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Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies. Disease
association, transmission and natural history
Renwick, N.M.
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Citation for published version (APA):
Renwick, N. M. (2001). Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies. Disease
association, transmission and natural history
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Download date: 12 Aug 2017
VEGFF levels in serum do not increase
followingg HIV-1 and HHV8 seroconversion
andd lack correlation with AIDS-KS
NeilNeil Remvick, Gerritjan Weverlin& joke Brouwer,
MargreetMargreet Bakker, Thomas F. Schulp Jaap Goudsmit
ManuscriptManuscript submitted
119 9
Abstract t
Objective:: To determine the utility of vascular endothelial growth factor (VEGF) concentrations in serum as a predictive
markerr for AIDS-KS.
Design:: Sera were obtained from 40 homosexual men who seroconverted for HIV-1 and HHV8 prior to or during their participationn in the Amsterdam Cohort Studies (1984-2000).
Methods:: We designed an ELISA to detect V E G F and measured VEGF prior to either infection, at HIV-1 and HHV8
seroconversion,, after both infections, at AIDS-KS diagnosis (n-11) and in the most recently available scrum sample.
Results:: The geometric mean serum VEGF' concentration was 81.5 pg/mL in those with AIDS-KS and 80,4 pg/mL in those
withh AIDS but without KS. Median serum VF,GF concentrations did not differ between the time points described above.
Higherr V E G F concentrations in serum were observed at higher CD4+ ceil counts.
Conclusions:: Serum concentrations of VE.GF were not influenced by HIV-1 or HHV8 infection orbv the conditions leading
too AIDS-KS. Sequential measurement of V E G F in serum is not expected to contribute to the prediction or therapeutic monitoringg of AIDS-KS.
INTRODUCTION N
Vascularr endothelial g r o w t h factor ( V E G F ) is a dimeric
ceptorr and viral interleukin-6, increase V E G F expression
glycoproteinn that acts via V E G F receptors ( V E G F R s ) to in-
(8-11).. V E G F receptors are also upregulated by H H V 8 in-
creasee vascular permeability and stimulate endothelial cell
fectionn in vitro; V E G F R - 2 expression is enhanced by an
g r o w t hh (1). A I D S related Kaposi's sarcoma ( A I D S - K S ) is a
H H V 8 - e n c o d e dd protein, KS-SM, and may be upregulated
t u m o rr that is characterised by disordered vascularisation
onn endothelial cells by H H V 8 infection (12,13). Binding
a n dd proliferation o f endothelial and spindle cells a n d is
andd activation of the angiogenic HIV-1 tat molecule to
causedd by H I V - 1 and H H V 8 coinfection (2,3). T h e expres-
V E G F R - 22 forms an intriguing link between HIV-1 infec-
sionn o f V E G F
tionn and angiogenesis (14,15).
mRNA
in A I D S - K S tissue suggests a
p a t h o g e n e t i cc role for this cytokine however its i m p o r t a n c e
iss d i s p u t e d d u e to conflicting reports o n the degree of
Nothingg is known o f V E G F concentrations in serum or
V E G FF expression in spindle cells (4-6). T h e presence of
plasmaa in relation to HIV-1 and H H V 8 seroconversion al-
V E G F R - 11 and -2 o n endothelial and spindle cells from
thoughh V E G F concentrations are reported to be higher in
A I D S - K SS lesions is considered
further
evidence of a
p a t h o g e n e t i cc role for this g r o w t h factor (4,7).
thee sera of those with A I D S - K S than without A I D S - K S
andd the plasma level of V E G F is thought to increase as
CD4 ++ cell counts decline (8,16). In this prospective study
V E G FF is p r o d u c e d in r e s p o n s e to HIV-1 and H H V 8 infec-
wee examined V E G F concentrations in the sera of 40 indi-
tionn in vitro; H I V - 1 infected T cells release V E G F and two
vidualss w h o seroconverted for HIV-1 and H I IV8 infection.
H HH V 8 - e n c o d e d proteins, namely the G protein-coupled re-
120 0
MATERIALSS AND METHODS
Participantss and study design
Thee individuals (n—40) in this study are homosexual men
whoo participated in the Amsterdam Cohort Studies
(1984-2000);; 32 individuals seroconverted for both viruses
duringg their participation in the cohort and 8 were HIV-1
seropositivee at study entry and seroconverted for HHV8
duringg the study period. The collection of serum samples,
serologicall methods and establishment of the moment of
HIV-11 and HHV8 seroconversion have been described in a
previouss publication (17). Twenty six individuals seroconvertedd for HIV-1 before HHV8, 11 seroconverted for
HHV88 before HIV-1 and 3 seroconverted simultaneously.
Elevenn participants were diagnosed with KS in the study period. riod.
VEGFF ELISA
Microtiterr plates (Greiner BV, Alphen a/d Rijn, The Netherlands)) were coated with 50 |^1 monoclonal anti-human
VEGFF antibody (ITK Diagnostics, Uithoorn, The Netherlands),, diluted to 1.0 Hg/ml in PBS Dulbecco's (Gibco
BRL,, Life Technologies Ltd, Scotland), and incubated overnightt at room temperature; all reaction volumes were 50 ul
perr well unless indicated.
Thee coating solution was discarded and plates were washed
66 times in PBS-Tween 20 (0.05%); all wash steps were identical.. Each well was blocked with 100 JJ.1 of commercially
availablee blocking buffer (CLB, Amsterdam, The Netherlands)) and the plates were incubated at 37°C for 1 hour;
blockingg buffer was discarded and plates were washed. Recombinantt human VEGF standards (ITK Diagnostics) or
255 (_il serum mixed with 25 )Lll commercially available dilu-
tionn buffer (CLB) were added to each well; incubation and
washh steps were performed as above. Biotinylated anti-humann VEGF antibody (ITK Diagnostics), diluted to 200
ng/mll in dilution buffer, was added to each well; incubation
andd wash steps were repeated. Streptavidin-HRP conjugate
(CLB),, diluted 1:10000 in dilution buffer, was added and the
platess were incubated at 37°C for 30 mins before washing.
Ü P DD substrate (Abbott Laboratories, Abbott Park, Illinois)
wass added and plates were incubated for 20 mins in the dark
att room temperature. The reaction was stopped using IN
H2SO44 and optical densities were read at a wavelength of
490nm. .
VEGFF concentrations were measured in serum samples, if
available,, at seven time points (see below). One hundred
andd fifty nine serum samples were available from all 40 participantss (range: 2 to 5 samples per subject, median: 4 samples).. To study the relationship between CD4+ cell counts
andd VEGF concentrations, CD4 + cell counts were available
forr 86 (54%) of 159 serum samples; 36 (90%) of 40 participantss had 2 or more CD4 + cell counts.
Statisticall analyses
Thee following time points were distinguished in this study;
priorr to either infection, at the moment of HIV-1 or HHV8
seroconversion,, after both infections and at AIDS-KS diagnosis.. Two additional time points, representing the most recentt serum sample in those with and without AIDS-KS,
weree also recognised. VEGF concentrations were logarithmicallyy transformed to obtain normal distribution and the
sevenn time points were compared using analysis of variance.
Thee relationship between CD4 + cell count and VEGF concentrationn was investigated using linear regression analysis.
121 1
RESULTS S
Thee geometric mean concentration of VEGF in serum was
88.66 pg/ml before infection with either virus, 97.5 pg/ml at
HIV-11 seroconversion, 101.6 pg/ml at HHV8 seroconversion,, 95.0 pg/ml after both infections, and 69.4 pg/ml at
AIDS-KSS diagnosis (Figure 1). The geometric mean concentrationn of V E G F in the most recent sample was 81.5
pg/mll in those with AIDS-KS and 80.4 pg/ml among those
withoutt AIDS-KS (Figure 1). The geometric means did not
0000
differr statistically between the seven time points (p=0.73).
VEGFF concentrations remained stable over time, as investigatedd using analysis of repeated measurements (p= 0.45).
Thee median of the individual regression coefficients of
VEGFF concentrations in relation to CD4 + cells was 0.9
(interquartilee range -7.9 - 7.5) pg/100 cells. When analysed
ass a group and not stratified per patient, the overall regressionn coefficient was 4.7 pg/100 cells (p=0.05, Figure 2).
-
f"" T
1000 -
-- T
"ll T
rX X
L
---
1 11 1
1 9 --
HIV V
HHV8 8
KS S
Figuree 1. Distribution (median, interquartile range and extremes)
off VEGF concentration in serum at seven timepoints (from left to
right);; prior to either infection, at HIV-1 seroconversion, at HHV8
seroconversion,, after both infections, at AIDS-KS diagnosis, the
mostt recent sample for those with AIDS-KS and the most recent
samplee for those without AIDS-KS. Numbers per group vary due
too the number of serum samples available.
122 2
5000
1000
CD44 cell counts [cells/mm1]
Figuree 2. Relationship between serum VEGF concentration and
CD4++ cell count. The solid line represent the linear regression line
andd the dashed lines indicate the 95% confidence interval of the
regressionn line
DISCUSSION N
Wee modified an ELISA that detects VEGF in serum at concentrationss similar to those measured in two previous studies;; Ascherl et al. reported VEGF concentrations of 357.1
(+/-197.9)) pg/mlin the AIDS-KS group and 186 (+/-91.7)
pg/mll in HIV-1 uninfected men. 8 Mercie et al. reported
VE.GFF plasma concentrations of 62.4 (+/-40.8) pg/ml in
thee AIDS-KS group and 51 (+/-21.9) pg/ml in the control
group. 166 Interassay variability was minimised as our study
wass performed on two microtiter plates and the optical densitiess from the standard curve of recombinant human
VEGFF were almost identical on each plate.
VEGFF was detectable in the serum of healthy individuals
andd the mean concentration remained stable throughout
thee period of observation in which both HIV-1 and HHV8
infectionn were acquired. VEGF concentration was no
higherr in the serum of individuals at the time of AIDS-KS
diagnosiss than for healthy individuals in our study. This
findingg contrasts with that by Ascherl et al. in which serum
VEGFF concentration was higher in persons with AIDS-KS
thann in HIV-1 uninfected controls. 8
Thee plasma concentration of VEGF has been reported to
increasee as the CD4 + cell count declines.16 In contrast, we
observedd a small increase in serum VEGF concentration at
higherr CD4 + cell counts and interpret this to mean that se-
rumm V E G F concentration is proportional to CD4 + cell
count. .
Measurementt of VEGF concentration in serum does not
clarifyy whether VEGF has a role in AIDS-KS pathogenesis;
serumm VEGF concentration may be sufficient to allow vascularr proliferation in the presence of HIV-1 and HHV8 infectionn or may be an inaccurate reflection of V E G F concentrationn or activity at the site of the lesion. As we found
noo relationship between serum VEGF concentration and
eitherr HIV-1 progression or AIDS-KS diagnosis, it seems
unlikelyy that V E G F is suitable for predicting AIDS-KS or
monitoringg therapeutic response.
AA substantial volume of work indicates that other inflammatoryy cytokines, such as basic fibroblast growth factor,
havee angiogenic roles in AIDS-KS.18 More recent studies
havee implicated VEGF-C and VEGF-D and VEGFR-2
andd -3 in AIDS-KS pathogenesis; VEGF-C is expressed in
endotheliall cells surrounding AIDS-KS lesions and spindle
cellss express VEGFR-2 and -3.19"21 Other growth factors
forr vascular endothelium such as angiopoietin-2 and its receptors,, Tie-1 and Tie-2, are also expressed in AIDS-KS
spindlee cells.22 Studies on such angiogenic molecules may
yieldd a marker that is suitable for predicting the appearance
andd monitoring the progression of AIDS-KS.
123 3
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