http://jhc.sagepub.com/ Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/content/50/6/767 The online version of this article can be found at: DOI: 10.1177/002215540205000603 2002 50: 767 J Histochem Cytochem Antonella N. Witmer, Jiapei Dai, Herbert A. Weich, Gijs F.J.M. Vrensen and Reinier O. Schlingemann Expression of Vascular Endothelial Growth Factor Receptors 1, 2, and 3 in Quiescent Endothelia Published by: http://www.sagepublications.com On behalf of: Official Journal of The Histochemical Society can be found at: Journal of Histochemistry & Cytochemistry Additional services and information for http://jhc.sagepub.com/cgi/alerts Email Alerts: http://jhc.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? - Jun 1, 2002 Version of Record >> by guest on June 2, 2013 jhc.sagepub.com Downloaded from
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http://jhc.sagepub.com/Journal of Histochemistry & Cytochemistry
http://jhc.sagepub.com/content/50/6/767The online version of this article can be found at:
DOI: 10.1177/002215540205000603
2002 50: 767J Histochem CytochemAntonella N. Witmer, Jiapei Dai, Herbert A. Weich, Gijs F.J.M. Vrensen and Reinier O. Schlingemann
Expression of Vascular Endothelial Growth Factor Receptors 1, 2, and 3 in Quiescent Endothelia
Published by:
http://www.sagepublications.com
On behalf of:
Official Journal of The Histochemical Society
can be found at:Journal of Histochemistry & CytochemistryAdditional services and information for
Volume 50(6): 767–777, 2002The Journal of Histochemistry & Cytochemistry
http://www.jhc.org
Expression of Vascular Endothelial Growth Factor Receptors 1, 2, and 3 in Quiescent Endothelia
Antonella N. Witmer, Jiapei Dai, Herbert A. Weich, Gijs F.J.M. Vrensen,and Reinier O. Schlingemann
Ocular Angiogenesis Group, Department of Ophthalmology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (ANW,JD,ROS); National Research Center for Biotechnology, Braunschweig, Germany (HAW); and Lens and Cornea Research Unit, Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands (ANW,JD,GFJMV).
SUMMARY
The vascular endothelial growth factor (VEGF) family is involved in angiogen-esis, and therefore VEGFs are considered as targets for anti-angiogenic therapeutic strate-gies against cancer. However, the physiological functions of VEGFs in quiescent tissues areunclear and may interfere with such systemic therapies. In pathological conditions, in-creased levels of expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 accom-pany VEGF activity. In this study we investigated normal human and monkey tissues for ex-pression patterns of these receptors. Immunohistochemical staining methods at the lightand electron microscopic level were applied to normal human and monkey tissue samples,using monoclonal antibodies (MAbs) against the three VEGFRs and anti-endothelial MAbsPAL-E and anti-CD31 to identify blood and lymph vessels. In human and monkey, similardistribution patterns of the three VEGFRs were found. Co-expression of VEGFR-1, -2, and -3was observed in microvessels adjacent to epithelia in the eye, gastrointestinal mucosa,liver, kidney, and hair follicles, which is in line with the reported preferential expression ofVEGF-A in some of these epithelia. VEGFR-1, -2, and -3 expression was also observed inblood vessels and sinusoids of lymphoid tissues. Furthermore, VEGFR-1, but not VEGFR-2and -3, was present in microvessels in brain and retina. Electron microscopy showed thatVEGFR-1 expression was restricted to pericytes and VEGFR-2 to endothelial cells in normalvasculature of tonsils. These findings indicate that VEGFRs have specific distribution pat-terns in normal tissues, suggesting physiological functions of VEGFs that may be disturbedby systemic anti-VEGF therapy. One of these functions may be involvement of VEGF inparacrine relations between epithelia and adjacent capillaries.
(J Histochem Cytochem 50:767–777, 2002)
V
ascular endothelial growth factor
(VEGF), apermeability factor (Senger et al. 1983; Dvorak et al.1999) and a potent inducer of angiogenesis (Ferrara1999), is considered to be a potential target for anti-angiogenic therapy in the treatment of cancer. VEGF,its localization, and its role in pathophysiological pro-cesses have been studied extensively over recent years
(reviewed in Carmeliet and Jain 2000; Yancopoulos etal. 2000; Dor et al. 2001). However, controversy ex-ists on the role of VEGF in orthophysiology in tissuesthat do not exhibit active angiogenesis. Physiologicalangiogenesis, such as during embryonic development(Breier 2000; Drake et al. 2000; Hamada et al. 2000),corpus luteum and endometrium formation (Ferrara etal. 1998; Smith 2001), and wound healing (Folkman1995), is rather well documented in relation to VEGFfunctions. Nevertheless, VEGF mRNA is also ex-pressed in various tissues in the non-diseased adult(Breier et al. 1992; Ferrara et al. 1992; Monacci et al.1993; Ferrara 1999), indicating that VEGF may be lo-
Correspondence to: Dr. R.O. Schlingemann, Dept. of Ophthal-mology, Academic Medical Center, PO Box 22660, 1100 DD Am-sterdam, The Netherlands. E-mail: [email protected]
Received for publication August 26, 2001; accepted January 4,2002 (1A5623).
KEY WORDS
endothelial growth factors
electron microscopy
endothelial growth factor
receptors
human
immunohistochemistry
monkey
tissue distribution
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cally required for maintenance of differentiated bloodvessels (Ferrara et al. 1992), a notion supported by theobservation of alveolar cell apoptosis due to chronicblockade of VEGFRs (Kasahara et al. 2000). On theother hand, other studies of inhibition of VEGF activ-ity in adult rodents, either by blocking its receptors orby postnatal VEGF inactivation using Cre-loxP-medi-ated VEGF gene deletion, did not result in gross ef-fects on tissue morphology or vasculature, arguingagainst this hypothesis (Fong et al. 1999; Gerber et al.1999; Laird et al. 2000; Robinson et al. 2001). Never-theless, observations in rodents cannot always be ex-trapolated to humans, and it is therefore important toalso investigate orthophysiological roles of the VEGFfamily in the normal human adult, e.g., by definingexpression patterns of VEGFRs in normal tissues. Thismay help us to understand whether and how VEGFRsregulate physiological VEGF activity and may predictpotential side effects of systemic anti-VEGF and/oranti-VEGFR therapies.
VEGFRs are tyrosine kinases that mediate signalingin endothelial cells of blood and lymph vessels, whichinduce cell proliferation, survival, or differentiation.This signaling is required for normal development andmaintenance of the vascular bed and for angiogenicresponses under (patho)physiological conditions. VEG-FRs are members of a receptor tyrosine kinase familythat is rather specific for endothelial cells, consistingof at least three members: VEGFR-1 (Flt-1), VEGFR-2(KDR), and VEGFR-3 (Flt-4). However, VEGFR-1 isalso present in microvascular pericytes (Takagi et al.1996) and in hematopoietic cells, such as monocytes(Mustonen and Alitalo 1995; Barleon et al. 1996). Inaddition to the transmembrane form of VEGFR-1, asoluble form of VEGFR-1 (sVEGFR-1) exists and isbelieved to act as an antagonist of VEGF activity.Ligands that bind to these VEGFRs are members ofthe VEGF family, which consists at present of sixmembers: VEGF-A, VEGF-B, VEGF-C, VEGF-D, theviral VEGF homologue VEGF-E, and placental growthfactor (Senger et al. 1983; Joukov et al. 1996; Achenet al. 1998; Olofsson et al. 1998; Witzenbichler et al.1998). VEGF-A binds with high affinity to VEGFR-1and VEGFR-2 (Vaisman et al. 1990; Mustonen andAlitalo 1995), VEGF-B and placental growth factorbind to VEGFR-1, VEGF-C and VEGF-D bind toVEGFR-2 and VEGFR-3, and VEGF-E binds to VEGFR-2.In pathological conditions such as cancer, VEGFactivity is invariably associated with high levels ofVEGFR expression (Brown et al. 1993b). High levelsof expression of VEGFRs in a given tissue or anatomicsite were therefore suggested to be a marker of VEGFactivity (Veikkola et al. 2000).
VEGF-A mRNA is constitutively expressed by kid-ney glomerular epithelium, choroid plexus epitheliumand retinal pigment epithelium (RPE) in mice, rats, and
humans (Breier et al. 1992; Simon et al. 1995; Yi et al.1998), and constitutive expression of VEGFR-1 and -2has been found in endothelial cells of human adultkidney glomeruli (Simon et al. 1998). We have re-cently shown that RPE cells in vitro secrete VEGF-Aat their basolateral (choroidal) side, whereas all threeVEGFRs were expressed in vivo at the side of endo-thelial cells of the choriocapillaris that faces the RPE(Blaauwgeers et al. 1999). VEGF-A and the VEGFR-3ligand VEGF-C may act here to induce a permeablephenotype and act as survival factors in a paracrinerelation between RPE and the choriocapillaris, be-cause endothelial cells of the choriocapillaris have fen-estrations only at their side facing the RPE (Blaauw-geers et al. 1999) and experimental destruction of RPEleads to choriocapillaris atrophy (Korte et al. 1984).
On the basis of these findings, we hypothesized thatthe VEGF family and its receptors are involved inparacrine functions of epithelia regulating survivaland permeability of adjacent endothelium in physio-logical conditions. To find further evidence for thishypothesis, we investigated the tissue distribution pat-terns of VEGFRs in the adult human and monkey,with special emphasis on endothelia adjacent to epi-thelia.
Materials and Methods
Tissue Samples
Normal human tissue samples were obtained from fresh sur-gical specimens and autopsies performed within 10 hr ofdeath (Table 1). Cryostat sections of the tissue samples werejudged by a certified pathologist and no indications of dis-ease were found. Normal human eyes from 10 donors werekindly provided by the Corneabank Amsterdam. For donoreyes and autopsy tissues, the cause of death was unrelated tothe tissues used. For electron microscopy, human tonsil sam-ples (
n
�
3) were obtained with informed consent within 30min after surgical removal to keep good morphology at the
Table 1 List of organs used for immunohistochemical localization of VEGFRs in normal human and monkey tissues
ultrastructural level. The use of human material was in ac-cordance with the Declaration of Helsinki on the use of hu-man material for research.
Normal tissue samples from two cynomolgus monkeys(
Macaca fascicularis
) (Table 1) were used for this study.Both animals had been used in behavioral studies in the past.All experiments were carried out in accordance with theguidelines established for animal care by the University ofNijmegen, The Netherlands.
All tissue samples were snap-frozen in liquid nitrogenand stored at
�
70C until used.
Light Microscopic Immunohistochemistry
Air-dried serial cryostat sections (8
�
m thick) were fixed incold acetone for 10 min and stained by an indirect immu-noperoxidase procedure (Witmer et al. 2001), using the fol-lowing antibodies: monoclonal antibodies (MAb) Flt-19(against VEGFR-1, dilution 1:400) and KDR-1 (againstVEGFR-2, dilution 1:400) (Simon et al. 1998), MAb 9D9F9(against VEGFR-3, dilution 1:1500) (Lymboussaki et al.1998), and the anti-endothelial MAbs PAL-E (dilution1:1000) (Schlingemann et al. 1985) and EN-4 (againstCD31; dilution 1:500) (Ruiter et al. 1989). Flt-19 and KDR-1were kindly provided by Dr. H.A. Weich (National ResearchCenter for Biotechnology; Braunschweig, Germany), 9D9F9by Prof. K. Alitalo (Haartman Institute; Helsinki, Finland),and EN-4 was purchased from Sanbio (Uden, The Nether-lands). In negative control incubations, the primary antibod-ies were omitted or replaced by an antibody against anon-human bacterial protein (mouse negative control immu-noglobulins; Dako, Glostrup, Denmark) in the same dilu-tions. Peroxidase activity was visualized by incubation in asolution of 3-amino-9-ethyl carbazole (AEC, red color) or3,3-diaminobenzidine (DAB, brown color) with 0.01%H
2
O
2
as substrates in 0.1 M PBS (pH 7.4) for 10 min. Coun-terstaining was performed with hematoxylin.
To determine the extent of vascular staining and the typeof positive vessels, the vascular markers PAL-E and anti-CD31 were used. Staining of serial sections with PAL-E wasperformed to identify capillaries and venules that are not in-volved in a blood–tissue barrier, whereas this antigen is ab-sent in lymphatic endothelium. Anti-CD31 was used as amarker for all blood and lymph vessel endothelium (Schlinge-mann et al. 1985,1997; Ruiter et al. 1989).
All sections were examined by two independent observ-ers. Microvascular staining was graded as follows: no stain-ing (
�
), weak staining (
�
), distinct staining (
�
), intensestaining (
��
), very intense staining (
���
).
Electron Microscopic Immunohistochemistry
For electron microscopy, samples of tonsil tissue were fixedfor 40 min at room temperature (RT) in 2% paraformalde-hyde in Sørensen’s phosphate buffer (pH 7.4) (Ruiter et al.1989; Schlingemann et al. 1991). After fixation, tissue sam-ples were washed in 0.1 M PBS, pH 7.4, incubated overnightin a 20% sucrose–PBS solution at 4C, snap-frozen in liquidnitrogen, and stored at
�
70C until use.A pre-embedding immunoperoxidase technique was used
to demonstrate subcellular distribution patterns of VEGFR-1and VEGFR-2 in tonsil tissue as described previously (Ruiter
et al. 1989; Schlingemann et al. 1990; Witmer et al. 2001).Cryostat sections (60
�
m thick) were cut from the parafor-maldehyde-fixed samples, washed in PBS, and incubated for1 hr in a blocking buffer containing normal horse serum(Vector; Burlingame, CA) with 0.05% saponin. Thenfree-floating sections were incubated with MAbs againstVEGFR-1 and VEGFR-2 (see above) for 1 hr at RT and thenovernight at 4C. Control sections were incubated with MAbPAL-E (see above) as a positive control, or with the antibodyagainst the non-human bacterial protein in the same dilu-tions as the negative control (see above). After extensivewashings in PBS, sections were incubated with biotinylatedhorse anti-mouse immunoglobulins for 1 hr at RT. Sectionswere washed and incubated with a streptavidin–horseradishperoxidase complex for 90 min at RT. Sections were washedfor 2 hr in Tris-HCl buffer (pH 7.8) and peroxidase activitywas demonstrated by incubation in a solution of DAB and0.01% H
2
O
2
for 10 min. The incubation was terminated byrinsing with distilled water. Sections were then postfixed in1% OsO
4
supplemented with 1% ferricyanide in 0.1 M so-dium cacodylate buffer for 30 min, dehydrated in a series ofgraded ethanols, and flat-embedded in epoxy resin. Ul-trathin sections (90–160 nm thick) were cut and photo-graphed using a Philips 201 electron microscope (Philips;Eindhoven, The Netherlands) at 80 kV. Counterstaining wasomitted.
Results
Light Microscopy
Staining patterns of all VEGFRs were similar in humanand monkey tissues and are summarized in Table 2.
VEGFR-1.
Weak to intense granular staining ofVEGFR-1 was found in capillaries adjacent to epithe-lia such as the choriocapillaris, capillaries of ciliaryprocesses in the eye, capillaries in the lamina propriaof gastrointestinal mucosa (Figure 1), kidney glomer-uli, capillaries surrounding hair follicles and withinthe papilla of hair follicles (Figure 2), capillaries inportal tracts close to bile ducts in liver, and capillariesof choroid plexus (Table 2). In addition, capillaries inthe CNS showed intense staining (Figure 3). Capillar-ies not adjacent to epithelia, and larger vessels, such asarterioles, venules, arteries, and veins, were negativeor only weakly stained for VEGFR-1. In lymphoid tis-sues, intense staining of VEGFR-1 was observed incapillaries, high endothelial venules, and sinusoids(Figure 4). The distribution pattern of the VEGFR-1staining product in this tissue gave the impression of alocalization of this receptor in microvascular pericytesand/or at the abluminal side of endothelial cells ofblood vessels. In these cases, unstained endothelialcells were found at the luminal side of small and highendothelial venules (Figure 4). Furthermore, non-vas-cular VEGFR-1 staining was observed in basementmembranes surrounding kidney tubuli, bile ductepithelium in portal tracts of the liver, in glassy mem-
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branes of hair follicles (Figure 2), which is the base-ment membrane separating epithelium from connectivetissue of the follicle, and in the inner limiting mem-brane of the retina (Witmer et al. 2002).
VEGFR-2.
Similar to VEGFR-1, larger vessels suchas arterioles, venules, arteries, and veins were negativeor weakly stained for VEGFR-2. Granular staining ofVEGFR-2 was always found in VEGFR-1-positivecapillaries adjacent to epithelia and lymphoid tissues(Figures 1, 2, and 4; Table 2). In lymphoid tissues,staining of VEGFR-2 was weak in arterioles andvenules and distinct in sinusoidal endothelium. In theCNS, staining of VEGFR-2 was absent in blood ves-sels, in contrast to VEGFR-1 (Figure 3).
VEGFR-3.
Co-localization of VEGFR-3 with VEGFR-1and VEGFR-2 was found in capillaries adjacent to ep-ithelia and capillaries of lymphoid tissues (Figures 1,2, and 4; Table 2). In lymphoid tissues, staining ofVEGFR-3 was weak in arterioles, distinct in venulesand veins, and intense in sinusoidal endothelium.Staining of VEGFR-3 in blood vessels displayed a finegranular intracellular pattern. VEGFR-3 staining wasabsent in blood vessels in the CNS (Figure 3).
Very intense staining of VEGFR-3 was observed inthin-walled lymphatic vessels in portal tracts of theliver, gastrointestinal villi (Figure 1), kidney, skin, andlymphoid tissues. These vessels were recognized in se-rial sections on the basis of their PAL-E negativity.
Non-vascular staining of VEGFR-3 was present incerebral parenchyma (Figure 4), consistent with dif-fuse VEGFR-3 staining in neural elements of the retina(Witmer et al. 2002).
Electron Microscopy
In tonsil tissue, staining of both VEGFR-1 and VEGFR-2was found in capillaries. Staining of VEGFR-1 was
Table 2 Staining patterns of VEGFs in normal human and monkey tissues
VEGFR-1 VEGFR-2 VEGFR-3 PAL-E CD31
Cerebral cortexCapillaries
�� � � � ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � � ���
Choroid plexus
a
Capillaries
� � � ��� ���
EyeChoriocapillaris
b
� �� �� ��� ���
Capillaries in ciliary process
� � �� ��� ���
Retina
c
�� � � � ���
Inner limiting membrane
c
�� � � � �
Duodenum
a
Capillaries in top of villus
� �� �� ��� ���
Other capillaries
� � � ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Lymphatics
� � �� � ���
Appendix
d
Capillaries in top of villus
�� �� �� ��� ���
Other capillaries
� � � ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Lymphatics
� � ��� � ���
LiverCapillaries close to bile
duct
� �� �� ��� ���
Sinusoids
� � �� �� ���
Arterioles
� � � �
or
� ���
Veins
� � � ��� ���
Lymphatics
� � �� � ���
Basement membrane ofbile duct
�� � � � �
KidneyGlomerular capillaries
� � �� � ���
Other capillaries
� � � ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Lymphatics
� � ��� � ���
Basement membrane oftubuli
� � � � �
Lymph node
a
Capillaries
��� ��� �� ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Sinusoids
�� � ��� � ���
Lymphatics
� � ��� � ���
Tonsil
a
Capillaries
��� ��� �� ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Sinusoids
�� � ��� �� ���
Lymphatics
� � ��� � ���
SpleenCapillaries
� � �� ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Sinusoids
�� � �� �� ���
Lymphatics
� � ��� � ���
(Continued)
Table 2 (Continued)
VEGFR-1 VEGFR-2 VEGFR-3 PAL-E CD31
SkinCapillaries near hair follicle
� �� �� ��� ���
Other capillaries
� � � ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
Lymphatics
� � ��� � ���
Glassy membrane
�� � � � �
MuscleCapillaries
� � � ��� ���
Arterioles, arteries
� � � � ���
Venules, veins
� � � ��� ���
a
Tested only in human tissue.
b
Blaauwgeers et al. 1999.
c
Witmer et al. in press.
d
Tested only in monkey tissue.
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found in pericytes (Figure 4E), whereas staining ofVEGFR-2 was restricted to endothelial cells (Figure4). VEGFR-1 and VEGFR-2 staining was found in adot-like configuration in the cytoplasm (Figures 4Eand 4F), which is in agreement with the granularstaining pattern of VEGFRs at the light microscopiclevel (Figures 4A and 4C). In addition, a more diffuseprecipitation in the cytoplasm was observed.
Discussion
The present study demonstrates (a) co-localization ofVEGFR-1, -2, and -3 in capillaries adjacent to epithe-lia in various normal human and monkey tissues, inaddition to VEGFR-3 expression in lymphatic vessels,(b) expression of VEGFR-1, -2, and -3 in microvesselsin lymphoid tissues, (c) expression of VEGFR-1, butnot of VEGFR-2 and VEGFR-3, in capillaries in brain
Figure 1 Photomicrographs of cryostat sections of human duodenum stained for VEGFR-1 (A), VEGFR-2 (B), VEGFR-3 (C), and PAL-E (D).Capillaries in the lamina propria of gastrointestinal mucosa are positive for all three VEGFRs (arrows). In the center of the villus, PAL-E-neg-ative lymph vessels are strongly positive for VEGFR-3 (arrowheads). Bars � 25 �m.
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and retina, (d) non-vascular soluble VEGFR-1 expres-sion in basement membranes of bile ducts and hairfollicles, and (e) at the ultrastructural level, expressionof VEGFR-1 in pericytes and expression of VEGFR-2in endothelial cells in capillaries of tonsils.
In addition to our previous observations of VEGFRexpression in the choriocapillaris in the eye (Blaauw-geers et al. 1999), we found expression of VEGFR-1,
-2, and -3 in capillaries adjacent to epithelia through-out the human and monkey body. In tumors in hu-mans and experimental animals, high levels of expres-sion of VEGFRs usually accompany VEGF activity(Plate et al. 1992; Brown et al. 1993a,b,1995; Hatvaet al. 1995,1996; Takahashi et al. 1995; Warren et al.1995). Expression of VEGFRs may therefore indicatelocal VEGF activity. In the present study of expression
Figure 2 Photomicrographs of serialcryostat sections of skin of the humanscalp stained for VEGFR-1 (A), VEGFR-2(B), VEGFR-3 (C) and PAL-E (D). Capil-laries (arrows) inside the papilla (p) ofthe hair follicle and adjacent to thehair follicle (arrows) are positive forall three VEGFRs. In addition, non-vascular staining for VEGFR-1 is presentin the glassy membrane of the hairfollicle (asterisk). hf, hair follicle; sat,subcutaneous adipose tissue. Bar �50 �m.
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of VEGFRs under orthophysiological conditions, lo-calization of VEGFR-1 and -2 in endothelia adjacentto epithelia corresponded with expression of VEGF-AmRNA that has been described in some of theseepithelia, such as choroid plexus, kidney glomeruli(Breier et al. 1992; Esser et al. 1998), gastrointestinalepithelium (Shifren et al. 1994; Terris et al. 1998), andepithelium of hair follicles (Shifren et al. 1994; Koz-lowska et al. 1998; Yano et al. 2001). Localization ofVEGFR-3 in endothelium of gastrointestinal mucosais in line with expression of VEGF-C mRNA that wasfound in small intestine (Joukov et al. 1996). Our ob-servations are also in line with expression patterns ofmRNA of VEGFR-1 and -2 in endothelial cells ofadult choroid plexus and kidney glomeruli (Millaueret al. 1993; Simon et al. 1995).
Taken together, our results and data available inthe literature suggest that VEGFs have physiologicalfunctions in these human tissues. This is important be-cause such functions may be disturbed by therapeuticanti-VEGF strategies in patients with cancer or eyedisease. It was recently shown that chronic VEGF in-
hibition indeed leads to apoptosis of alveolar cells inthe lungs of experimental animals (Kasahara et al.2000). However, other experimental studies of effectsof VEGF-A inhibition in rodents were unable to dem-onstrate gross toxicity, arguing against a physiologicalrole of VEGF-A in adult rodents (Fong et al. 1999;Gerber et al. 1999; Robinson et al. 2001), findingsthat may, however, not be relevant to humans.
Studies of VEGFR expression patterns at the pro-tein level in normal human tissues are scarce. Simon etal. (1998) demonstrated expression of VEGFR-1 andVEGFR-2 in kidney glomerular capillaries, and ex-pression of VEGFR-3 has been found almost exclu-sively in lymphatic vessels, with the exception of a fewfenestrated endothelia (Partanen et al. 2000; Lym-boussaki et al. 1998). Our observations on VEGFR-3expression are largely in agreement with previous re-ports, but we found a more widespread distributionpattern of VEGFR-3, both in endothelia adjacentto epithelia and in lymphoid tissues. Interestingly,VEGFR-3 expression in blood vessels always co-local-ized with VEGFR-2 expression, allowing a synergistic
Figure 3 Photomicrographs of cryostat sections of human cerebral cortex stained for VEGFR-1 (A), VEGFR-2 (B), VEGFR-3 (C) and CD31 (D).Only VEGFR-1 (C) is present in blood vessels of the CNS in a fine, granular intracellular pattern. VEGFR-2 and VEGFR-3 are absent in capillar-ies. Non-vascular staining of VEGFR-3 is present in cerebral parenchyma. Arrows indicate capillaries. Bars � 30 �m.
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Figure 4 Photomicrographs (A–D) and electron micrographs (E,F) of human tonsil stained for VEGFR-1 (A,E), VEGFR-2 (C,F), VEGFR-3 (D),and PAL-E (B). Staining of VEGFR-1 is suggestive for localization of this receptor in microvascular pericytes and/or the abluminal side of en-dothelial cells in high endothelial venules, because unstained endothelial cells are present at the luminal side (A,B). All three VEGFRs arestained in a granular pattern at the light microscopic level, corresponding to the dot-like configuration at the electron microscopic level (ar-rowheads). VEGFR-1 is expressed in pericytes (p), whereas VEGFR-2 is expressed in endothelial cells (e). ecm, extracellular matrix; L, lumen.Bars: A–D � 20 �m; E,F � 2 �m.
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role for different members of the VEGF family in thesesites. On the other hand, both receptors are notpresent in microvessels of brain and retina, whereasVEGFR-1 is constitutively expressed in these capillar-ies. Other authors have similarly shown that duringangiogenesis in the embryonic brain, high initial ex-pression of VEGFR-2 is present, whereas in the adultbrain, when angiogenesis has ceased, VEGFR-2 ex-pression is not detectable (Kremer et al. 1997).
In addition to a localization near epithelia, we alsoobserved increased VEGFR expression in lymphoidtissues. VEGF-A mRNA expression was previously re-ported in normal tonsil by in situ hybridization (Fosset al. 1997). In normal lymph nodes, low expressionof VEGFs was also found (Niki et al. 2000; Nishi andMaruyama 2000). Our observation of distinct VEGFRexpression in lymph nodes and tonsils suggests thatthe VEGF/VEGFR signaling system plays a permanentrole in these highly vascularized and dynamic tissues.
At the light microscopic level, VEGFR-1 staining ofcapillaries was found in a tube-like pattern, suggestingstaining of pericytes and/or the abluminal side ofendothelial cells. At the electron microscopic level,we found staining of VEGFR-1 in tonsil pericytesonly and not in endothelial cells, whereas staining ofVEGFR-2 was localized in endothelial cells only, as re-ported by others (Feng et al. 2000). These findings areconsistent with observations of VEGFR-1 expressionin pericytes in vitro (Takagi et al. 1996) and indicatethat, in vivo, VEGFR-1 is predominantly a pericyte re-ceptor for VEGF. Subcellular expression of VEGFR-3was previously found only in endothelial cells (Witmeret al. 2001). Staining patterns at the electron micro-scopic level of VEGFR-1 and VEGFR-2 were found tobe in a dot-like configuration in the cytoplasm of cells,consistent with the granular staining pattern in capil-laries at the light microscopic level. These intracellularstructures could not be identified but they may repre-sent storage vesicles containing VEGFR under quies-cent conditions, rather than vesiculo–vacuolar organelles(VVOs) (Feng et al. 2000) on the basis of morphology.In the case of pericyte or endothelial cell stimulation,these vesicles may be “shuttled” to the cell membranewhere VEGFR is redistributed to be able to bind itsligands (Qu Hong et al. 1995).
In addition to vascular VEGFR-1 expression, non-vascular extracellular VEGFR-1 expression was ob-served in the basement membranes of kidney tubuli,bile ducts, and hair follicles, and in the inner limitingmembrane of the retina, i.e., the basement membraneof Müller cells (Witmer et al. 2002). This may repre-sent the soluble form of VEGFR-1, which was sug-gested to regulate availability of VEGF (Hornig et al.2000).
Non-vascular VEGFR-3 expression was observedin cerebral parenchyma. It remains to be elucidated
whether this represents a specific localization of thisreceptor in neural elements or crossreactivity of theantibody used. Similar non-vascular VEGFR-3 expres-sion was found in neural elements of the retina (Wit-mer et al. 2002).
The results of the present study provide importantclues with respect to the role of the VEGF family un-der physiological conditions. In the CNS, initial sig-naling of VEGF may occur via VEGFR-1 on pericytesand/or endothelial cells, allowing tight control ofVEGF activity. Via this signaling pathway, VEGFR-2and VEGFR-3 may be switched on under pathologicalconditions to enhance vascular permeability or to in-duce angiogenesis (Ortega et al. 1997,1999), as wehave recently shown in the retina (Witmer et al.2002). Epithelia may require permanent VEGF signal-ing in a paracrine relation to maintain a permeable orfenestrated phenotype in adjacent capillaries, and thusthe need of constitutive VEGFR-2 and -3 expression.
Several anti-angiogenic agents, alone or in combi-nation with conventional therapies, are now in clinicaltrials (see the Internet site http://cancertrials.nci.nih.gov), including drugs that block VEGF and VEGFRsignaling. However, long-term side effects are not yetknown and it has already been implied that these ther-apies could affect normal tissues and physiological an-giogenesis (Carmeliet and Jain 2000; Oosthuyse et al.2001; Yano et al. 2001). In the present study, weshow that VEGFRs are indeed expressed at specificsites, suggesting orthophysiological functions of VEGFsthat may be disturbed by anti-VEGF therapy.
AcknowledgmentsSupported by the Haagsch Oogheelkundig Fonds, the
Landelijke Stichting voor Blinden en Slechtzienden, theDonders Fonds Utrecht, the Edmond and Marianne Blaauw-fonds, and the Diabetes Fonds Nederland (grants 95.103and 99.050).
We wish to thank Prof Dr K. Alitalo for providing the an-tibody against VEGFR-3, Prof Dr C.J.F. van Noorden forcritically reading the manuscript, the Department of Pathol-ogy, Academic Medical Center, Amsterdam, The Nether-lands (Head Prof Dr J.J. Weening), and Dr D. Nijdam (De-partment of Otolaryngology, Academic Medical Center,Amsterdam) for their assistance in obtaining the tissues, andW. Meun and T. Put for preparing the microphotographs.
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