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1 From the Ocular Angiogenesis Group, Department of Ophthalmology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; the 3 National Research Center for Biotechnology, Braunschweig, Germany; the 4 Department of Pathology, Haartman Institute, Helsinki, Finland; and the 2 Lens and Cornea Research Unit, the Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. In retinas of 27 eyes of diabetic donors, 18 eyes of nondiabetic control donors, and 4 monkey eyes injected with PBS or VEGF-A, expression patterns of VEGFR-1, -2, and -3 in relation to leaky microvessels, as identified by the marker pathologische anatomie Leiden-endothelium (PAL-E) were studied by immunohistochemistry.
RESULTS. In control human retinas and retinas of PBS-injected monkey eyes, all three VEGFRs were expressed in nonvascular areas, but only VEGFR-1 was constitutively expressed in retinal microvessels. In diabetic eyes, increased microvascular VEGFR-2 expression was found in association with PAL-E expression, whereas microvascular VEGFR-3 was present in a subset of PAL-E–positive cases. In VEGF-A–injected monkey eyes, VEGFR-1, -2, and -3 and PAL-E were expressed in retinal microvessels.
CONCLUSIONS. The VEGFR-1, -2, and -3 expression patterns in control retinas suggest physiological functions of VEGFs that do not involve the vasculature. Initial vascular VEGF signaling may act primarily through VEGFR-1. In diabetic eyes, expression of retinal VEGFR-2 and -3 is increased, mainly in leaky microvessels, and VEGF-A induces vascular expression of the VEGF-A receptor VEGFR-2 and the VEGF-C/D receptor VEGFR-3. These findings indicate a dual role of VEGFs in the physiology and pathophysiology of the retina and suggest that microvascular VEGFR-2 and -3 signaling by VEGFs occurs late in the pathogenesis of DR, possibly initiated by high levels of VEGF-A in established nonproliferative DR.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent literature on the possible role of VEGF in ocular angiogenesis and DR is complex. On the one hand, extensive evidence is available suggesting that VEGF is the main cytokine causing vascular leakage and angiogenesis in various conditions involving retinal ischemia.15 16 17 18 19 In nonproliferative DR, leakage of retinal microvessels is probably caused by local VEGF production in areas of capillary nonperfusion.15 20 In addition, VEGF mRNA levels are increased in the ischemic retina of patients with advanced DR,21 and proliferative DR is associated with high levels of VEGF in the vitreous.1 22 23 On the other hand, various reports show mRNA and protein expression of VEGF-A and VEGFR-1 and -2 in the normal retina,24 25 26 27 suggesting physiological functions of VEGF.
Because differential expression of its receptors on vascular cells may be an important mechanism of regulating the activity of VEGF, it has been hypothesized that distribution patterns and/or levels of VEGFR are major determinants of VEGF activity in the normal retina or in pathologic conditions such as DR.28 However, few data are available on expression patterns of VEGFRs in the retinal vasculature of control or DR eyes.24 27 29 30 It is also unknown how VEGFR expression in the retina is regulated. Increased VEGFR-2 expression has been described in relation to retinal ischemia in mice,31 and in background DR in rats,29 both inside and outside the vasculature. VEGFR-2 and -3 expression in human retinal microvessels is associated with proliferative DR.27 However, these studies did not include histologic markers of vascular changes related to DR. We have recently shown that local loss of the blood–retinal barrier and specific morphologic changes in capillaries in DR are highly correlated with the expression of the antigen pathologische anatomie Leiden-endothelium (PAL-E),32 a marker for nonbarrier endothelium.33 Expression of PAL-E and staining of permeability markers were found in specific areas in a pattern similar to that of DR lesions in clinical angiography. Proliferative DR caused widespread staining of PAL-E in retinas. Therefore, the PAL-E antigen can be considered an immunohistochemical marker of areas with vascular leakage related to DR.32
In the present study, we investigated (1) the expression patterns of VEGFRs in retinas of control and diabetic eyes, in relation to staining of both the entire vasculature using CD31 antibodies and leaky microvessels using the antibody PAL-E, and (2) the expression patterns of VEGFRs in retinal microvessels in a model of VEGF-induced retinopathy in monkeys.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monkey Eyes
Two cynomolgus monkeys (Macaca fascicularis), a
15-year-old male and a 5-year-old female, were used for the
experiments. All experiments were performed in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research and
in accordance with the guidelines for animal care at the University of
Nijmegen, The Netherlands. Both animals had been used for behavioral
studies unrelated to visual function and without alteration of the
eyes. The animals received four injections of phosphate-buffered saline
(50 µL PBS, pH 7.4) in the left eye through the pars plana into the
center of the vitreous, with a 30-gauge needle (at days 0, 2, 4, and
7), and four injections with bioactive human recombinant VEGF-A (0.5
µg in 50 µL PBS; Harbor Bio-Products, Norwood, MA) in the right eye
on the same days. Before the intravitreal injections, 20 mg/kg ketamine
hydrochloride, 0.005 mg/kg acepromazine, and 0.03 mg/kg atropine
sulfate were administered intramuscularly for general anesthesia. Ten
minutes after injection, intraocular pressure was measured using a
Schiotz tonometer (Sklar Manufacturing, New York, NY). The animals were
killed at day 9 with an overdose of intravenous pentobarbital and
subsequently perfused through the abdominal aorta with PBS (37°C, pH
7.4) for 10 minutes at a perfusion pressure of 70 to 80 mm Hg before
the eyes were enucleated. Eyes were dissected, and the posterior
segment was snap frozen in liquid nitrogen and stored at -70°C until
used.
Immunohistochemistry
Tissue blocks of the posterior half of the frozen human and
monkey globes were cut according to a standard protocol. Air-dried
serial cryostat sections (10 µm thick) of one tissue block containing
the midperipheral retina and posterior pole of one eye of each patient
and of both eyes of both monkeys were fixed in cold acetone for 10
minutes, postfixed for 2 minutes in Zamboni fixative (2%
paraformaldehyde in a saturated picric acid solution), and stained by
an indirect immunoperoxidase procedure. For this purpose, sections were
incubated for 20 minutes in PBS containing 0.1% sodium azide and
0.03% H2O2 to quench
endogenous peroxidase activity. To reduce nonspecific staining,
sections were incubated with a broad-spectrum serum blocking solution
(Histostain Plus kit; Zymed, San Francisco, CA) in PBS containing
0.05% saponin (Sigma, St. Louis, MO) for 15 minutes. Subsequently,
sections were incubated overnight at 4°C with a solution of the after
antibodies: monoclonal antibodies Flt-19 (against VEGFR-1, 1:400) and
KDR-1 (against VEGFR-2, 1:400),34
monoclonal antibody
9D9F9 (against VEGFR-3, 1:1500),35
and the
anti-endothelial monoclonal antibodies PAL-E (1:1000)36
and EN-4 (against CD31, 1:500).37
Flt-19 and KDR-1 were
kindly provided by Herbert A. Weich, National Research Center
for Biotechnology, Braunschweig, Germany; 9D9F9 was provided by Kari
Alitalo, Haartman Institute, Helsinki, Finland; and EN-4 by Sanbio,
Uden, The Netherlands. For negative control incubations, primary
antibody was omitted, or sections were stained with an antibody against
a nonhuman bacterial protein (mouse negative control immunoglobulins;
Dako, Glostrup, Denmark). Sections were subsequently incubated with
biotinylated goat anti-mouse immunoglobulins for 15 minutes, followed
by streptavidin-horseradish peroxidase complex for 15 minutes.
Peroxidase activity was visualized using 3-amino-9-ethylcarbazole (AEC,
red color) or 3,3'-diaminobenzidine (DAB, brown color) with 0.01%
H2O2 as substrate. The
reaction was terminated by rinsing the sections with distilled water.
Counterstaining was performed with hematoxylin.
Data Analysis
For each antibody, three masked sections taken from a
standardized sample, encompassing the midperipheral to the central part
of the retina of each donor, were examined by two independent
observers. When the observers were not in agreement, the section was
scored again by the two observers together. The total number of
microvessels over a 5-mm length of sections of retina was determined on
the basis of CD31-positive staining. Retinal microvascular staining of
VEGFRs and PAL-E was graded semiquantitatively as follows: 0, no
staining; 1, sporadic staining (less than three positive microvessels
per 5-mm section); 2, patchy staining (localized areas with staining
microvessels, but <50% of vessels positive); or 3, uniform staining
(>50% of vessels positive). For statistical analysis of the
association between ordinal variables (PAL-E staining and VEGFR
staining), we used the conventional nonparametric rank order 
-test,
the outcome of which can be interpreted as the correlation coefficient
(SPSS ver. 8.0; section Crosstabs; SPSS, Chicago, IL). This test
provides, in the absence of a gold standard for both parameters, the
same weight to the error of PAL-E grade 3 staining when VEGFR staining
is assigned grade 2, versus the error of PAL-E grade 2 staining when
VEGFR staining is assigned grade 3.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGFR-3.
In control eyes, retinal vascular staining of VEGFR-3 was absent (16/18
cases; Fig. 1E
, Table 2
) or sporadic (2/18 cases), whereas VEGFR-3 staining was observed in
microvessels of retinas of 11 of 27 diabetic eyes (Table 2)
. VEGFR-3
was expressed in microvessels of the inner nuclear layer and the
ganglion cell layer (Fig. 2G)
. VEGFR-3 staining was mainly found in
retinas with distinct PAL-E expression (Table 2)
, although a number of
PAL-E–positive cases did not show any vascular staining of VEGFR-3. In
addition, the association between distribution patterns of VEGFR-3 and
-2 staining were high (Table 3)
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VEGF-Induced Retinopathy in Monkeys
Changes in monkey eyes due to PBS and VEGF injection have been
described.38
Briefly, in both VEGF-injected eyes, retinal
venous dilation was observed by funduscopic evaluation of the eyes on
day 9. The eyes were without biomicroscopic signs of inflammation, and
intraocular pressures remained at less than 25 mm Hg at all time
points. Hematoxylin-eosin staining of sections of the central retina of
the eyes injected with VEGF demonstrated edematous changes.
CD31 staining revealed a normal distribution pattern of microvessels throughout the retinas of both PBS- and VEGF-injected eyes. However, staining of CD31 was stronger in the VEGF-injected eyes (Figs. 3C 3D) . The vessels in these eyes were hypertrophic, more dilated, and slightly more tortuous than in PBS-injected eyes. Staining of PAL-E was not present in retinas of PBS-injected eyes, whereas it was uniformly distributed in retinas of VEGF-injected eyes (data not shown).
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VEGFR-2.
In PBS-injected eyes, retinal microvessels were negative for VEGFR-2
(Fig. 3G)
, whereas retinal microvascular staining of VEGFR-2 was
localized in a granular pattern in VEGF-injected eyes (Fig. 3H)
.
VEGFR-2–positive microvessels were mainly localized in the ganglion
and nerve fiber layers, whereas most microvessels in the inner nuclear
layer were negative (Fig. 3H) .
Variable weak granular staining of VEGFR-2 was observed in all monkey eyes outside the vasculature in neural or glial elements of the ganglion cell layer, the inner plexiform and nuclear layers, and the outer plexiform layer (Figs. 3G 3H) , which was consistent with findings in human eyes.
VEGFR-3.
Retinal microvascular staining of VEGFR-3 was observed in the ganglion
and nerve fiber layers, whereas microvessels in the inner nuclear layer
were negative in VEGF-injected eyes (Fig. 3J) . In eyes injected with
PBS, staining of VEGFR-3 was absent in the retinal microvessels (Fig. 3I)
.
In all eyes, staining of VEGFR-3 was observed outside the retinal vasculature in neural elements of the inner plexiform layer and the inner part of the outer plexiform layer in a pearl-necklace–like configuration (Fig. 3I 3J) .
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have studied human donor eyes without information on the type of diabetes mellitus or the presence and/or degree of DR, for reasons of privacy. However, we were able to investigate the relation between VEGFR expression and vascular leakage, by staining adjacent sections for the endothelial antigen PAL-E, a specific marker for capillary leakage in diabetic retina.32 Although PAL-E cannot be regarded as a direct marker for DR, microvascular leakage is associated clinically (and thus histologically) with other features of DR.
VEGF is a major cytokine that causes vascular leakage and angiogenesis, and its expression has been demonstrated in ischemic retina, in line with the clinical association of retinal ischemia with retinal leakage and ocular angiogenesis.16 17 39 However, in situ hybridization and immunohistochemistry with polyclonal antibodies25 have shown mRNA and protein expression of VEGF-A and VEGFR-1, -2, and -3 also in the normal retina, where vascular leakage and angiogenesis do not occur.24 26 27 Our present data help to explain these expression patterns. By the use of highly specific monoclonal antibodies,34 35 we found all three VEGFRs to be expressed in nonvascular cells in retinas of human control eyes and PBS-treated monkey eyes, suggesting that VEGFs have a physiological function in retinas with respect to neural elements, which is in agreement with the neurotrophic function of VEGF-A.40 41 42 Only VEGFR-1, a receptor that mediates migration but not permeability or mitosis, was ubiquitously localized in microvascular walls in retinas. VEGFR-1 is expressed by retinal pericytes in vitro.43 44 VEGFR-1 expression by pericytes in vivo may corroborate our observations of VEGFR-1 expression in the walls of retinal microvessels, where it may enable pericytes rather than endothelial cells to respond initially to VEGF signaling under quiescent conditions. Alternatively, the vascular staining pattern of VEGFR-1 may represent a localization of the soluble form of this receptor in the basement membrane of retinal microvessels. This possibility is supported by our observation of VEGFR-1 staining in the inner limiting membrane (i.e., the basement membrane of Müller cells), which indicates a localization of VEGFR-1 in the extracellular matrix, rather than on cells. sVEGFR-1 has been suggested to act as a scavenger for VEGF-A,14 45 thereby protecting vessels from becoming leaky or proliferative at low levels of VEGF-A. Ultrastructural studies are needed to further investigate these interesting possibilities, in that our light microscopic analysis did not allow an exact localization of VEGFR-1 in endothelial cells, pericytes, and/or extracellular matrix elements of microvessels.
Sporadic PAL-E expression with weak staining intensity was found in a number of control retinas, as previously described.32 Sporadic VEGFR-2 and -3 expression with weak staining intensity was found in a few control eyes as well (5/18 and 2/18 eyes, respectively), in some cases associated with sporadic PAL-E expression (3/18 and 1/18 eyes, respectively). The functional relevance of this finding in control eyes remains to be elucidated, but a striking phenotypic shift of expression of PAL-E and VEGFRs was observed in diabetic eyes. In these human eyes and in two monkey eyes treated with a prolonged regimen of VEGF-A, we observed microvascular expression of VEGFR-1, -2, and -3. Microvascular expression of VEGFR-2 was associated with leaky vessels in the diabetic retina, as can be concluded from the significant association with PAL-E expression. The observations in monkey eyes injected with VEGF-A indicate that high levels of VEGF-A and/or prolonged exposure to VEGF induces VEGFR-2 expression in retinal vascular cells. In brain capillaries, a similar mechanism of VEGFR-2 induction has been suggested.12 46 This may be a direct effect of VEGF-A on endothelial cells through their constitutive expression of VEGFR-1, a very low-level baseline expression of VEGFR-2 that remained undetectable by immunohistochemistry, or an indirect effect of VEGF-A through another route that induces a secondary signal acting on endothelial cells.
In light of our previous demonstration of a relation between PAL-E expression and established vascular leakage in DR and together with our current observations, the following hypothetical scenario in the course of DR may be proposed: Retinal ischemia causes overexpression of VEGF-A, which triggers expression of VEGFR-2 on microvascular cells, possibly through VEGFR-1, leading to vascular leakage (PAL-E expression) and eventually to angiogenesis caused by VEGF-A’s signaling this receptor. Our detailed observations in human diabetic eyes support this scenario. We found VEGFR-2 expression mainly in retinal areas with microvascular leakage (suggesting the presence of established DR), as recognized by vascular staining of the PAL-E antigen.32 It has been demonstrated that VEGF-A is overexpressed in such areas.28 39 47 48 In addition, expression of VEGFR-3 correlated well with expression of VEGFR-2 in diabetic eyes, suggesting induction of VEGFR-2 and -3 and a role for their respective ligands in such areas.
In 9 of 27 diabetic eyes (Table 2) and in monkey eyes injected with VEGF-A, we observed VEGFR-3 upregulation associated with increased PAL-E staining in retinal microvessels. In diabetic eyes, VEGFR-3 staining was observed in vessels in the deeper layers of the retina, in which VEGF-A is overexpressed in DR.21 28 39 48 In the monkey retina, VEGFR-3 expression was localized in vessels of the inner layers of the retina, suggesting a dose–response effect of exogenous VEGF-A, as is the case with VEGFR-2. VEGF-A is not a ligand for VEGFR-3, and VEGFR-3 and its ligand VEGF-C were previously considered to be involved in lymphangiogenesis in the adult,35 49 50 51 and in angiogenesis in embryos and tumors.51 52 53 Our findings indicate distinct and differing roles of VEGFR-3 and its ligands VEGF-C and/or -D in the normal and diabetic retina, and our studies in monkey eyes suggest that its expression is induced by VEGF-A. Outside the vasculature, VEGFR-3 staining was observed unexpectedly in a distinct pearl-necklace–like configuration in the outer plexiform layer in both control and diabetic retinas, a pattern highly suggestive of staining of synaptic complexes of cone photoreceptors.
Finally, it has been suggested that VEGF-A upregulation also plays a role in preclinical human DR28 48 and in early retinopathy in rat models of streptozotocin-induced diabetes29 54 and that preclinical increased vascular expression of VEGFR-2 mediates this role.29 In our study, we did not find evidence for such early expression, in that diabetic eyes without PAL-E staining did not differ from the nondiabetic control eyes in distribution patterns of VEGFR staining. The discrepancy between studies in rat models of experimental DR29 54 and our study may exist because small rodents with experimental diabetes do not represent a good model of human preclinical DR, and/or because vascular expression of VEGFR-2 in control eyes and preclinical DR is below the detection level of our immunohistochemical method.
In summary, our findings indicate a dual role of VEGFs in the physiology and pathophysiology of the retina, and suggest that VEGFR-2 and -3 signaling of vascular cells occurs relatively late in the diabetic retina, in areas with established microvascular leakage associated with DR, possibly initiated by high levels of VEGF-A produced in these areas.28 39 47 48
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Acknowledgements |
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Footnotes |
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Submitted for publication June 6, 2001; revised October 31, 2001; accepted November 14, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Reinier O. Schlingemann, Department of Ophthalmology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands; r.schlingemann{at}amc.uva.nl
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