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Abstract

The vascular endothelial growth factor (VEGF) family is involved in angiogenesis, and therefore VEGFs are considered as targets for anti-angiogenic therapeutic strategies against cancer. However, the physiological functions of VEGFs in quiescent tissues are unclear and may interfere with such systemic therapies. In pathological conditions, increased levels of expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 accompany VEGF activity. In this study we investigated normal human and monkey tissues for expression patterns of these receptors. Immunohistochemical staining methods at the light and electron microscopic level were applied to normal human and monkey tissue samples, using monoclonal antibodies (MAbs) against the three VEGFRs and anti-endothelial MAbs PAL-E and anti-CD31 to identify blood and lymph vessels. In human and monkey, similar distribution patterns of the three VEGFRs were found. Co-expression of VEGFR-1, −2, and −3 was 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 of VEGF-A in some of these epithelia. VEGFR-1, −2, and −3 expression was also observed in blood vessels and sinusoids of lymphoid tissues. Furthermore, VEGFR-1, but not VEGFR-2 and −3, was present in microvessels in brain and retina. Electron microscopy showed that VEGFR-1 expression was restricted to pericytes and VEGFR-2 to endothelial cells in normal vasculature of tonsils. These findings indicate that VEGFRs have specific distribution patterns in normal tissues, suggesting physiological functions of VEGFs that may be disturbed by systemic anti-VEGF therapy. One of these functions may be involvement of VEGF in paracrine relations between epithelia and adjacent capillaries.

Keywords

endothelial growth factors electron microscopy endothelial growth factor receptors human immunohistochemistry monkey tissue distribution

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), a permeability factor (Senger et al. 1983; Dvorak et al. 1999) and a potent inducer of angiogenesis (Ferrara 1999), is considered to be a potential target for anti-angiogenic therapy in the treatment of cancer. VEGF, its localization, and its role in pathophysiological processes have been studied extensively over recent years (reviewed in Carmeliet and Jain 2000; Yancopoulos et al. 2000; Dor et al. 2001). However, controversy exists on the role of VEGF in orthophysiology in tissues that do not exhibit active angiogenesis. Physiological angiogenesis, such as during embryonic development (Breier 2000; Drake et al. 2000; Hamada et al. 2000), corpus luteum and endometrium formation (Ferrara et al. 1998; Smith 2001), and wound healing (Folkman 1995), is rather well documented in relation to VEGF functions. Nevertheless, VEGF mRNA is also expressed 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 locally required for maintenance of differentiated blood vessels (Ferrara et al. 1992), a notion supported by the observation of alveolar cell apoptosis due to chronic blockade of VEGFRs (Kasahara et al. 2000). On the other hand, other studies of inhibition of VEGF activity in adult rodents, either by blocking its receptors or by postnatal VEGF inactivation using Cre-loxP-mediated VEGF gene deletion, did not result in gross effects on tissue morphology or vasculature, arguing against this hypothesis (Fong et al. 1999; Gerber et al. 1999; Laird et al. 2000; Robinson et al. 2001). Nevertheless, observations in rodents cannot always be extrapolated to humans, and it is therefore important to also investigate orthophysiological roles of the VEGF family in the normal human adult, e.g., by defining expression patterns of VEGFRs in normal tissues. This may help us to understand whether and how VEGFRs regulate physiological VEGF activity and may predict potential side effects of systemic anti-VEGF and/or anti-VEGFR therapies.

VEGFRs are tyrosine kinases that mediate signaling in endothelial cells of blood and lymph vessels, which induce cell proliferation, survival, or differentiation. This signaling is required for normal development and maintenance of the vascular bed and for angiogenic responses under (patho)physiological conditions. VEGFRs are members of a receptor tyrosine kinase family that is rather specific for endothelial cells, consisting of at least three members: VEGFR-1 (Flt-1), VEGFR-2 (KDR), and VEGFR-3 (Flt-4). However, VEGFR-1 is also present in microvascular pericytes (Takagi et al. 1996) and in hematopoietic cells, such as monocytes (Mustonen and Alitalo 1995; Barleon et al. 1996). In addition to the transmembrane form of VEGFR-1, a soluble form of VEGFR-1 (sVEGFR-1) exists and is believed to act as an antagonist of VEGF activity. Ligands that bind to these VEGFRs are members of the VEGF family, which consists at present of six members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, the viral VEGF homologue VEGF-E, and placental growth factor (Senger et al. 1983; Joukov et al. 1996; Achen et al. 1998; Olofsson et al. 1998; Witzenbichler et al. 1998). VEGF-A binds with high affinity to VEGFR-1 and VEGFR-2 (Vaisman et al. 1990; Mustonen and Alitalo 1995), VEGF-B and placental growth factor bind to VEGFR-1, VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3, and VEGF-E binds to VEGFR-2. In pathological conditions such as cancer, VEGF activity is invariably associated with high levels of VEGFR expression (Brown et al. 1993b). High levels of expression of VEGFRs in a given tissue or anatomic site were therefore suggested to be a marker of VEGF activity (Veikkola et al. 2000).

VEGF-A mRNA is constitutively expressed by kidney glomerular epithelium, choroid plexus epithelium and 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 −2 has been found in endothelial cells of human adult kidney glomeruli (Simon et al. 1998). We have recently shown that RPE cells in vitro secrete VEGF-A at their basolateral (choroidal) side, whereas all three VEGFRs were expressed in vivo at the side of endothelial cells of the choriocapillaris that faces the RPE (Blaauwgeers et al. 1999). VEGF-A and the VEGFR-3 ligand VEGF-C may act here to induce a permeable phenotype and act as survival factors in a paracrine relation between RPE and the choriocapillaris, because endothelial cells of the choriocapillaris have fenestrations only at their side facing the RPE (Blaauwgeers et al. 1999) and experimental destruction of RPE leads to choriocapillaris atrophy (Korte et al. 1984).

On the basis of these findings, we hypothesized that the VEGF family and its receptors are involved in paracrine functions of epithelia regulating survival and permeability of adjacent endothelium in physiological conditions. To find further evidence for this hypothesis, we investigated the tissue distribution patterns of VEGFRs in the adult human and monkey, with special emphasis on endothelia adjacent to epithelia.

Materials and Methods

Tissue Samples

Normal human tissue samples were obtained from fresh surgical specimens and autopsies performed within 10 hr of death (Table 1). Cryostat sections of the tissue samples were judged by a certified pathologist and no indications of disease were found. Normal human eyes from 10 donors were kindly provided by the Corneabank Amsterdam. For donor eyes and autopsy tissues, the cause of death was unrelated to the tissues used. For electron microscopy, human tonsil samples (n = 3) were obtained with informed consent within 30 min after surgical removal to keep good morphology at the ultrastructural level. The use of human material was in accordance with the Declaration of Helsinki on the use of human material for research.

Table 1

List of organs used for immunohistochemical localization of VEGFRs in normal human and monkey tissues

Tissues tested	Number	Tissues tested	Number
Human		Monkey
Cerebral cortex	3	Appendix	2
Choroid plexus	1	Cardiac muscle	2
Duodenum	8	Cerebral cortex	2
Eye	10	Eye	2
Kidney	7	Kidney	2
Liver	6	Liver	2
Lymph node	4	Spleen	2
Muscle		Skin	2
Cardiac muscle	1
Skeletal muscle	1
Striated muscle	4
Skin	3
Tonsil	3

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 the guidelines established for animal care by the University of Nijmegen, The Netherlands.

All tissue samples were snap-frozen in liquid nitrogen and stored at −70C until used.

Light Microscopic Immunohistochemistry

Air-dried serial cryostat sections (8 μm thick) were fixed in cold acetone for 10 min and stained by an indirect immunoperoxidase procedure (Witmer et al. 2001), using the following antibodies: monoclonal antibodies (MAb) Flt-19 (against VEGFR-1, dilution 1:400) and KDR-1 (against VEGFR-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 (dilution 1:1000) (Schlingemann et al. 1985) and EN-4 (against CD31; dilution 1:500) (Ruiter et al. 1989). Flt-19 and KDR-1 were kindly provided by Dr. H.A. Weich (National Research Center for Biotechnology; Braunschweig, Germany), 9D9F9 by Prof. K. Alitalo (Haartman Institute; Helsinki, Finland), and EN-4 was purchased from Sanbio (Uden, The Netherlands). In negative control incubations, the primary antibodies were omitted or replaced by an antibody against a non-human bacterial protein (mouse negative control immunoglobulins; Dako, Glostrup, Denmark) in the same dilutions. Peroxidase activity was visualized by incubation in a solution of 3-amino-9-ethyl carbazole (AEC, red color) or 3,3-diaminobenzidine (DAB, brown color) with 0.01% H₂O₂ as substrates in 0.1 M PBS (pH 7.4) for 10 min. Counterstaining was performed with hematoxylin.

To determine the extent of vascular staining and the type of positive vessels, the vascular markers PAL-E and anti-CD31 were used. Staining of serial sections with PAL-E was performed to identify capillaries and venules that are not involved in a blood-tissue barrier, whereas this antigen is absent in lymphatic endothelium. Anti-CD31 was used as a marker for all blood and lymph vessel endothelium (Schlingemann et al. 1985, 1997; Ruiter et al. 1989).

All sections were examined by two independent observers. Microvascular staining was graded as follows: no staining (-), weak staining (±), distinct staining (+), intense staining (+ +), very intense staining (+ + +).

Electron Microscopic Immunohistochemistry

For electron microscopy, samples of tonsil tissue were fixed for 40 min at room temperature (RT) in 2% paraformaldehyde in Sørensen's phosphate buffer (pH 7.4) (Ruiter et al. 1989; Schlingemann et al. 1991). After fixation, tissue samples were washed in 0.1 M PBS, pH 7.4, incubated overnight in a 20% sucrose-PBS solution at 4C, snap-frozen in liquid nitrogen, and stored at −70C until use.

A pre-embedding immunoperoxidase technique was used to demonstrate subcellular distribution patterns of VEGFR-1 and VEGFR-2 in tonsil tissue as described previously (Ruiter et al. 1989; Schlingemann et al. 1990; Witmer et al. 2001). Cryostat sections (60 μm thick) were cut from the paraformaldehyde-fixed samples, washed in PBS, and incubated for 1 hr in a blocking buffer containing normal horse serum (Vector; Burlingame, CA) with 0.05% saponin. Then free-floating sections were incubated with MAbs against VEGFR-1 and VEGFR-2 (see above) for 1 hr at RT and then overnight at 4C. Control sections were incubated with MAb PAL-E (see above) as a positive control, or with the antibody against the non-human bacterial protein in the same dilutions as the negative control (see above). After extensive washings in PBS, sections were incubated with biotinylated horse anti-mouse immunoglobulins for 1 hr at RT. Sections were washed and incubated with a streptavidin-horseradish peroxidase complex for 90 min at RT. Sections were washed for 2 hr in Tris-HCl buffer (pH 7.8) and peroxidase activity was demonstrated by incubation in a solution of DAB and 0.01% H₂O₂ for 10 min. The incubation was terminated by rinsing with distilled water. Sections were then postfixed in 1% OsO₄ supplemented with 1% ferricyanide in 0.1 M sodium cacodylate buffer for 30 min, dehydrated in a series of graded ethanols, and flat-embedded in epoxy resin. Ultrathin sections (90–160 nm thick) were cut and photographed using a Philips 201 electron microscope (Philips; Eindhoven, The Netherlands) at 80 kV. Counterstaining was omitted.

Results

Light Microscopy

Staining patterns of all VEGFRs were similar in human and monkey tissues and are summarized in Table 2.

VEGFR-1. Weak to intense granular staining of VEGFR-1 was found in capillaries adjacent to epithelia such as the choriocapillaris, capillaries of ciliary processes in the eye, capillaries in the lamina propria of gastrointestinal mucosa (Figure 1), kidney glomeruli, capillaries surrounding hair follicles and within the papilla of hair follicles (Figure 2), capillaries in portal tracts close to bile ducts in liver, and capillaries of choroid plexus (Table 2). In addition, capillaries in the CNS showed intense staining (Figure 3). Capillaries not adjacent to epithelia, and larger vessels, such as arterioles, venules, arteries, and veins, were negative or only weakly stained for VEGFR-1. In lymphoid tissues, intense staining of VEGFR-1 was observed in capillaries, high endothelial venules, and sinusoids (Figure 4). The distribution pattern of the VEGFR-1 staining product in this tissue gave the impression of a localization of this receptor in microvascular pericytes and/or at the abluminal side of endothelial cells of blood vessels. In these cases, unstained endothelial cells were found at the luminal side of small and high endothelial venules (Figure 4). Furthermore, non-vascular VEGFR-1 staining was observed in basement membranes surrounding kidney tubuli, bile duct epithelium in portal tracts of the liver, in glassy membranes of hair follicles (Figure 2), which is the basement membrane separating epithelium from connective tissue of the follicle, and in the inner limiting membrane of the retina (Witmer et al. 2002).

Table 2

Staining patterns of VEGFs in normal human and monkey tissues

	VEGFR-1	VEGFR-2	VEGFR-3	PAL-E	CD31
Cerebral cortex
Capillaries	+ +	−	−	−	+ + +
Arterioles, arteries	−	−	+ + +
Venules, veins	± −	−	−	+ + +
Choroid plexus a
Capillaries	+	+	+		+ + +
Eye
Choriocapillaris 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	−	−	+ + +	−	+ + +
Liver
Capillaries close to bile	+	++	++	+ + +	+ + +
duct
Sinusoids	−	+	++	+ +	+ + +
Arterioles	±	±	±	+or-	+ + +
Veins	±	+	±	+ + +	+ + +
Lymphatics	−	−	++	−	+ + +
Basement membrane of	+ +	−	−	−	−
bile duct
Kidney
Glomerular capillaries	+	+	++	−	+ + +
Other capillaries	±	±	+	+ + +	+ + +
Arterioles, arteries	−	−	−	−	+ + +
Venules, veins	±	±	−	+ + +	+ + +
Lymphatics	−	−	+ + +	−	+ + +
Basement membrane of	+	−	−	+	−
tubuli
Lymph node a
Capillaries	+ + +		+ + +	+ +	+ + +	+ + +
Arterioles, arteries	± ± ±	±	+ + +
Venules, veins	±	±	+	+ + +	+ + +
Sinusoids	+ +	+	+ + +	+	+ + +
Lymphatics	−	−	+ + +	−	+ + +
Tonsil a
Capillaries	+ + +		+ +	+ + +	+ + +
Arterioles, arteries	±	±	±	±	+ + +
Venules, veins	±	±	+	+ + +	+ + +
Sinusoids	++	+	+ + +	+ +	+ + +
Lymphatics	−	−	+ + +	−	+ + +
Spleen
Capillaries	+	+	++	+ + +	+ + +
Arterioles, arteries	± −	−	−	+ + +
Venules, veins	±	−	−	+ + +	+ + +
Sinusoids	++	+	++	+ +	+ + +
Lymphatics	−	−	+ + +	−	+ + +
Skin
Capillaries near hair follicle	+	+ +	+ +	+ + +	+ + +
Other capillaries	±	±	−	+ + +	+ + +
Arterioles, arteries	−	−	−	−	+ + +
Venules, veins	−	−	±	+ + +	+ + +
Lymphatics	−	−	+ + +	−	+ + +
Glassy membrane	+ +	−	−	−	−
Muscle
Capillaries	±	±	±	+ + +	+ + +
Arterioles, arteries	−	−	−	−	+ + +
Venules, veins	±	−	−	+ + +	+ + +

Tested only in human tissue.

Blaauwgeers et al. 1999.

Witmer et al. in press.

Tested only in monkey tissue.

VEGFR-2. Similar to VEGFR-1, larger vessels such as arterioles, venules, arteries, and veins were negative or weakly stained for VEGFR-2. Granular staining of VEGFR-2 was always found in VEGFR-1-positive capillaries adjacent to epithelia and lymphoid tissues (Figures 1, 2, and 4; Table 2). In lymphoid tissues, staining of VEGFR-2 was weak in arterioles and venules and distinct in sinusoidal endothelium. In the CNS, staining of VEGFR-2 was absent in blood vessels, in contrast to VEGFR-1 (Figure 3).

VEGFR-3. Co-localization of VEGFR-3 with VEGFR-1 and VEGFR-2 was found in capillaries adjacent to epithelia and capillaries of lymphoid tissues (Figures 1, 2, and 4; Table 2). In lymphoid tissues, staining of VEGFR-3 was weak in arterioles, distinct in venules and veins, and intense in sinusoidal endothelium. Staining of VEGFR-3 in blood vessels displayed a fine granular intracellular pattern. VEGFR-3 staining was absent in blood vessels in the CNS (Figure 3).

Very intense staining of VEGFR-3 was observed in thin-walled lymphatic vessels in portal tracts of the liver, gastrointestinal villi (Figure 1), kidney, skin, and lymphoid tissues. These vessels were recognized in serial sections on the basis of their PAL-E negativity.

Non-vascular staining of VEGFR-3 was present in cerebral parenchyma (Figure 4), consistent with diffuse 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-2 was found in capillaries. Staining of VEGFR-1 was found in pericytes (Figure 4E), whereas staining of VEGFR-2 was restricted to endothelial cells (Figure 4). VEGFR-1 and VEGFR-2 staining was found in a dot-like configuration in the cytoplasm (Figures 4E and 4F), which is in agreement with the granular staining pattern of VEGFRs at the light microscopic level (Figures 4A and 4C). In addition, a more diffuse precipitation in the cytoplasm was observed.

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-negative lymph vessels are strongly positive for VEGFR-3 (arrowheads). Bars = 25 μm.

Discussion

The present study demonstrates (a) co-localization of VEGFR-1, −2, and −3 in capillaries adjacent to epithelia in various normal human and monkey tissues, in addition to VEGFR-3 expression in lymphatic vessels, (b) expression of VEGFR-1, −2, and −3 in microvessels in lymphoid tissues, (c) expression of VEGFR-1, but not of VEGFR-2 and VEGFR-3, in capillaries in brain and retina, (d) non-vascular soluble VEGFR-1 expression in basement membranes of bile ducts and hair follicles, and (e) at the ultrastructural level, expression of VEGFR-1 in pericytes and expression of VEGFR-2 in endothelial cells in capillaries of tonsils.

Figure 2

Photomicrographs of serial cryostat sections of skin of the human scalp stained for VEGFR-1 (A), VEGFR-2 (B), VEGFR-3 (C) and PAL-E (D). Capillaries (arrows) inside the papilla (p) of the hair follicle and adjacent to the hair follicle (arrows) are positive for all three VEGFRs. In addition, nonvascular staining for VEGFR-1 is present in the glassy membrane of the hair follicle (asterisk). hf, hair follicle; sat, subcutaneous adipose tissue. Bar = 50 μm.

In addition to our previous observations of VEGFR expression in the choriocapillaris in the eye (Blaauwgeers et al. 1999), we found expression of VEGFR-1, −2, and −3 in capillaries adjacent to epithelia throughout the human and monkey body. In tumors in humans and experimental animals, high levels of expression of VEGFRs usually accompany VEGF activity (Plate et al. 1992; Brown et al. 1993a, b, 1995; Hatva et al. 1995, 1996; Takahashi et al. 1995; Warren et al. 1995). Expression of VEGFRs may therefore indicate local VEGF activity. In the present study of expression of VEGFRs under orthophysiological conditions, localization of VEGFR-1 and −2 in endothelia adjacent to epithelia corresponded with expression of VEGF-A mRNA that has been described in some of these epithelia, such as choroid plexus, kidney glomeruli (Breier et al. 1992; Esser et al. 1998), gastrointestinal epithelium (Shifren et al. 1994; Terris et al. 1998), and epithelium of hair follicles (Shifren et al. 1994; Kozlowska et al. 1998; Yano et al. 2001). Localization of VEGFR-3 in endothelium of gastrointestinal mucosa is in line with expression of VEGF-C mRNA that was found in small intestine (Joukov et al. 1996). Our observations are also in line with expression patterns of mRNA of VEGFR-1 and −2 in endothelial cells of adult choroid plexus and kidney glomeruli (Millauer et al. 1993; Simon et al. 1995).

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 capillaries. Non-vascular staining of VEGFR-3 is present in cerebral parenchyma. Arrows indicate capillaries. Bars = 30 μm.

Taken together, our results and data available in the literature suggest that VEGFs have physiological functions in these human tissues. This is important because such functions may be disturbed by therapeutic anti-VEGF strategies in patients with cancer or eye disease. It was recently shown that chronic VEGF inhibition indeed leads to apoptosis of alveolar cells in the lungs of experimental animals (Kasahara et al. 2000). However, other experimental studies of effects of VEGF-A inhibition in rodents were unable to demonstrate gross toxicity, arguing against a physiological role of VEGF-A in adult rodents (Fong et al. 1999; Gerber et al. 1999; Robinson et al. 2001), findings that may, however, not be relevant to humans.

Studies of VEGFR expression patterns at the protein level in normal human tissues are scarce. Simon et al. (1998) demonstrated expression of VEGFR-1 and VEGFR-2 in kidney glomerular capillaries, and expression of VEGFR-3 has been found almost exclusively in lymphatic vessels, with the exception of a few fenestrated endothelia (Partanen et al. 2000; Lymboussaki et al. 1998). Our observations on VEGFR-3 expression are largely in agreement with previous reports, but we found a more widespread distribution pattern of VEGFR-3, both in endothelia adjacent to epithelia and in lymphoid tissues. Interestingly, VEGFR-3 expression in blood vessels always co-localized with VEGFR-2 expression, allowing a synergistic role for different members of the VEGF family in these sites. On the other hand, both receptors are not present in microvessels of brain and retina, whereas VEGFR-1 is constitutively expressed in these capillaries. Other authors have similarly shown that during angiogenesis in the embryonic brain, high initial expression of VEGFR-2 is present, whereas in the adult brain, when angiogenesis has ceased, VEGFR-2 expression is not detectable (Kremer et al. 1997).

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 endothelial cells in high endothelial venules, because unstained endothelial cells are present at the luminal side (A, B). All three VEGFRs are stained in a granular pattern at the light microscopic level, corresponding to the dot-like configuration at the electron microscopic level (arrowheads). 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 μm; E, F = 2 μm.

In addition to a localization near epithelia, we also observed increased VEGFR expression in lymphoid tissues. VEGF-A mRNA expression was previously reported in normal tonsil by in situ hybridization (Foss et al. 1997). In normal lymph nodes, low expression of VEGFs was also found (Niki et al. 2000; Nishi and Maruyama 2000). Our observation of distinct VEGFR expression in lymph nodes and tonsils suggests that the VEGF/VEGFR signaling system plays a permanent role in these highly vascularized and dynamic tissues.

At the light microscopic level, VEGFR-1 staining of capillaries was found in a tube-like pattern, suggesting staining of pericytes and/or the abluminal side of endothelial cells. At the electron microscopic level, we found staining of VEGFR-1 in tonsil pericytes only and not in endothelial cells, whereas staining of VEGFR-2 was localized in endothelial cells only, as reported by others (Feng et al. 2000). These findings are consistent with observations of VEGFR-1 expression in pericytes in vitro (Takagi et al. 1996) and indicate that, in vivo, VEGFR-1 is predominantly a pericyte receptor for VEGF. Subcellular expression of VEGFR-3 was previously found only in endothelial cells (Witmer et al. 2001). Staining patterns at the electron microscopic level of VEGFR-1 and VEGFR-2 were found to be in a dot-like configuration in the cytoplasm of cells, consistent with the granular staining pattern in capillaries at the light microscopic level. These intracellular structures could not be identified but they may represent storage vesicles containing VEGFR under quiescent 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 membrane where VEGFR is redistributed to be able to bind its ligands (Qu Hong et al. 1995).

In addition to vascular VEGFR-1 expression, non-vascular extracellular VEGFR-1 expression was observed in the basement membranes of kidney tubuli, bile ducts, and hair follicles, and in the inner limiting membrane of the retina, i.e., the basement membrane of Müller cells (Witmer et al. 2002). This may represent the soluble form of VEGFR-1, which was suggested to regulate availability of VEGF (Hornig et al. 2000).

Non-vascular VEGFR-3 expression was observed in cerebral parenchyma. It remains to be elucidated whether this represents a specific localization of this receptor in neural elements or crossreactivity of the antibody used. Similar non-vascular VEGFR-3 expression was found in neural elements of the retina (Wit-mer et al. 2002).

The results of the present study provide important clues with respect to the role of the VEGF family under physiological conditions. In the CNS, initial signaling of VEGF may occur via VEGFR-1 on pericytes and/or endothelial cells, allowing tight control of VEGF activity. Via this signaling pathway, VEGFR-2 and VEGFR-3 may be switched on under pathological conditions to enhance vascular permeability or to induce angiogenesis (Ortega et al. 1997, 1999), as we have recently shown in the retina (Witmer et al. 2002). Epithelia may require permanent VEGF signaling in a paracrine relation to maintain a permeable or fenestrated phenotype in adjacent capillaries, and thus the need of constitutive VEGFR-2 and −3 expression.

Several anti-angiogenic agents, alone or in combination with conventional therapies, are now in clinical trials (see the Internet site http://cancertrials.nci.nih.gov), including drugs that block VEGF and VEGFR signaling. However, long-term side effects are not yet known and it has already been implied that these therapies could affect normal tissues and physiological angiogenesis (Carmeliet and Jain 2000; Oosthuyse et al. 2001; Yano et al. 2001). In the present study, we show that VEGFRs are indeed expressed at specific sites, suggesting orthophysiological functions of VEGFs that may be disturbed by anti-VEGF therapy.

Footnotes

Acknowledgments

Supported by the Haagsch Oogheelkundig Fonds, the Landelijke Stichting voor Blinden en Slechtzienden, the Donders Fonds Utrecht, the Edmond and Marianne Blaauwfonds, and the Diabetes Fonds Nederland (grants 95.103 and 99.050).

We wish to thank Prof Dr K. Alitalo for providing the antibody against VEGFR-3, Prof Dr C.J.F. van Noorden for critically reading the manuscript, the Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands (Head Prof Dr J.J. Weening), and Dr D. Nijdam (Department of Otolaryngology, Academic Medical Center, Amsterdam) for their assistance in obtaining the tissues, and W. Meun and T. Put for preparing the microphotographs.

References

Achen

Jeltsch

Kukk

Makinen

Vitali

Wilks

Alitalo

Stacker

(1998) Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc Natl Acad Sci USA 95:548–553.

Barleon

Sozzani

Zhou

Weich

Mantovani

Marme

(1996) Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87:3336–3343.

Blaauwgeers

Holtkamp

Rutten

Witmer

Koolwijk

Partanen

Alitalo

Kroon

Kijlstra

van Hinsbergh

Schlingemann

(1999) Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am J Pathol 155:421–428.

Breier

(2000) Functions of the VEGF/VEGF receptor system in the vascular system. Semin Thromb Hemost 26:553–559.

Breier

Albrecht

Sterrer

Risau

(1992) Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 114:521–532.

Brown

Berse

Jackman

Tognazzi

Guidi

Dvorak

Senger

Connolly

Schnitt

(1995) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol 26:86–91.

Brown

Berse

Jackman

Tognazzi

Manseau

Dvorak

Senger

(1993a) Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am J Pathol 143:1255–1262.

Brown

Berse

Jackman

Tognazzi

Manseau

Senger

Dvorak

(1993b) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res 53:4727–4735.

Carmeliet

Jain

(2000) Angiogenesis in cancer and other diseases. Nature 407:249–257.

10.

Dor

Porat

Keshet

(2001) Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis. Am J Physiol 280:C1367–1374.

11.

Drake

LaRue

Ferrara

Little

(2000) VEGF regulates cell behavior during vasculogenesis. Dev Biol 224:178–188.

12.

Dvorak

Nagy

Feng

Brown

Dvorak

(1999) Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol 237:97–132.

13.

Esser

Wolburg

Breier

Kurzchalia

Risau

(1998) Vascular endothelial growth factor induces endothelial fenestrations in vitro. J Cell Biol 140:947–959.

14.

Feng

Nagy

Brekken

Pettersson

Manseau

Pyne

Mulligan

Thorpe

Dvorak

(2000) Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors. J Histochem Cytochem 48:545–556.

15.

Ferrara

(1999) Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol 237:1–30.

16.

Ferrara

Chen

Davis-Smyth

Gerber

Nguyen

Peers

Chisholm

Hillan

Schwall

(1998) Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nature Med 4:336–340.

17.

Ferrara

Houck

Jakeman

Leung

(1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Rev 13:18–32.

18.

Folkman

(1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1:27–31.

19.

Fong

Shawver

Sun

Tang

App

Powell

Kim

Schreck

Wang

Risau

Ullrich

Hirth

McMahon

(1999) SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res 59:99–106.

20.

Foss

Araujo

Demel

Klotzbach

Hummel

Stein

(1997) Expression of vascular endothelial growth factor in lymphomas and Castleman's disease. J Pathol 183:44–50.

21.

Gerber

Hillan

Ryan

Kowalski

Keller

Rangell

Wright

Radtke

Aguet

Ferrara

(1999) VEGF is required for growth and survival in neonatal mice. Development 126:1149–1159.

22.

Hamada

Oike

Takakura

Ito

Jussila

Dumont

Alitalo

Suda

(2000) VEGF-C signaling pathways through VEGFR-2 and VEGFR-3 in vasculoangiogenesis and hematopoiesis. Blood 96:3793–3800.

23.

Hatva

Bohling

Jaaskelainen

Persico

Haltia

Alitalo

(1996) Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas. Am J Pathol 148:763–775.

24.

Hatva

Kaipainen

Mentula

Jaaskelainen

Paetau

Haltia

Alitalo

(1995) Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors. Am J Pathol 146:368–378.

25.

Hornig

Barleon

Ahmad

Vuorela

Ahmed

Weich

(2000) Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids. Lab Invest 80:443–454.

26.

Joukov

Pajusola

Kaipainen

Chilov

Lahtinen

Kukk

Saksela

Kalkkinen

Alitalo

(1996) A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J 15:290–298.

27.

Kasahara

Tuder

Taraseviciene-Stewart

Le Cras

Abman

Hirth

Waltenberger

Voelkel

(2000) Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 106:1311–1319.

28.

Korte

Reppucci

Henkind

(1984) RPE destruction causes choriocapillary atrophy. Invest Ophthalmol Vis Sci 25:1135–1145.

29.

Kozlowska

Blume-Peytavi

Kodelja

Sommer

Goerdt

Majewski

Jablonska

Orfanos

(1998) Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle. Arch Dermatol Res 290:661–668.

30.

Kremer

Breier

Risau

Plate

(1997) Up-regulation of flk-1/vascular endothelial growth factor receptor 2 by its ligand in a cerebral slice culture system. Cancer Res 57:3852–3859.

31.

Laird

Vajkoczy

Shawver

Thurnher

Liang

Mohammadi

Schlessinger

Ullrich

Hubbard

Blake

Fong

Strawn

Sun

Tang

Hawtin

Tang

Shenoy

Hirth

McMahon

Cherrington

(2000) SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res 60:4152–4160.

32.

Lymboussaki

Partanen

Olofsson

Thomas-Crusells

Fletcher

De Waal

Kaipainen

Alitalo

(1998) Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors. Am J Pathol 153:395–403.

33.

Millauer

Wizigmann-Voos

Schnurch

Martinez

Moller

Risau

Ullrich

(1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72:835–846.

34.

Monacci

Merrill

Oldfield

(1993) Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues. Am J Physiol 264:C995–1002.

35.

Mustonen

Alitalo

(1995) Endothelial receptor tyrosine kinases involved in angiogenesis. J Cell Biol 129:895–898.

36.

Niki

Iba

Tokunou

Yamada

Matsuno

Hirohashi

(2000) Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma. Clin Cancer Res 6:2431–2439.

37.

Nishi

Maruyama

(2000) Increased expression of vascular endothelial growth factor (VEGF) in Castleman's disease: proposed pathomechanism of vascular proliferation in the affected lymph node. Leukemia Lymphoma 38:387–394.

38.

Olofsson

Korpelainen

Pepper

Mandriota

Aase

Kumar

Gunji

Jeltsch

Shibuya

Alitalo

Eriksson

(1998) Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc Natl Acad Sci USA 95:11709–11714.

39.

Oosthuyse

Moons

Storkebaum

Beck

Nuyens

Brusselmans

Dorpe

Hellings

Gorselink

Heymans

Theilmeier

Dewerchin

Laudenbach

Vermylen

Raat

Acker

Vleminckx

Bosch

Cashman

Fujisawa

Drost

Sciot

Bruyninckx

Hicklin

Ince

Gressens

Lupu

Plate

Robberecht

Herbert

Collen

Carmeliet

(2001) Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nature Genet 28:131–138.

40.

Ortega

Hutchings

Plouet

(1999) Signal relays in the VEGF system. Front Biosci 4:D141–152.

41.

Ortega

Jonca

Vincent

Favard

Ruchoux

Plouet

(1997) Systemic activation of the vascular endothelial growth factor receptor KDR/flk-1 selectively triggers endothelial cells with an angiogenic phenotype. Am J Pathol 151:1215–1224.

42.

Partanen

Arola

Saaristo

Jussila

Ora

Miettinen

Stacker

Achen

Alitalo

(2000) VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J 14:2087–2096.

43.

Plate

Breier

Weich

Risau

(1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359:845–848.

44.

Hong

Nagy

Senger

Dvorak

(1995) Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium. J Histochem Cytochem 43:381–389.

45.

Robinson

Shih

McMahon

Caldwell

Smith

(2001) Nonvascular role for VEGF: VEGFR-1, 2 activity is critical for neural retinal development. FASEB J 15:1215–1217.

46.

Ruiter

Schlingemann

Rietveld

De Waal

(1989) Monoclonal antibody-defined human endothelial antigens as vascular markers. J Invest Dermatol 93:25S–32S.

47.

Schlingemann

Dingjan

Emeis

Blok

Warnaar

Ruiter

(1985) Monoclonal antibody PAL-E specific for endothelium. Lab Invest 52:71–76.

48.

Schlingemann

Hofman

Anderson

Troost

Van der Gaag

(1997) Vascular expression of endothelial antigen PAL-E indicates absence of blood-ocular barriers in the normal eye. Ophthalmic Res 29:130–138.

49.

Schlingemann

Rietveld

Kwaspen

van de Kerkhof

De Waal

Ruiter

(1991) Differential expression of markers for endothelial cells, pericytes, and basal lamina in the microvasculature of tumors and granulation tissue. Am J Pathol 138:1335–1347.

50.

Schlingemann

Rietveld

De Waal

Bradley

Skene

Davies

Greaves

Denekamp

Ruiter

(1990) Leukocyte antigen CD34 is expressed by a subset of cultured endothelial cells and on endothelial abluminal microprocesses in the tumor stroma. Lab Invest 62:690–696.

51.

Senger

Galli

Dvorak

Perruzzi

Harvey

Dvorak

(1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219:983–985.

52.

Shifren

Doldi

Ferrara

Mesiano

Jaffe

(1994) In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action. J Clin Endocrinol Metab 79:316–322.

53.

Simon

Grone

Johren

Kullmer

Plate

Risau Fuchs

(1995) Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am J Physiol 268:F240–250.

54.

Simon

Rockl

Hornig

Grone

Theis

Weich

Fuchs

Yayon

Grone

(1998) Receptors of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in fetal and adult human kidney: localization and [125I]VEGF binding sites. J Am Soc Nephrol 9:1032–1044.

55.

Smith

(2001) Regulation of angiogenesis in the endometrium. Trends Endocrinol Metab 12:147–151.

56.

Takagi

King

Aiello

(1996) Identification and characterization of vascular endothelial growth factor receptor (Flt) in bovine retinal pericytes. Diabetes 45:1016–1023.

57.

Takahashi

Kitadai

Bucana

Cleary

Ellis

(1995) Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 55:3964–3968.

58.

Terris

Scoazec

Rubbia

Bregeaud

Pepper

Ruszniewski

Belghiti

Flejou

Degott

(1998) Expression of vascular endothelial growth factor in digestive neuroendocrine tumours. Histopathology 32:133–138.

59.

Vaisman

Gospodarowicz

Neufeld

(1990) Characterization of the receptors for vascular endothelial growth factor. J Biol Chem 265:19461–19466.

60.

Veikkola

Karkkainen

Claesson-Welsh

Alitalo

(2000) Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60:203–212.

61.

Warren

Yuan

Matli

Gillett

Ferrara

(1995) Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. J Clin Invest 95:1789–1797.

62.

Witmer

Blaauwgeers

Weich

Alitalo

Vrensen

GFJM

Schlingemann

(2002) Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey. Invest Ophthalmol Vis Sci 43:849–857.

63.

Witmer

Van Blijswijk

Hofman

Partanen

Vrensen

GFJM

Schlingemann

(2001) VEGFR-3 in adult angiogenesis. J Pathol 195:490–497.

64.

Witzenbichler

Asahara

Murohara

Silver

Spyridopoulos

Magner

Principe

Kearney

Isner

(1998) Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J Pathol 153:381–394.

65.

Yancopoulos

Davis

Gale

Rudge

Wiegand

Holash

(2000) Vascular-specific growth factors and blood vessel formation. Nature 407:242–248.

66.

Yano

Brown

Detmar

(2001) Control of hair growth and follicle size by VEGF-mediated angiogenesis. J Clin Invest 107:409–417.

67.

Mai

Uyama

Yew

(1998) Time-course expression of vascular endothelial growth factor as related to the development of the retinochoroidal vasculature in rats. Exp Brain Res 118:155–160.

Expression of Vascular Endothelial Growth Factor Receptors 1,2,and 3 in Quiescent Endothelia

Abstract

Keywords

Materials and Methods

Tissue Samples

Light Microscopic Immunohistochemistry

Electron Microscopic Immunohistochemistry

Results

Light Microscopy

Electron Microscopy

Discussion

Footnotes

Acknowledgments

References