
44
VEGF levels were not measured at
the time of admission in the dogs of this study, but
baseline levels might have proved valuable in these
dogs. Additionally, the presence or absence of shock
at admission was not recorded, nor was the total uid
volume administered within the rst 24 hours, but
this might be useful in future studies. Elevated VEGF
levels were also found in 18 adult human patients
with severe sepsis; serum albumin levels were used as
an indirect measure of vascular leak and found to be
decreased in septic patients. The VEGF level at study
entry was correlated to the severity of multiple organ
dysfunction and the peak VEGF levels were signi-
cantly higher in the nonsurvivors.
37
Although the
dogs in our study were not all septic, 16 dogs in-
cluded in this study had a documented infection and
15 of 16 had at least 1 positive VEGF serum level.
Further focused investigations of VEGF levels in dogs
with sepsis or a known infectious process are neces-
sary to determine if VEGF represents a biomarker
of sepsis or a potential prognostic indicator in this
population.
Experimentally, mice with cecal ligation-puncture-in-
duced sepsis had a time-dependent increase in plasma
VEGF concentrations, with peak levels at 24 hours.
45,46
Similarly, the systemic administration of lipopoly-
saccharide to human volunteers caused an elevation
in circulating VEGF levels with peak levels occurring at
4 hours.
45
Although it is possible that the release and
function of VEGF is different in dogs versus mice or
humans, both of these studies suggest that it might be
benecial to measure VEGF levels earlier in the course
of disease in order to truly appreciate the peak value.
Similarly, 10 humans with severe sepsis had elevated
levels of VEGF compared with controls, and the levels
remained elevated in most patients during their ICU
stay.
45
However, although this did not appear to be the
case in the critically ill dogs of this study.
Because a heterogenous group of critically ill dogs
was included in this study, it may prove worthwhile to
narrow the study population or timing of sample
acquisition in future studies. The rst VEGF sample
in the current study was obtained upon admission
to ICU (which is typically 1224 h from the time of
hospital admission). Evaluation of VEGF levels at the
time of presentation to the emergency room might
prove more informative because it is possible that the
true baseline or peak were missed or inuenced by
treatment before ICU admission. The half-life of VEGF
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00457.x 463
VEGF levels and impedance in critically ill dogs
is only 33.7 13 minutes in humans,
47
and it is there-
fore possible that timing of sampling is critical for this
biomarker to be of value. The value of VEGF may
also be enhanced as part of a panel of mediators,
including nitric oxide, TNF-a, IL-6, procalcitonin, and
C-reactive protein, similar to the markers of inamma-
tion that are commonly examined in people with SIRS
and sepsis.
48
Recently, a new mRNA splice variant of VEGF has
been found that produces an inhibitory isoform of
VEGF
165
named VEGF
165
b. Theoretically, inhibitory
variants of all 7 isoforms are possible.
49
Available
anti-VEGF antibodies do not distinguish between
VEGF and the inhibitory isoforms, making interpreta-
tion of our ELISA results potentially difcult.
34,49
This
would be one explanation for the 8 dogs with a positive
delta VEGF in the absence of clinical edema formation.
Alternatively, low VEGF levels with evidence of edema
may be due to an upregulation of the VEGF receptors
present without a signicant upregulation of VEGF
production. Serum albumin was not measured daily
in the animals of this study, but might also have con-
tributed to edema formation in animals with clinical
evidence of edema and a low VEGF, despite the
COP, because it is the rate of decrease in COP that
determines transvascular ux, rather than just the ab-
solute number.
50,51
The small sample size in this study is a possible rea-
son for the lack of a statistical relationship between
VEGF levels and clinically evident vascular leak states
or other parameters. Similarly, a statistical relationship
between VEGF levels and the WBC count or type of
disease may be more likely when examining a greater
number of dogs with a single underlying disease pro-
cess (eg, pancreatitis). VEGF may still prove to be a
valuable marker of specic disease states or prognosis
when used in combination with other markers of vas-
cular leak and disease severity. Larger studies that in-
corporate concurrent measurements of the closely
related placental growth factor and cytokines such as
TNF-a, IL-1, and IL-6 may also improve our under-
standing of the relationship and time course of the var-
ious mediators of inammation. The dynamics of
critical illness are not well appreciated, but under-
standing patient-specic responses may allow treat-
ment decisions to be tailored to individuals and thus
improve outcome. Obtaining VEGF and cytokine levels
upon admission to the hospital and serially (eg, q12 h)
thereafter may prove critical for this type of individu-
alized care. In addition, investigating the ability of the
serum from animals with high VEGF levels to induce
endothelial hyperpermeability, as measured by albu-
min leakage through harvested endothelial cells, might
provide further information concerning the relationship
between elevated VEGF levels and vascular leak
syndromes.
Vascular leak syndromes are especially challenging
to diagnose early or treat effectively after extravasation
has occurred. New strategies to minimize the develop-
ment of uid extravasation from the vascular space in
critically ill animals might include strategies aimed at
modifying VEGF and other mediators, although this
approach requires appropriate timing and dose re-
sponse so the benecial effects of VEGF in the body are
not compromised.
It is difcult to assess or measure endothelial func-
tion in clinical patients. Dysfunction and subsequent
vascular leak states are typically recognized by the
presence of interstitial edema on physical examination.
Objective, quantitative measures of edema are not
readily available; therefore, MFBIA was used in this
study to try and determine changes in body water
compartmentalization. This method has been validated
for use in healthy dogs and is a rapid, noninvasive
technique.
16
However, MFBIA was not a reliable mon-
itoring tool for assessing daily volume shifts in the
critically ill dogs of this study. There are several pos-
sible reasons for the day to day variability including
variation in patient position, change in the surface on
which the patient was lying, and subtle alterations in
electrode placement. Changes in lean body mass or
skin temperature can affect MFBIA measurements in
humans.
52
Many of the animals in the current study
had signicant changes in body temperature from day
to day. Additionally, critically ill patients might have
changes in reactance and resistance due to disruptions
in cell membrane integrity or an increased space be-
tween adjacent cells that will contribute to daily
changes in MFBIA measurements. Further research
comparing MFBIA in critically ill dogs to gold standard
techniques such as deuterium oxide (for TBW) and so-
dium bromide (for ECW) might prove helpful for fu-
ture studies.
Even if MFBIA measurements had proven more re-
liable in this study, the technology still does not allow a
direct measurement of the interstitial or intravascular
space, but rather only measures TBW, ICW, and ECW.
Other techniques used to measure vascular leak have
included close measurements of net uid balance,
44
extravasation of Evans blue dye into specic organs
(experimental),
45
and changes in serum albumin
levels.
37
An association between VEGF and the development
of ARDS has been found in humans,
38,53
but this is
controversial
54
and has not yet been studied in com-
panion animals. However, the lung expression of VEGF
delivered via adenovirus vector leads to pulmonary
edema and increased vascular permeability in the lungs
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00457.x 464
D.C. Silverstein et al.
of mice.
55
Humans with dengue hemorrhagic fever
were also found to have elevated VEGF levels, in
addition to a signicant association between VEGF and
D-dimer levels.
56
Future studies might therefore inves-
tigate the relationship between VEGF and the coagula-
tion and brinolytic system in dogs.
In summary, this study was unable to demonstrate a
signicant relationship between VEGF levels in the
blood of dogs with inammatory disease and their
SPI2, the presence of edema on physical examination,
the WBC count, or presence of sepsis. Dogs with a
VEGF level 470 pg/mL were less likely to survive.
Further studies are needed to determine the diagnostic
and prognostic value of this cytokine in critically ill
dogs. Additionally, the use of MFBIA to monitor
changes in TBW and ECW requires further investiga-
tion in critically ill dogs with dynamic systemic
derangements.
Acknowledgement
The authors would like to acknowledge Virginia Good
for her technical assistance and expertise.
Footnotes
a
HYDRA ECF/ICF System Bioimpedance Analyzer Model 4200, Xitron
Technologies, San Diego, CA.
b
Vacutainer tubes, Beckton Dickinson, Vacutainer Systems, Franklin
Lakes, NJ.
c
Human VEGF ELISA Kit, Quantikine, R&D Systems, Minneapolis, MN.
d
Wescor Colloid Osmometer Model 4400, Wescor, Logan, UT.
e
I-STAT Analyzer, Heska Corporation, Fort Collins, CO.
f
Ohmeda Pulse Oximeter Biox 3700, BOC Healthcare, BOC Group Inc,
Boulder, CO.
g
SAS Statistical Software, Version 9.1, SAS Institute, Cary, NC.
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