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1 From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico; and the 2 Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria.
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Abstract |
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METHODS. Sixteen-kDa PRL and basic fibroblast growth factor (bFGF) or anti-PRL antibodies were placed into rat cornea micropockets and neovascularization evaluated by the optical density associated with capillaries stained by the peroxidase reaction and by the number of vessels growing into the implants. Prolactin receptors in corneal epithelium were investigated by immunocytochemistry.
RESULTS. bFGF induced a dose-dependent stimulation of corneal neovascularization. This effect was inhibited by coadministration of 16-kDa PRL, as indicated by a 65% reduction in vessel density and a 50% decrement in the incidence of angiogenic responses. Corneal angiogenic reactions of different intensities were induced by implantation of polyclonal and monoclonal anti-PRL antibodies. Corneal epithelial cells were labeled by several anti-PRL receptor monoclonal antibodies.
CONCLUSIONS. These findings show that exogenous 16-kDa PRL inhibits bFGF-induced corneal neovascularization and suggest that PRL-like molecules with antiangiogenic actions function in the cornea. PRL receptors in the corneal epithelium may imply that PRL in the cornea derives from lacrimal PRL internalized through an intracellular pathway. These observations are consistent with the notion that members of the PRL family are potential regulators of corneal angiogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several stimulators and inhibitors of angiogenesis have been identified that modulate the ability of endothelial cells to digest the basement membrane and proliferate, migrate, and/or associate into a new capillary network.3 4 The balance between naturally occurring inducers and inhibitors of angiogenesis determines the active neovascularization observed during embryogenesis as well as the vascular quiescence maintained by most tissues in adult life.4 5 The cornea and the cartilage are normally avascular. Their resistance to vascular invasion appears to involve a predominance of inhibitors over stimulators of angiogenesis.4 5 6
Among a long list of active peptides, members of the prolactin (PRL) hormonal family have been proposed as potential regulators of angiogenesis.7 Fragments of PRL of 14 kDa and 16 kDa bind to endothelial cell receptors and inhibit endothelial cell proliferation, migration, and tube formation.7 8 9 10 11 12 Similarly, the 16-kDa amino terminal fragment of PRL inhibits the in vivo development of the microvasculature of the chick chorioallantoic membrane (CAM).10 In addition proliferin and proliferin-related protein (PRP), considered members of the PRL family on the basis of primary sequence homology, compete with 16-kDa PRL for endothelial cell binding9 and have opposite effects on angiogenesis.13 Proliferin stimulates and PRP inhibits endothelial cell migration and corneal neovascularization, respectively.13
The cornea is a powerful model for investigating the in vivo regulation of blood vessel growth.14 In the present study, we used rat corneas to analyze the potential contribution of PRLs to the regulation of angiogenesis. Our findings extend those previously reported of in vivo antiangiogenic action of 16-kDa PRL10 and provide evidence to suggest that PRL-like molecules with inhibitory actions on blood vessel growth, may be involved in the maintenance of corneal avascularity. A preliminary report of these findings has been presented.15 In all facets of the study, animals were managed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purification of IgG
Antibodies from rabbit antisera, nonimmune sera, or ascites fluid
were purified on a protein A Sepharose column (Sigma, St. Louis, MO),
as described.19
Briefly, samples were added to columns
equilibrated with phosphate-buffered saline (PBS; pH 8). Columns were
washed extensively with PBS (pH 8) and IgG eluted with 0.1 M sodium
acetate (pH 3) into tubes with 1 M Tris hydrochloride (pH 8) to
neutralize the acid. The concentration of antibodies was determined by
the Bradford method (Bio-Rad, Richmond, CA).
Enzyme-linked Immunosorbent Assay
Polyclonal antibodies directed against 23-kDa PRL or 16-kDa PRL
and the INN-rPRL-1 anti-23-kDa PRL monoclonal antibody were
characterized by enzyme-linked immunosorbent assay (ELISA), performed
as described.20
For these assays, wells were coated with
10 ng of either 23-kDa PRL or 16-kDa PRL. Primary polyclonal (1:2000)
or monoclonal (1:100) antibodies, with or without serial dilutions of
23-kDa or 16-kDa PRLs were added to the coated wells. Horseradish
peroxidase–conjugated anti-rabbit IgG or anti-mouse IgG (Vector
Laboratories, Burlingame, CA) was used at 1:2000 or 1:1000 dilution,
respectively. Bound horseradish peroxidase–conjugated antibodies were
revealed by reaction with 2 mg of ortho-phenylenediamine
(dihydrochloride) in 0.1 M citrate buffer (pH 5.0) with 0.1% hydrogen
peroxide. Reaction was stopped with 4 N sulfuric acid and optical
density (OD) determined at 490 nm.
Western Blot Analysis
Twenty-three-kDa and 16-kDa PRLs were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15%
acrylamide separating and 4% stacking laboratory gels, under reducing
conditions using the buffer system of Laemmli.21
Proteins
were blotted into nitrocellulose membranes and probed with polyclonal
(1 µg/ml) or monoclonal (2 µg/ml) antibodies. Antigen–antibody
reaction was developed with the use of an alkaline phosphatase
secondary antibody kit (Bio-Rad, Hercules, CA).
Rat Corneal Bioassay
The bioassay was performed with male Wistar rats (200–250 g), as
previously described.22
Briefly, rats were anesthetized
with sodium pentobarbital. A corneal pocket was made by inserting a
27-gauge needle, with the pocket’s base 1 mm from the limbus. Implants
containing the test substances were made using
poly-2-hydroxylethylmethacrylate (Hydron; Interferon Sciences, New
Brunswick, NJ), stained with india ink, and combined (1:1) with the
sample substance. The Hydron substance solution was pipetted onto a
sterile Teflon surface and air dried to produce a 2-mm-diameter disc.
Approximately one eighth of the disc was implanted in each corneal
pocket. Animals were killed 6 days after implantation and corneas
dissected, flattened by cuts leaving three to four regions of at least
4 mm2 each, and fixed for 2 hours with 4%
paraformaldehyde-1.5% glutaraldehyde in 0.1 M phosphate buffer (pH
7.4). Corneal capillary network was delineated by staining the enclosed
red blood cells by a peroxidase reaction, as described.10
Accordingly, visualization of vessels followed corneal incubation in
0.05% 3,3'-diaminobenzidine in the presence of 0.01%
H2O2 in 0.1 M phosphate
buffer. Subsequently, corneas were dehydrated, mounted (Entellan;
Merck, Darmstadt, Germany), coverslipped, and analyzed in a light-field
microscope (model 104; Nikon, Garden City, NY). Images were digitized
with a video camera (model CCD72; Dage–MTI, Michigan City, IN) and
subjected to analysis (NIH Image ver. 1.62, National Institutes of
Health, Bethesda, MD). A mask of a region of interest, with a
semitriangular shape and approximately 4.0 mm2,
was generated and centered at the implant on each corneal digitized
image. This was the smaller common area to be contained within the
segments delimited by the cuts performed to flattened the corneas.
Corneal angiogenesis was quantitated by recording the OD associated
with pixel gray values of stained capillaries within the region of
interest mask. Image background was subtracted to ensure OD uniformity
on the image. Only angiogenic reactions reaching an OD value equal to
or higher than the double of the average control level were considered
to be substantial enough for the evaluation of the incidence of
neovascularization. In addition, the number of long vessels reaching
the implant from perilimbal vessels was quantitated on the printed
digitized corneal images by double-blind, independent observers.
Immunocytochemistry
Male Wistar rats (200–250 g) were killed by decapitation. Eyes
were enucleated and fixed for 2 hours with 4% paraformaldehyde-1.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), transferred to 20%
(wt/vol) sucrose until saturation, and then cut at 15 mm thickness in a
cryostat (-16°C). Sections were incubated for 30 minutes in methanol
containing 30% H2O2 and 1
hour in 10% bovine serum albumin (BSA), 3% normal calf serum, and
0.3% Triton X-100 in PBS. Subsequently, sections were incubated
overnight in a humid atmosphere at 4°C with each anti-PRL receptor
monoclonal antibody (100 µg/ml) in 0.3% Triton X-100 PBS. The
sections were rinsed and incubated with secondary biotinylated
anti-mouse IgG followed by avidin-biotin-peroxidase (ABC kit, Vector
Laboratories). Bound anti-PRL receptor antibodies were revealed with
0.05% 3,3'-diaminobenzidine and 0.15%
H2O2 for 2 to 3 minutes.
Sections were dehydrated, mounted (Entellan; Merck), coverslipped, and
analyzed in a light-field microscope (model BX60; Olympus, Lake
Success, NY).
Statistical Analysis
Data reported as ODs and number of vessels (mean ± SEM) were
analyzed by two-way analysis of variance (replicate x treatment)
to partition out variation due to experimental replicates. Mean values
for each treatment were compared using Duncan’s new multiple-range
test. Differences were considered significant at P <
0.05. Data reported as incidence of angiogenesis were analyzed for
statistical significance by the nonparametric Mann–Whitney rank sum
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of Rat Corneal Angiogenesis by Anti-PRL Antibodies
Implants containing anti-16-kDa PRL or anti-23-kDa PRL polyclonal
antibodies induced corneal angiogenesis (Fig. 5)
. Although anti-16-kDa PRL antibodies induced substantial angiogenic
responses in 20% of the corneas treated, anti-23-kDa PRL antibodies
did the same in 40% (Fig. 5)
. On average, these responses were
equivalent in magnitude to those induced by doses of bFGF between 60
and 120 ng (Fig. 2)
. Although OD determinations adequately monitor the
moderate and strong neovascularization responses induced by bFGF, they
could underestimate mild vessel growth, such as that occurring in
response to PRL antibodies. Thus the number of leading vessels reaching
the implant was used as an additional score for proangiogenic actions
of anti-PRL antibodies. Both anti-16-kDa PRL and anti-23-kDa PRL
antibodies increased the number of these vessels over preimmune
antibody values. To control for nonspecific reactions to
antigen–antibody interactions, 36 corneas were treated with 2 µg
BSA, anti-BSA antibodies, or their combination, with no positive
responses detected (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several angiogenesis inhibitors have been described, some of which correspond to fragments of larger proteins.5 The latter include angiostatin, a 36-kDa internal fragment of plasminogen28 ; endostatin, the 20-kDa C-terminal fragment of collagen XVIII29 ; an internal fragment of platelet factor 430 ; fragments of laminin31 ; peptides derived from thrombospondin32 ; and the 14-kDa and 16-kDa N-terminal fragments of PRL.10 12
PRL fragments of 14 kDa and 16 kDa inhibit endothelial cell proliferation.8 9 10 Similarly, 16-kDa PRL inhibits the activation of urokinase,11 a protease involved in endothelial cell migration, and tube formation by endothelial cells in vitro.10 Moreover, 16-kDa PRL inhibits the in vivo formation of blood vessels in the chick embryo CAM.10 PRL fragments with antiangiogenic effects appear to be produced by the enzymatic proteolysis of 23-kDa PRL in both the anterior and posterior lobes of the pituitary gland and are found in the circulation.11 12 33 Moreover, endothelial cells from different blood vessels and species have been shown to express the PRL gene,34 35 36 and endothelia-derived PRL molecules have been found to affect endothelial cell proliferation.34 The placenta produces proliferin and PRP, two PRL-like proteins that stimulate and inhibit angiogenesis, respectively.13 Both proteins have been implicated in the regulation of placental neovascularization.13 Accordingly, various lines of evidence provide support to the hypothesis that members of the PRL family function as potential regulators of angiogenesis.
In the present study we used the rat cornea to investigate this hypothesis. The cornea provides a well characterized bioassay for angiogenesis, in which the elicitation of an angiogenic reaction is a convincing demonstration of true neovascularization, because this organ is normally avascular.14 With this assay we have evaluated the in vivo antiangiogenic action of 16-kDa PRL on the well-known angiogenic effect of bFGF. In addition, we have treated corneas with anti-PRL antibodies in an attempt to unmask possible PRL-like proteins involved in tissue avascularity. Antibody administration to corneas has been a valuable tool for the detection of molecules active in angiogenesis.37
Corneal angiogenesis was quantitated through measurement of densitometric (OD) values associated with stained capillaries, which also allowed a qualitative determination of the incidence of angiogenesis. These parameters provided reliable dose–response quantitations of bFGF-induced angiogenesis. Active doses of bFGF were similar to effective ones previously described in this assay.37 Addition of 16-kDa PRL reduced bFGF-induced angiogenesis by 65%, when judged by the ODs of the treated area. Moreover, 16-kDa PRL reduced by 50% the incidence of bFGF-induced angiogenesis. Sixteen-kDa PRL was active at a dose five times lower than the effective one in the CAM assay.10 This in vivo inhibitory effect of 16-kDa PRL is consistent with the previously described antiangiogenic effect of PRP,13 the placental PRL-like protein that competes for 16-kDa PRL receptor in endothelial cells.9 PRP was shown to inhibit bFGF-induced vessel growth in rat corneas.13 However, 23-kDa PRL did not modify bFGF angiogenic action. This absence of effect is to be expected because 23-kDa PRL does not bind to 16-kDa PRL receptors in endothelial cells.9 Receptors mediating the antiangiogenic actions of PRL fragments differ from the classic (cloned) PRL receptors, both structurally and functionally.9 Thus, 23-kDa PRL has been shown to be inactive in angiogenesis, in endothelial cells both in vitro and in vivo, and in the microvasculature of the CAM.8 10
The in vivo antiangiogenic action of 16-kDa PRL was originally described in the CAM.10 The CAM in the early chick embryo (days 6–9) is a rapidly growing membrane where angiogenesis actively occurs in response to multiple stimulatory factors. In the present study we showed that 16-kDa PRL inhibited the in vivo effect of a defined stimulator of angiogenesis, bFGF. Inhibition by 16-kDa PRL of the in vivo angiogenic effect of bFGF matches the reported 16-kDa PRL inhibition of bFGF-induced proliferation of endothelial cells in culture.8 10 Actually, it is known that this PRL fragment inhibits the mitogenic effect of bFGF and vascular endothelial growth factor by acting distal to their receptors and proximal to the mitogen-activated protein kinases, specifically by inhibition of the activation of Raf-1.38 39 The fact that 16-kDa PRL inhibits the in vivo effect of bFGF is consistent with the notion that PRL-like proteins can counteract the net stimulatory effect of angiogenic factors.
In an attempt to investigate whether endogenous PRLs are involved in the control of cornea avascularity, we studied the effect of neutralizing the action of endogenous PRL with PRL-directed antibodies on corneal neovascularization. The polyclonal and monoclonal PRL antibodies used reacted with both 23-kDa and 16-kDa PRL standards by ELISA and western blot. All these antibodies resulted in corneal angiogenic responses. The number of long capillary vessels was increased in the area surrounding antibody-releasing implants. Accordingly, treatment with anti-PRL antibodies was associated with higher ODs in these areas, and therefore, with a substantial stimulation of the incidence of angiogenesis. Specificity of antibody action was supported by significant differences between nonimmune rabbit and mouse IgG. However, mouse IgG produced a moderate angiogenic reaction, perhaps because of inflammation. Nevertheless, the anti-PRL monoclonal antibody induced a significant response three times higher than this control. The possibility that inflammation related to antigen–antibody interaction contributed to the angiogenic effect of anti-PRL antibodies is unlikely, because no angiogenic response followed cotreatment of corneas with BSA and anti-BSA antibodies.
The possibility that angiogenesis induced by PRL antibodies is caused by blockage of the antiangiogenic effect of endogenous PRL-like molecules, raises various intriguing issues. This novel proposal requires that PRL-like molecules, specifically PRL fragments and/or other antiangiogenic PRL forms, be present in the cornea. Although these PRLs must be determined and identified, their presence in the cornea is likely. We have recently detected immunoreactive PRL in the aqueous fluid of humans,40 and rat retinal endothelial cells have been found to express the PRL gene.36 Moreover, PRL receptors have been identified in the retina,41 and PRL has been proposed to play a role in the metamorphosis of visual pigments in amphibians.42 In addition, PRL has been shown to be synthesized by acinar cells of lacrimal glands.43 These cells express the PRL mRNA and locate PRL-like immunoreactivity in secretory vesicles.43 Consistent with this, PRL has been detected in human tears,23 and evidence has been provided to suggest that its presence in tears is under systemic hormonal control.44 Actually, it has been proposed that this hormone may have a role in the physiology of the cornea, the conjunctiva, or the conjunctiva-associated lymphoid tissue.43 In this regard, it can be hypothesized that antiangiogenic PRLs present in the cornea could originate from lacrimal PRL and thus could have an extraocular origin. Consistent with this proposal, immunocytochemistry with four well-characterized PRL receptor monoclonal antibodies, showed that receptors for prolactin seemed to be located throughout the corneal epithelium. Because PRL is known to be taken up by receptor-mediated endocytosis in several cell types,45 46 it is possible to hypothesize that epithelial cells in the cornea may bind and internalize the PRL present in tears, and thus provide an avenue for the incorporation of lacrimal PRL into this organ. Therefore, these results add support to the presence of PRL in the cornea and provide an insight into the possible extraocular origin of this hormone.
No conclusions can be drawn on the nature of the possible contribution of PRLs to cornea avascularity. Sixteen-kDa PRL may be a candidate, because it inhibits corneal neovascularization, and it reacts with all the antibodies used. Alternatively, other PRL-like proteins may be involved, as suggested by the fact that the 16-kDa PRL-directed antibodies were less active in corneal blood vessel growth than antibodies raised against the whole 23-kDa PRL molecule.
The present study provides observations consistent with the hypothesis that members of the PRL family may function as regulators of angiogenesis, specifically in the maintenance of corneal avascularity. The presence of PRL receptors in ocular tissues similar to those of the cornea further supports the eye as a novel target for PRL actions. Identification of PRL in ocular tissues and fluids and its possible ocular and/or extraocular origin are under current investigation.
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Acknowledgements |
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Footnotes |
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Supported by Grants 3662P,27950-N from the National Council of Science and Technology of Mexico, IN201895 from the National University of Mexico, 75197-554801 from The Howard Hughes Medical Institute, and RGA 96-097 from The Third World Academy of Sciences.
Submitted for publication November 24, 1998; revised March 24, 1999; accepted May 20, 1999.
Commercial relationships policy: N.
Corresponding author: Carmen Clapp, Centro de Neurobiología, Universidad Nacional Autónoma de México, Apdo Postal 1-1141, 76001 Querétaro, Qro., Mexico. E-mail: clapp{at}servidor.unam.mx
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)
or 23-kDa (•) PRL standards and anti-23-kDa PRL polyclonal antibodies
(
23-kDa PRL), monoclonal antibodies (
