ARTICLE |
Correspondence to: Reinier O. Schlingemann, Dept. of Ophthalmology, Academic Medical Center, PO Box 22660, 1100 DD Amsterdam, The Netherlands. E-mail: r.schlingemann@amc.uva.nl
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Summary |
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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.
(J Histochem Cytochem 50:767777, 2002)
Key Words: endothelial growth factors, electron microscopy, endothelial growth factor, receptors, human, immunohistochemistry, monkey, tissue distribution
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Introduction |
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VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), a permeability factor (
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 (
VEGF-A mRNA is constitutively expressed by kidney glomerular epithelium, choroid plexus epithelium and retinal pigment epithelium (RPE) in mice, rats, and humans (
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.
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Materials and Methods |
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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.
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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 (
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 bloodtissue barrier, whereas this antigen is absent in lymphatic endothelium. Anti-CD31 was used as a marker for all blood and lymph vessel endothelium (
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) (
A pre-embedding immunoperoxidase technique was used to demonstrate subcellular distribution patterns of VEGFR-1 and VEGFR-2 in tonsil tissue as described previously (
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Results |
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Light Microscopy
Staining patterns of all VEGFRs were similar in human and monkey tissues and are summarized in Table 2.
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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 (Fig 1), kidney glomeruli, capillaries surrounding hair follicles and within the papilla of hair follicles (Fig 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 (Fig 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 (Fig 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 (Fig 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 (Fig 2), which is the basement membrane separating epithelium from connective tissue of the follicle, and in the inner limiting membrane of the retina (
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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 (Fig 1, Fig 2, and Fig 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 (Fig 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 (Fig 1, Fig 2, and Fig 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 (Fig 3).
Very intense staining of VEGFR-3 was observed in thin-walled lymphatic vessels in portal tracts of the liver, gastrointestinal villi (Fig 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 (Fig 4), consistent with diffuse VEGFR-3 staining in neural elements of the retina (
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 (Fig 4E), whereas staining of VEGFR-2 was restricted to endothelial cells (Fig 4). VEGFR-1 and VEGFR-2 staining was found in a dot-like configuration in the cytoplasm (Fig 4E and Fig 4F), which is in agreement with the granular staining pattern of VEGFRs at the light microscopic level (Fig 4A and Fig 4C). In addition, a more diffuse precipitation in the cytoplasm was observed.
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Discussion |
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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.
In addition to our previous observations of VEGFR expression in the choriocapillaris in the eye (
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 (
Studies of VEGFR expression patterns at the protein level in normal human tissues are scarce.
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 (
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 (
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 (
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 (
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 (
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 (
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Acknowledgments |
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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.
Received for publication August 26, 2001; accepted January 4, 2002.
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