UvA-DARE (Digital Academic Repository)
Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies.
Disease association, transmission and natural history
Renwick, N.M.
Publication date
2001
Link to publication
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.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)
and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open
content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please
let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material
inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter
to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You
will be contacted as soon as possible.
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
Abstract t
Objective:: T o 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 par-ticipationn 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 p g / m L 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 moni-toringg of AIDS-KS.
INTRODUCTION N
V a s c u l a rr endothelial g r o w t h factor ( V E G F ) is a dimeric g l y c o p r o t e i nn t h a t acts via V E G F receptors ( V E G F R s ) to in-creasee vascular permeability and stimulate endothelial cell g r o w t hh (1). A I D S related K a p o s i ' s sarcoma ( A I D S - K S ) is a t u m o rr t h a t is characterised by disordered vascularisation a n dd proliferation o f endothelial a n d spindle cells a n d is c a u s e dd by H I V - 1 a n d H H V 8 coinfection (2,3). T h e expres-sionn o f V E G F m R N A 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 h o w e v e r its i m p o r t a n c e iss d i s p u t e d d u e t o conflicting reports o n the d e g r e e of V E G FF e x p r e s s i o n in spindle cells (4-6). T h e p r e s e n c e of V E G F R - 11 and -2 o n endothelial and spindle cells from A I D S - K SS lesions is c o n s i d e r e d 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).
V E G FF is p r o d u c e d in r e s p o n s e t o H I V - 1 and H H V 8 infec-tionn in vitro; H I V - 1 infected T cells release V E G F and t w o H HH V 8 - e n c o d e d p r o t e i n s , namely the G p r o t e i n - c o u p l e d
re-ceptorr and viral interleukin-6, increase V E G F expression (8-11).. V E G F receptors are also upregulated by H H V 8 in-fectionn in vitro; V E G F R - 2 expression is enhanced by an H H V 8 - e n c o d e dd protein, K S - S M , and may be upregulated o nn endothelial cells by H H V 8 infection (12,13). Binding andd activation of the angiogenic H I V - 1 tat molecule to V E G F R - 22 forms an intriguing link between H I V - 1 infec-tionn and angiogenesis (14,15).
N o t h i n gg is k n o w n o f V E G F c o n c e n t r a t i o n s in serum or plasmaa in relation to H I V - 1 and H H V 8 seroconversion al-thoughh V E G F c o n c e n t r a t i o n s are reported to be higher in 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 t h o u g h t to increase as C D 4++ cell c o u n t s decline (8,16). In this prospective study wee examined V E G F c o n c e n t r a t i o n s in the sera of 40 indi-vidualss w h o s e r o c o n v e r t e d for H I V - 1 and H I IV8 infection.
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 serocon-vertedd 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 pe-riod. pe-riod.
VEGFF ELISA
Microtiterr plates (Greiner BV, Alphen a/d Rijn, The Neth-erlands)) were coated with 50 |^1 monoclonal anti-human VEGFF antibody (ITK Diagnostics, Uithoorn, The Nether-lands),, diluted to 1.0 Hg/ml in PBS Dulbecco's (Gibco BRL,, Life Technologies Ltd, Scotland), and incubated over-nightt 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 iden-tical.. Each well was blocked with 100 JJ.1 of commercially availablee blocking buffer (CLB, Amsterdam, The Nether-lands)) and the plates were incubated at 37°C for 1 hour; blockingg buffer was discarded and plates were washed. Re-combinantt human V E G F 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-hu-mann V E G F 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 I N H2SO44 and optical densities were read at a wavelength of 490nm. .
V E G FF 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 par-ticipantss (range: 2 to 5 samples per subject, median: 4 sam-ples).. To study the relationship between CD4+ cell counts andd V E G F concentrations, CD4+ cell counts were available forr 86 (54%) of 159 serum samples; 36 (90%) of 40 partici-pantss 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 diag-nosis.. Two additional time points, representing the most re-centt serum sample in those with and without AIDS-KS, weree also recognised. VEGF concentrations were logarith-micallyy transformed to obtain normal distribution and the sevenn time points were compared using analysis of variance. Thee relationship between CD4+ cell count and V E G F con-centrationn was investigated using linear regression analysis.
RESULTS S
Thee geometric mean concentration of VEGF in serum was88.66 p g / m l before infection with either virus, 97.5 pg/ml at HIV-11 seroconversion, 101.6 pg/ml at HHV8 seroconver-sion,, 95.0 p g / m l after both infections, and 69.4 pg/ml at AIDS-KSS diagnosis (Figure 1). The geometric mean con-centrationn of V E G F in the most recent sample was 81.5 p g / m ll in those with AIDS-KS and 80.4 pg/ml among those withoutt AIDS-KS (Figure 1). The geometric means did not
differr statistically between the seven time points (p=0.73). VEGFF concentrations remained stable over time, as investi-gatedd 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 regres-sionn coefficient was 4.7 pg/100 cells (p=0.05, Figure 2).
HIV V HHV8 8 KS S 0000 1000 -1 9
--f"" T
-- T
L
"ll T rX X
---
1 1 1 1
5000 1000 CD44 cell counts [cells/mm1]
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.
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 V E G F in serum atcon-centrationss similar to those measured in two previous stud-ies;; Ascherl et al. reported V E G F 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 den-sitiess from the standard curve of recombinant human V E G FF were almost identical on each plate.
V E G FF 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. V E G F 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 V E G FF concentration was higher in persons with AIDS-KS thann in HIV-1 uninfected controls.8
Thee plasma concentration of V E G F has been reported to increasee as the CD4+ cell count declines.16 In contrast, we observedd a small increase in serum V E G F 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 V E G F concentration in serum does not clarifyy whether V E G F has a role in AIDS-KS pathogenesis; serumm V E G F concentration may be sufficient to allow vas-cularr proliferation in the presence of HIV-1 and HHV8 in-fectionn or may be an inaccurate reflection of V E G F con-centrationn 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 inflam-matoryy 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 re-ceptors,, 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.
REFERENCES S
11 Yancopoulos G D , Davis S, Gale NVC', Rudge JS, Vuegand S|, Holashh J. \'ascular-specific growth factors and blood vessel for-mation.. Katun 2000;407:242-8.
22 Ruszczak Z, Maver-Da Silva A, Orfanos CH. Kaposi's sarcoma inn AIDS. Multicentric angioneoplasia in early skin lesions. Am]
11 hrmatopathol 1987;9:388-98.
33 Martin J N , O s m o n d D H . Kaposi's sarcoma-associated herpesviruss and sexual transmission of cancer risk. Curr Opin
Om-olOm-ol 1999;11:508-15.
44 Brown 1.1% Tognazzi K, Dvorak H F , Harrist T). Strong expres-sionn of kinase insert domain-containing receptor, a vascular per-meabilityy factor/ vascular endothelial growth factor receptor in AIDS-associatedd Kaposi's sarcoma and cutaneous angiosarcoma.. Am] Putho/1996; 148:1065-74.
55 Oornali F , Zietz C, Benelli R, et a/. Vascular endothelial growth factorr regulates angiogenesis and vascular permeability in Kaposi'ss sarcoma. Am J Pathol 1996;149:1851-69.
66 Samaniego F, Markham P D , Gendelman R, et al. Vascular endo-theliall growth factor and basic fibroblast growth factor present in Kaposi'ss sarcoma (KS) are induced by inflammatory cytokines andd synergize to promote vascular permeability and KS lesion development.. Am J Pathol 1998;152:1433-43.
77 Masood R, Cai J, Z h e n g T , Smith DL, Naidu Y, Gill PS. Vascular endotheliall growth factor/ vascular permeability factor is an autocrinee growth factor for AIDS-Kaposi sarcoma. Proc Sat/
AcadAcad Sri USA 1997;94:979-84.
88 Ascherl G, Hohenadl C, Schatz O , et al. Infection with human immunodeficiencyy virus-1 increases expression of vascular en-dotheliall cell growth factor in T cells: implications for acquired immunodeficiencyy syndrome-associated vasculopathy. Rlood 1999;93:4232-41. .
99 Bais C, Santomasso B, Coso O , et al. G-protein-coupled receptor off Kaposi's sarcoma-associated herpesvirus is a viral oncogene andd angiogenesis activator. Nature 1998;391:86-9.
If)) Sodhi A, Montaner S, Patel V, et al. T h e Kaposi's sarcoma-associ-atedd herpes virus G protein-coupled receptor upregulates vascu-larr endothelial growth factor expression and secretion through mitogen-activatedd protein kinase and p38 pathways acting on hypoxia-induciblee factor 1 alpha. Cancer Res 2000;60:4873-80. 111 Aoki Y, )affe F.S, Chang Y, et al. Angiogenesis and
hacmatopoiesiss induced bv Kaposi's sarcoma-associated herpesviruss encoded interleukin-6. Blood 1999;93:4034-43.
122 Gupta AK, Ruvolo V, Patterson C, Swaminathan S. The human herpesviruss 8 homolog of P'pstein-Barr virus SM protein (KS-SM)) is a posttranscriptional activator of gene expression. / II 'trol 2000;74:1038-44.
111 Flore (). Rafii S. Fly S, O'Feary JJ. Hyjek I'M, Cesarman F. Transformationn of primary human endothelial cells bv Kaposi's sarcoma-associatedd herpesvirus. Xaturr 1998;394:588-92. 144 Alhini A, Soldi R, Giunciuglio D , et al. The angiogenesis induced
byy H1V-1 tat protein is mediated bv the Flk-1/KDR receptor on vascularr endothelial cells. Xat Med: 1996;2:1371-5.
155 Ganju RK, Munshi N, Nair BC, Fiu ZY, Gill P, Groopman J F . 11 luman immunodeficiency yirus tat modulates the Flk-1/KDR receptor,, mitogen-activated protein kinases, and components of focall adhesion in Kaposi's sarcoma cells. / I 'in/1998;72:6131-7. 166 Mercie P, Devianne 1, Viallard JF, tt al. Vascular endothelial
growthh factor ( Y F X J F 1 6 5 ) plasma level increase with immunodepressionn in AIDS patients with Kaposi's sarcoma.
MicrovascMicrovasc Res 1999;57:208-10.
1?? Renwick N , Halabv T, Weverling GJ, et al. Seroconversion for humann herpesvirus 8 during HIV infection is highly predictive of Kaposi'ss sarcoma. AIDS 1998;12:2481-8.
188 Knsoli B, Sturzl M, Monini P. Cytokine-mediated growth pro-motionn of Kaposi's sarcoma and primary effusion lymphoma.
Sem'mSem'm Cancer Blot 2000;10:367-81.
199 (ussila F, Valtola R, Partanen TA, tt al. Lymphatic endothelium andd Kaposi's sarcoma spindle cells detected by antibodies against thee vascular endothelial growth factor reccptor-3. Cancer Res 1988;58:1599-1604. .
200 Dupin K, Fisher C, Kellam P, et al Distribution of human herpesvirus-88 latently infected cells in Kaposi's sarcoma, multicentricc Castleman's disease, and primary effusion lym-phoma.. Prm-Xatl. leadSci 1'SA 1999;96:4546-51.
211 Skobe M, Brown FF, Tognazzi K, et al. Vascular endothelial growthh factor-C ( Y F G F - Q and its receptors KDR and fit—4- arc-expressedd in AIDS-associated Kaposi's sarcoma. / Invest Demiatol 1999;113:1047-53. .
222 Brown FF, Dezube B|, Tognazzi, Dvorak HF, Yancopoulos G D .. Fxpression of Tie-1, Tie-2, and angiopoietins 1,2, and 4 in Kaposi'ss sarcoma and cutaneous angiosarcoma. Am ƒ Pathol 2000;; 156:2179-83.
