June 2001
Volume 42, Issue 7
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Retinal Cell Biology  |   June 2001
Expression of Prolactin Gene and Secretion of Prolactin by Rat Retinal Capillary Endothelial Cells
Author Affiliations
  • Alejandra Ochoa
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
  • Pável Montes de Oca
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
  • Jose Carlos Rivera
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
  • Zulma Dueñas
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
  • Gabriel Nava
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
  • Gonzalo Martínez de la Escalera
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
  • Carmen Clapp
    From the Neurobiology Center, National Autonomous University of Mexico, Querétaro, Mexico.
Investigative Ophthalmology & Visual Science June 2001, Vol.42, 1639-1645. doi:
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      Alejandra Ochoa, Pável Montes de Oca, Jose Carlos Rivera, Zulma Dueñas, Gabriel Nava, Gonzalo Martínez de la Escalera, Carmen Clapp; Expression of Prolactin Gene and Secretion of Prolactin by Rat Retinal Capillary Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2001;42(7):1639-1645.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Prolactin fragments inhibit blood vessel formation, whereas anti-prolactin antibodies induce angiogenesis in the cornea. Endothelial cells from brain capillaries and the umbilical vein produce prolactin, and this study was undertaken to determine whether retinal capillary endothelial cells could be a source for prolactin in the eye.

methods. Primary cultures of rat retinal endothelial cells were investigated for the expression of prolactin mRNA by reverse transcription–polymerase chain reaction (RT-PCR) and Southern blot analysis and by in situ hybridization. The prolactin protein was analyzed by immunocytochemistry, enzyme-linked immunoabsorbent assay, Western blot analysis, and the Nb2-cell bioassay. The effect of prolactin and the 16-kDa prolactin fragment on retinal endothelial cell proliferation was investigated, and the expression of the cloned prolactin receptor was analyzed by RT-PCR and Southern blot analysis.

results. Retinal endothelial cells expressed prolactin mRNA and full-length 23-kDa prolactin. Prolactin was observed in the cytoplasm of cells and in their conditioned medium at levels 300 times those described in endothelial cells from other vessels and species. Exogenous 16-kDa prolactin inhibited rat retinal endothelial cell proliferation, whereas 23-kDa prolactin was inactive. No evidence was obtained for the expression of the cloned prolactin receptor in these cells, but the prolactin receptor was amplified in whole rat retina.

conclusions. Endothelial cells from the microcirculation of rat retina produce and release prolactin. That the cloned prolactin receptor was not expressed in these cells argues against direct autocrine effects of prolactin. Possible paracrine effects are suggested by the expression of the prolactin receptor in retinal tissue.

Angiogenesis, the formation of new capillary blood vessels, is a leading cause of blindness worldwide and occurs in diseases such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, corneal conjunctivalization, and ocular trachoma. 1 Ocular angiogenesis may result from an imbalance between stimulatory and inhibitory factors presumed to occur from an elevated expression of local angiogenic factors induced by hypoxia. 2 3  
Various angiogenic factors have been proposed to mediate vasoproliferative eye diseases, including basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1 and, most importantly, vascular endothelial growth factor (VEGF). 4 5 6 7 However, the imbalance responsible for pathologic angiogenesis may also result from downregulation of inhibitors of neovascularization. 8 9 10  
Several of the described inhibitors of angiogenesis are fragments of larger proteins, including angiostatin, a 36-kDa internal fragment of plasminogen 11 ; endostatin, the 20-kDa C-terminal fragment of collagen XVIII 12 ; an internal fragment of platelet factor 4 13 ; fragments of laminin 14 ; peptides derived from thrombospondin 15 ; and the 16-kDa and 14-kDa N-terminal fragments of prolactin (PRL). 16 17  
PRL is a pleiotropic protein that acts on functions that range from reproduction and osmoregulation to immunomodulation and angiogenesis. 18 PRL fragments of 16 and 14 kDa appear to be produced by a cathepsin-D–like protease 19 and have been reported in the anterior and posterior lobes of the pituitary gland and in the circulation. 17 20 These PRL fragments act as potent antiangiogenic factors in vivo and in vitro, inhibiting endothelial cell proliferation, 16 21 and stimulating type 1 plasminogen activator inhibitor expression. 22 These inhibitory actions appear to occur through a receptor distinct from cloned PRL receptors, because specific, high-affinity, saturable binding sites for 16-kDa PRL are found in endothelial cells. 23  
PRL fragments may be involved in the control of ocular angiogenesis. The 16-kDa PRL inhibits bFGF-induced corneal neovascularization, and implants containing anti-PRL antibodies induce a local angiogenic reaction in the cornea. 24 Similarly, PRL has been measured in the cornea and aqueous humor of the rat 25 and in the aqueous humor and subretinal fluid of patients with retinopathy of prematurity. 26 Some of this PRL may be produced locally within the eye. Reverse transcription–polymerase chain reaction (RT-PCR) has detected the expression of PRL mRNA in the retina of the rat 25 and in the vitreous fibrovascular membranes of patients with retinopathy of prematurity. 26 Endothelial cells from bovine brain capillaries and the human umbilical vein produce PRL, 27 28 and we investigated whether endothelial cells from the microcirculation of the retina secrete PRL and thus could constitute a source for ocular PRL. A preliminary report of these findings has been presented. 29  
Materials and Methods
Reagents
1,1′-Dioctadecyl-1,3,3,3′,3′-tetramethylindocarbocyanine perchlorate acetylated low-density lipoprotein (Dil-Ac-LDL) was purchased from Molecular Probes, Inc. (Eugene, OR); fluorescein isothiocyanate (FITC)–conjugated Bandeiraea simplicifolia I isolectin B4 (BSI) and tetramethylrhodamine isothiocyanate (TRITC)–labeled Ulex europaeus I (UEA I) lectin from Sigma Chemical Co. (St. Louis, MO); and monoclonal antibody against human von Willebrand protein from Accurate Chemical & Scientific Corp. (Westbury, NY). VEGF was a kind gift from Napoleone Ferrara (Genentech, San Francisco, CA), and human bFGF was kindly provided by Judith A. Abraham (Scios, Inc., Mountain View, CA). Normal rabbit serum and second antibodies against mouse or rabbit IgG coupled to TRITC were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Rat 23-kDa PRL (biological grade) and rat PRL antisera (S-9 and IC-5) were donated by Albert F. Parlow of the National Hormone and Pituitary Program (NHPP, Torrance, CA). Locally produced anti-PRL antiserum was raised in rabbits against rat 23-kDa PRL standard and characterized as described. 24 The 16-kDa PRL was generated after the enzymatic proteolysis of rat 23-kDa PRL with a particulate fraction from rat mammary glands, gel filtration, and carbamidomethylation, as reported. 30  
Isolation and Culture of Rat Retinal Capillary Endothelial Cells
Rat retinal capillary endothelial cells (RRCECs) were obtained from rat retinas using a modified method described in rabbits. 31 All animals were maintained and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eyeballs were hemisected, and the vitreous placed on fibronectin-coated plates (10 μg/ml; obtained as the rest of the reagents for tissue culture from Gibco BRL, Rockville, MD). Retinas cut into small pieces were placed in the vitreous-containing plates and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), porcine heparin (100 μg/ml), bFGF (2 ng/ml), and penicillin-streptomycin (100 U/ml). Because endothelial cells are quick to adhere, their selection versus other cell types was favored by frequent changes of culture medium—initially, 3 hours after the onset of culture and then every 12 hours. Retinal explants were removed on the third day of culture. On days 7 through 10, round cells forming patches with a cobblestone appearance were picked up using a micropipette containing 0.025% trypsin and transferred to a 48-well plate coated with fibronectin. The cells were grown and subsequently split 1:3. Typically, three to six rats (250 g each) provided enough material for one 60-mm plate. Cells were stored frozen by passage 4 or replated for experimental use between the 5th and the 12th passages. 
RRCEC Proliferation
RRCECs (2.5 × 103 cells/15-mm well) were cultured in serum-free Opti-MEM (Gibco BRL), except for VEGF proliferation experiments in which the Human Endothelial-Serum Free Medium System (Gibco BRL) supplemented with epidermal growth factor (EGF, 10 ng/ml) was used. Incubations were for 48 hours, with the growth factors or PRLs added twice: once at the time of seeding the cells and once again 24 hours later. At the end of the incubation, cells were pulsed for 20 hours with 0.6 μCi[ 3H]-thymidine/15-mm well, and[ 3H]-thymidine incorporation into DNA was measured as an index of cell proliferation. 21  
Immunocytochemistry, Lectin-Binding and Dil-Ac-LDL Uptake
RRCEC grown on glass coverslips previously coated with fibronectin in Opti-MEM were washed with PBS and fixed in 4% formaldehyde-PBS for 10 minutes, at room temperature (RT). Immunocytochemistry was performed as described 28 using a monoclonal antibody against the von Willebrand factor (1μ g/ml), or anti-PRL antiserum (1:1000; IC-5) and a 1:100 dilution of second antibodies coupled to rhodamine. For lectin-binding experiments, cells were incubated for 30 minutes with either BSI (25μ g/ml) or UEA-I (100 μg/ml) in PBS supplemented with 0.1 mg/ml CaCl2 and MgCl2, as described. 32 33 For Dil-Ac-LDL uptake, live cells on coverslips were incubated for 4 hours with Dil Ac-LDL (10 μg/ml) in 10% FBS-DMEM, as indicated, 34 and fixed as for immunocytochemistry. In all cases, cells were coverslipped using an anti-fade kit (Molecular Probes, Inc.) and examined under an epifluorescence microscope (model BX60; Olympus, Lake Success, NY). 
Endothelial Cell Networks
Formation of cell networks was investigated by plating RRCECs within type I collagen gels (Vitrogen 100; Collagen Corp., Palo Alto, CA), as reported. 16  
Reverse Transcription–Polymerase Chain Reaction
RT-PCR and Southern blot analysis were performed essentially as described. 35 For rat PRL detection, four primers complementary to exons 2 to 5 of the rat PRL gene, 30 cycles, and an annealing temperature of 65°C were used, as previously indicated. 17 For rat PRL receptors, primers and conditions were as previously reported. 36 Briefly, a common forward primer (5′-ATCCTGGGACAGATGGAGGAC-3′) and a common reverse primer (5′-ATCCACACGGTTGTGTCCTTC-3′) were used to detect the short, intermediate, and long isoforms. Reverse primers were used to specifically detect the short (5′-TGGCTGAGGCTGACAAAAGAG-3′) or long (5′-AGACAGTGGGGCTTTTCTCCT-3′) isoforms. Amplification was with 40 cycles and an annealing temperature of 56°C. 
In Situ Hybridization
Sense and antisense probes were transcribed in vitro from linearized plasmids (pcDNA3; Invitrogen, Carlsbad, CA) containing the cDNA for rat PRL with T7 and SP6 polymerases and labeled with digoxygenin-uridine triphosphate (Boehringer-Mannheim, Mannheim, Germany). RRCECs grown on fibronectin-coated coverslips were fixed with 4% formaldehyde, 5% acetic acid, and 0.9% NaCl-PBS at room temperature for 30 minutes. In situ hybridization was performed according to the manufacturer’s instructions. Briefly, cells were dehydrated with ethanol, washed in 100% xylene to remove residual lipids, and rehydrated. Cells were treated with 0.1% pepsin in 0.1 N HCl, postfixed with 1% formaldehyde, washed, and prehybridized for 1 hour at 37°C in hybridization buffer (4× SSC [1× 150 mM NaCl/15 mM sodium citrate, pH 7.0], 10% dextran sulfate, 1× Denhardt’s solution, 2 mM EDTA, 50% formamide, and 500 μg/ml herring sperm DNA). Probes were denatured at 80°C for 10 minutes and hybridization performed in hybridization buffer for 16 hours at 37°C. Cells were washed with 60% formamide in 0.2× SSC at 37°C and with 2× SSC at room temperature. Hybridized probe was determined by using the fluorescent antibody enhancer set for digoxigenin detection (Boehringer-Mannheim) and viewed with a microscope with an attached confocal system (PCM 2000; Nikon, Melville, NY). 
RRCEC Lysates and Conditioned Media
RRCECs (106 cells/100-mm well) were incubated for 24 hours in 10 ml serum-free Opti-MEM. Conditioned media were clarified by centrifugation (10 minutes at 1200g), concentrated 10 times (Centricon 3; Amicon, Beverly, MA) and stored at− 70°C. Cells were lysed in 1% Nonidet P-40, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1 μg/ml aprotinin, and 100 μg/ml phenylmethylsulfonyl fluoride (Sigma, Milwaukee, WI). 
Western Blot Analysis
Two micrograms of protein from RRCEC lysates and conditioned media were mixed and boiled in electrophoresis sample buffer containingβ -mercaptoethanol, and resolved in an SDS-polyacrylamide slab gel (15% acrylamide-bisacrylamide). Gels were blotted onto nitrocellulose membranes, probed with a 1:500 dilution of an anti-PRL antisera (NHPP, S-9 or locally produced), and developed using the alkaline phosphatase second antibody kit (Bio-Rad Laboratories, Hercules, CA). 
Enzyme-Linked Immunosorbent Assay
The ELISA was performed as described elsewhere, 24 using wells coated with 10 ng of 23-kDa PRL, a 1:8000 dilution of locally raised anti-PRL antiserum, and a 1:2000 dilution of horseradish peroxidase (HP)–conjugated second antibodies (Vector Laboratories, Burlingame, CA). Bound HP-conjugated antibodies were revealed by reaction with o-phenylenediamine dihydrochloride in the presence of hydrogen peroxide. Optical density was measured at 490 nm. 
Nb2-Cell Bioassay
Bioactive PRL was determined in RRCEC-conditioned media by using the Nb2-cell bioassay, as detailed previously. 37 Incubations were performed for 48 hours in the absence or presence of different dilutions of RRCEC-conditioned medium, nonconditioned medium, or 23-kDa PRL standard, with or without a 1:500 dilution of locally produced PRL antiserum. Nb2-cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl-2,5)-diphenyltetrazolium bromide (MTT) colorimetric assay. 38  
Statistical Analysis
Each experiment was an average of three or more replicates of each condition. Results are representative of three or more experiments. The data in each experiment were analyzed for statistical significance by Student’s t-test. 
Results
Characterization of RRCEC Cultures
RRCECs fulfilled various established criteria 39 for the identification of endothelial cells—that is, they formed monolayers with cobblestone morphology and showed positive immunofluorescent staining for von Willebrand protein and a strong uptake of Dil-Ac-LDL (Figs. 1A 1B) . Similarly, the RRCECs bound the endothelial cell–specific lectins BS-I and UEA-I and associated into cell networks when grown within type I collagen gels (Figs. 1C 1D 1E 1F) . Furthermore, RRCECs proliferated in response to increasing concentrations of the angiogenic factor bFGF and the specific endothelial cell mitogen VEGF (Fig. 2)
Expression of PRL mRNA by RRCECs
Total RNA from RRCECs was subjected to RT-PCR in which four combinations of primers were used with annealing sites within exons 2 to 5 of the rat PRL gene (Fig. 3A) . Amplification yielded fragments of 388, 586, 220, and 418 bp (Fig. 3B , lanes 6–9) that were consistent with the predicted sizes for the full-length PRL mRNA and similar to those amplified by the same primer combinations in the rat PRL cDNA positive control (Fig. 3B , lanes 2–5). No positive signal was detected in the absence of reverse transcriptase (Fig. 3B , lane 10) or in the negative control without RNA (Fig. 3B , lane 1). Expression of PRL mRNA in RRCECs was confirmed by in situ hybridization, using an antisense PRL RNA probe that positively labeled the perinuclear and/or nuclear areas of more than 90% of cells (Fig. 3C) . Specificity of PRL mRNA expression was confirmed by the absence of a positive reaction with the sense riboprobe (not shown). 
Expression of PRL Protein by RRCECs
Anti-PRL antiserum coupled to light immunofluorescence labeled the cytoplasm of more than 90% of RRCECs (Fig. 4A) . Specificity of immunostain was ascertained by its neutralization with 1 μM PRL and by the absence of reaction to normal rabbit serum (not shown). 
Western blot analysis probed with anti-PRL antiserum revealed a 23-kDa immunoreactive protein in both RRCEC lysates and conditioned media (Fig. 4B) . Specificity of antibody reaction was indicated by absence of protein after PRL antiserum was preabsorbed with 1 μM PRL (Fig. 4B) or after blots were probed with normal rabbit serum (not shown). ELISA determinations showed that the levels of immunoreactive PRL in media conditioned for 24 hours with RRCECs were two times those in cellular lysates (Fig. 4C)
In support of the PRL nature of the 23-kDa immunoreactive protein, RRCEC-conditioned media, but not nonconditioned media, stimulated the proliferation of Nb2 cells in a dose-dependent manner (Fig. 5B) . Stimulation by conditioned media and by PRL standard was abolished by PRL antiserum (Fig. 5A) . The level of activity in the conditioned media of RRCECs was equivalent to 10 ng/ml of 23-kDa PRL, as estimated by serial dose–response effects of the rat 23-kDa PRL standard and after correcting values for a 10× concentration factor. Accordingly, both the bioassay and the ELISA measured equivalent PRL levels in RRCEC-conditioned media. 
PRL Effects on RRCEC Proliferation
To investigate whether PRL is active on RRCECs, we tested the effect of 23-kDa and 16-kDa PRLs on RRCEC proliferation. Whereas 16-kDa PRL inhibited in a dose-dependent fashion the proliferation of RRCECs induced by bFGF, no effect followed treatment with 23-kDa PRL (Fig. 6A) . Similarly, rat 23-kDa PRL, human 23-kDa PRL, and lactogenic human growth hormone did not affect basal proliferation of RRCECs (Fig. 6B) . Consistent with the absence of effect, no evidence for the expression of the cloned PRL receptor could be obtained through RT-PCR in which primer combinations were used that were designed to amplify the long, medium, or short forms of the PRL receptor (Fig. 7 , lane 1). Conversely, amplification of total RNA isolated from whole rat retinas by using the same primers yielded a 588-bp transcript (Fig. 7 , lanes 3–6). The size of this transcript is consistent with the one predicted for the PRL receptor mRNA and is similar to that of products amplified by the same primer combination in Nb2 cells (Fig. 7 , lane 2), hypothalamus (Fig. 7 , lane 8), and the rat PRL receptor cDNA (Fig. 7 , lane 9), positive controls. 
Discussion
Endothelial cells play critical roles in a large number of physiologic and pathologic processes, such as leukocyte trafficking, inflammation, wound healing, tumor metastasis, and angiogenesis. The role of endothelial cells in these events varies between macrovascular and microvascular endothelium and is known to be affected by the anatomic location of the vascular bed. 40 41 In the present study, endothelial cells from the microcirculation of the retina produced and released PRL, a pleiotropic protein with effects on reproduction, osmoregulation, immunomodulation, and angiogenesis. 18  
Expression of PRL mRNA in RRCECs was demonstrated by the RT-PCR amplification of PRL transcripts of the size expected for the full-length PRL mRNA encoding a 23-kDa PRL, the predominant PRL isoform. Similarly, the expression of the PRL mRNA was confirmed in RRCECs by in situ hybridization. In addition, fluorescence immunocytochemistry and Western blot analysis provided evidence for the translation of PRL mRNA in RRCECs. Accordingly, the cytoplasm of RRCECs contained PRL-like antigens that associated with a 23-kDa PRL-like immunoreactive protein present in both RRCEC lysates and conditioned media. Because of its apparent molecular weight, the 23-kDa protein could correspond to native unmodified PRL. Altogether, these results indicate that retinal endothelium expresses the PRL gene and the major 23-kDa PRL isoform. 
Consistent with the release of 23-kDa PRL by retinal endothelial cells, RRCEC-conditioned medium stimulated the proliferation of Nb2 cells. Mitogenesis of the pre-T rat lymphoma Nb2 cells is dependent on lactogenic hormones, 42 and 23-kDa PRL is the ligand known to activate signal transduction by the PRL receptor in these cells. 43 In addition, the bioassay and the ELISA measured equivalent PRL concentrations in RRCEC-conditioned media. Because both assays were standardized using 23-kDa PRL (NHPP standard), the equivalent PRL values measured by the two assays further indicate that the PRL-like protein in RRCEC-conditioned media corresponds to 23-kDa PRL. 
Expression of the PRL gene in RRCECs confirms previous observations in endothelial cells from bovine brain capillaries and human umbilical veins. 27 28 However, dissimilarities in the type of PRL mRNA and protein expressed were noted between RRCECs and the other endothelial cells. Whereas RRCECs transcribed only the full-length PRL message and produced only the 23-kDa PRL isoform, the other endothelial cells express PRL mRNAs of different sizes and synthesize PRLs of 23, 21, 16, and 14 kDa. 27 28 In addition, RRCECs released more than 300 times the amount of bioactive PRL estimated to be secreted by endothelial cells from bovine brain capillaries (30 pg/ml), 27 or from human veins where PRL levels are too low to be quantitated. 28  
These differences in PRL production and secretion between rat retina and bovine brain and human umbilical cells illustrate the functional heterogeneity of endothelial cells. Functional dissimilarities between various endothelial cell types, including retinal endothelium, have been postulated both in vivo and in vitro. 41 For example, retinal endothelial cells are known to maintain in vitro some of their distinct characteristics associated with their in vivo blood–retinal barrier function 44 and stand among other endothelial cell subtypes in their ability to express VEGF under basal conditions. 45  
The functional implication of the PRL phenotype of retinal endothelium—that is, production and release of high levels of PRL, is unknown. Retinal endothelial cells may function as an ocular source for PRL, although its relation to PRL detected in the aqueous humor of rats 25 and in the aqueous humor and subretinal fluid of patients with retinopathy of prematurity 26 is unclear. PRL acts as a hormone or cytokine on functions that range from reproduction and osmoregulation to immunomodulation and angiogenesis. 18 Although antiangiogenic effects of PRL fragments are well documented, 16 17 20 21 22 23 24 the effects of 23-kDa PRL on angiogenesis are controversial. The 23-kDa PRL appears to stimulate neovascularization in late, but not in early, stages of formation of the chick chorioallantoic membrane. 16 46 Moreover, in vitro studies that failed to show 23-kDa PRL’s effects and PRL receptor expression in endothelial cells from different vessels and species 16 21 23 have been counteracted by a recent study showing that 23-kDa PRL can alter the actin cytoskeleton and adhesion properties of injured pulmonary artery endothelial cells and that these cells express the PRL receptor. 47 In this regard, the present work argues against direct effects of 23-kDa PRL on retinal endothelium, because no evidence was obtained for the expression of any of the known PRL receptor isoforms in RRCECs, and 23-kDa PRL did not modify bFGF-induced proliferation or basal proliferation of these cells. 
Nevertheless, 23-kDa PRL could affect endothelial cells indirectly by acting as the molecular precursor of fragments with antiangiogenic actions. In this regard, 16-kDa PRL inhibited bFGF-induced RRCEC proliferation, and recent evidence has suggested that antiangiogenic PRL fragments are present in ocular tissues, such as the cornea. The 16-kDa PRL inhibits bFGF-induced corneal neovascularization, and implants containing anti-PRL antibodies induce a local angiogenic reaction in the cornea. 24 Moreover, PRL can be cleaved into 16-kDa PRL by vitreous proteases and 16-kDa PRL can be detected in retinal homogenates (Dueñas and Clapp, unpublished observations, 2000). 
However, endothelium-derived PRL may act as a paracrine regulator of retinal cells. Our results show the expression of the PRL receptor mRNA in the retina, and early studies have provided evidence for PRL’s effects on the retina. These include putative effects on the metamorphosis of visual pigments in amphibians 48 and the regulation of thyrotropin-releasing hormone receptors 49 and photoreceptor destruction 50 in rats. 
In this study endothelial cells from the microcirculation of the retina actively produced and released PRL. Identification of its functional role and proteolytic processing by ocular tissues warrants further investigation. 
 
Figure 1.
 
Characterization of RRCEC cultures. RRCECs obtained after sequential seeding and cloning fulfilled the following endothelial cell criteria: they formed monolayers (A), showed positive immunostaining for the von Willebrand protein (B), incorporated fluorescent Dil-Ac-LDL (C), reacted with fluorescent endothelial cell–specific BSI (D) and UEA-I (E), and formed networks when grown on type I collagen gels (F). Scale bar, (A, F) 38 μm; (BE) 21 μm.
Figure 1.
 
Characterization of RRCEC cultures. RRCECs obtained after sequential seeding and cloning fulfilled the following endothelial cell criteria: they formed monolayers (A), showed positive immunostaining for the von Willebrand protein (B), incorporated fluorescent Dil-Ac-LDL (C), reacted with fluorescent endothelial cell–specific BSI (D) and UEA-I (E), and formed networks when grown on type I collagen gels (F). Scale bar, (A, F) 38 μm; (BE) 21 μm.
Figure 2.
 
bFGF- and VEGF-induced proliferation of RRCECs. RRCECs proliferated in response to increasing concentrations of bFGF (A) and the specific endothelial cell mitogen VEGF (B). Data are mean ± SEM of triplicate determinations. *P < 0.05 versus basal proliferation.
Figure 2.
 
bFGF- and VEGF-induced proliferation of RRCECs. RRCECs proliferated in response to increasing concentrations of bFGF (A) and the specific endothelial cell mitogen VEGF (B). Data are mean ± SEM of triplicate determinations. *P < 0.05 versus basal proliferation.
Figure 3.
 
Expression of PRL mRNA in RRCECs. (A) Schematic representation of expected PCR products using primers (arrows) complementary to exons 2 to 5 of the PRL gene. The predicted sizes of PCR products for each primer combination are given in base pairs. (B) Southern blot analysis of PCR products from reverse transcribed total RNA from RRCECs (lanes 6–9) amplified (30 cycles) with the four combinations of PRL primers shown in (A). Similar PCR products were amplified from PRL cDNA (lanes 2–5). Negative controls were without RNA (lane 1) and without reverse transcriptase (lane 10). (C) Presence of the PRL mRNA in RRCECs as revealed by in situ hybridization and confocal microscopy. Scale bar, (Ca) 6 μm; (Cb) 30 μm.
Figure 3.
 
Expression of PRL mRNA in RRCECs. (A) Schematic representation of expected PCR products using primers (arrows) complementary to exons 2 to 5 of the PRL gene. The predicted sizes of PCR products for each primer combination are given in base pairs. (B) Southern blot analysis of PCR products from reverse transcribed total RNA from RRCECs (lanes 6–9) amplified (30 cycles) with the four combinations of PRL primers shown in (A). Similar PCR products were amplified from PRL cDNA (lanes 2–5). Negative controls were without RNA (lane 1) and without reverse transcriptase (lane 10). (C) Presence of the PRL mRNA in RRCECs as revealed by in situ hybridization and confocal microscopy. Scale bar, (Ca) 6 μm; (Cb) 30 μm.
Figure 4.
 
Expression of PRL in RRCECs. (A) PRL expression was detected in more than 90% of RRCECs after fluorescence immunocytochemistry with anti-PRL antibodies. (B) Western blot analysis probed with anti-PRL antibodies showing a 23-kDa immunoreactive protein in cell lysates (CL) and conditioned medium (CM) from RRCECs. Specificity of antibody reaction was confirmed by absence of positive signal after preabsorption of anti-PRL antibodies with 1 μM PRL. (C) ELISA determination of PRL in CL and CM of RRCECs cultured for 24 hours. Data are mean ± SEM of three independent experiments. MW, molecular weight standard. Scale bar, 30 μm.
Figure 4.
 
Expression of PRL in RRCECs. (A) PRL expression was detected in more than 90% of RRCECs after fluorescence immunocytochemistry with anti-PRL antibodies. (B) Western blot analysis probed with anti-PRL antibodies showing a 23-kDa immunoreactive protein in cell lysates (CL) and conditioned medium (CM) from RRCECs. Specificity of antibody reaction was confirmed by absence of positive signal after preabsorption of anti-PRL antibodies with 1 μM PRL. (C) ELISA determination of PRL in CL and CM of RRCECs cultured for 24 hours. Data are mean ± SEM of three independent experiments. MW, molecular weight standard. Scale bar, 30 μm.
Figure 5.
 
Secretion of bioactive PRL by rat RRCECs. (A) Proliferation of Nb2 cells in response to PRL (NIH standard) alone or together with anti-PRL antibodies. (B) Proliferation of Nb2 cells in response to RRCEC-conditioned medium (CM) was blocked by anti-PRL antibodies to levels similar to those induced by nonconditioned medium (NCM). CM and NCM were concentrated 10-fold. Proliferation was determined by a colorimetric assay, followed by optical density measurement at 595 nm. Data are mean ± SEM of triplicate determinations. *P < 0.05 versus cells without PRL or CM.
Figure 5.
 
Secretion of bioactive PRL by rat RRCECs. (A) Proliferation of Nb2 cells in response to PRL (NIH standard) alone or together with anti-PRL antibodies. (B) Proliferation of Nb2 cells in response to RRCEC-conditioned medium (CM) was blocked by anti-PRL antibodies to levels similar to those induced by nonconditioned medium (NCM). CM and NCM were concentrated 10-fold. Proliferation was determined by a colorimetric assay, followed by optical density measurement at 595 nm. Data are mean ± SEM of triplicate determinations. *P < 0.05 versus cells without PRL or CM.
Figure 6.
 
Modulation of RRCEC proliferation by 16-kDa but not by 23-kDa PRL. (A) Effect of 16-kDa and 23-kDa PRL on bFGF-induced proliferation of RRCECs. RRCECs were cultured in the absence (▵) or presence (○) of bFGF alone or together with increasing concentrations of rat 16-kDa PRL (•) and rat 23-kDa PRL (▴). (B) Absence of effect of increasing concentrations of rat 23-kDa PRL (▴), human 23-kDa PRL (▪), and human growth hormone (♦) on RRCEC basal proliferation. Data are mean ± SEM of triplicate determinations.* P < 0.05 versus bFGF alone.
Figure 6.
 
Modulation of RRCEC proliferation by 16-kDa but not by 23-kDa PRL. (A) Effect of 16-kDa and 23-kDa PRL on bFGF-induced proliferation of RRCECs. RRCECs were cultured in the absence (▵) or presence (○) of bFGF alone or together with increasing concentrations of rat 16-kDa PRL (•) and rat 23-kDa PRL (▴). (B) Absence of effect of increasing concentrations of rat 23-kDa PRL (▴), human 23-kDa PRL (▪), and human growth hormone (♦) on RRCEC basal proliferation. Data are mean ± SEM of triplicate determinations.* P < 0.05 versus bFGF alone.
Figure 7.
 
Expression of PRL receptor mRNA in whole rat retinas. Southern blot analysis of PCR products using primers complementary to the three forms of the rat PRL receptor cDNA. A 588-bp transcript was amplified from reverse-transcribed total RNA from four different rat retinas (lanes 3–6) but not from rat retinal capillary endothelial cells (lane 1). The 588-bp transcript was also amplified from Nb2 cells (lane 2), rat hypothalamus (lane 8), and PRL receptor cDNA (lane 9) as positive controls. Negative control was without RNA (lane 7).
Figure 7.
 
Expression of PRL receptor mRNA in whole rat retinas. Southern blot analysis of PCR products using primers complementary to the three forms of the rat PRL receptor cDNA. A 588-bp transcript was amplified from reverse-transcribed total RNA from four different rat retinas (lanes 3–6) but not from rat retinal capillary endothelial cells (lane 1). The 588-bp transcript was also amplified from Nb2 cells (lane 2), rat hypothalamus (lane 8), and PRL receptor cDNA (lane 9) as positive controls. Negative control was without RNA (lane 7).
The authors thank Fernando López-Barrera, Olivia Vázquez, and Pilar Galarza for expert technical assistance. 
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Figure 1.
 
Characterization of RRCEC cultures. RRCECs obtained after sequential seeding and cloning fulfilled the following endothelial cell criteria: they formed monolayers (A), showed positive immunostaining for the von Willebrand protein (B), incorporated fluorescent Dil-Ac-LDL (C), reacted with fluorescent endothelial cell–specific BSI (D) and UEA-I (E), and formed networks when grown on type I collagen gels (F). Scale bar, (A, F) 38 μm; (BE) 21 μm.
Figure 1.
 
Characterization of RRCEC cultures. RRCECs obtained after sequential seeding and cloning fulfilled the following endothelial cell criteria: they formed monolayers (A), showed positive immunostaining for the von Willebrand protein (B), incorporated fluorescent Dil-Ac-LDL (C), reacted with fluorescent endothelial cell–specific BSI (D) and UEA-I (E), and formed networks when grown on type I collagen gels (F). Scale bar, (A, F) 38 μm; (BE) 21 μm.
Figure 2.
 
bFGF- and VEGF-induced proliferation of RRCECs. RRCECs proliferated in response to increasing concentrations of bFGF (A) and the specific endothelial cell mitogen VEGF (B). Data are mean ± SEM of triplicate determinations. *P < 0.05 versus basal proliferation.
Figure 2.
 
bFGF- and VEGF-induced proliferation of RRCECs. RRCECs proliferated in response to increasing concentrations of bFGF (A) and the specific endothelial cell mitogen VEGF (B). Data are mean ± SEM of triplicate determinations. *P < 0.05 versus basal proliferation.
Figure 3.
 
Expression of PRL mRNA in RRCECs. (A) Schematic representation of expected PCR products using primers (arrows) complementary to exons 2 to 5 of the PRL gene. The predicted sizes of PCR products for each primer combination are given in base pairs. (B) Southern blot analysis of PCR products from reverse transcribed total RNA from RRCECs (lanes 6–9) amplified (30 cycles) with the four combinations of PRL primers shown in (A). Similar PCR products were amplified from PRL cDNA (lanes 2–5). Negative controls were without RNA (lane 1) and without reverse transcriptase (lane 10). (C) Presence of the PRL mRNA in RRCECs as revealed by in situ hybridization and confocal microscopy. Scale bar, (Ca) 6 μm; (Cb) 30 μm.
Figure 3.
 
Expression of PRL mRNA in RRCECs. (A) Schematic representation of expected PCR products using primers (arrows) complementary to exons 2 to 5 of the PRL gene. The predicted sizes of PCR products for each primer combination are given in base pairs. (B) Southern blot analysis of PCR products from reverse transcribed total RNA from RRCECs (lanes 6–9) amplified (30 cycles) with the four combinations of PRL primers shown in (A). Similar PCR products were amplified from PRL cDNA (lanes 2–5). Negative controls were without RNA (lane 1) and without reverse transcriptase (lane 10). (C) Presence of the PRL mRNA in RRCECs as revealed by in situ hybridization and confocal microscopy. Scale bar, (Ca) 6 μm; (Cb) 30 μm.
Figure 4.
 
Expression of PRL in RRCECs. (A) PRL expression was detected in more than 90% of RRCECs after fluorescence immunocytochemistry with anti-PRL antibodies. (B) Western blot analysis probed with anti-PRL antibodies showing a 23-kDa immunoreactive protein in cell lysates (CL) and conditioned medium (CM) from RRCECs. Specificity of antibody reaction was confirmed by absence of positive signal after preabsorption of anti-PRL antibodies with 1 μM PRL. (C) ELISA determination of PRL in CL and CM of RRCECs cultured for 24 hours. Data are mean ± SEM of three independent experiments. MW, molecular weight standard. Scale bar, 30 μm.
Figure 4.
 
Expression of PRL in RRCECs. (A) PRL expression was detected in more than 90% of RRCECs after fluorescence immunocytochemistry with anti-PRL antibodies. (B) Western blot analysis probed with anti-PRL antibodies showing a 23-kDa immunoreactive protein in cell lysates (CL) and conditioned medium (CM) from RRCECs. Specificity of antibody reaction was confirmed by absence of positive signal after preabsorption of anti-PRL antibodies with 1 μM PRL. (C) ELISA determination of PRL in CL and CM of RRCECs cultured for 24 hours. Data are mean ± SEM of three independent experiments. MW, molecular weight standard. Scale bar, 30 μm.
Figure 5.
 
Secretion of bioactive PRL by rat RRCECs. (A) Proliferation of Nb2 cells in response to PRL (NIH standard) alone or together with anti-PRL antibodies. (B) Proliferation of Nb2 cells in response to RRCEC-conditioned medium (CM) was blocked by anti-PRL antibodies to levels similar to those induced by nonconditioned medium (NCM). CM and NCM were concentrated 10-fold. Proliferation was determined by a colorimetric assay, followed by optical density measurement at 595 nm. Data are mean ± SEM of triplicate determinations. *P < 0.05 versus cells without PRL or CM.
Figure 5.
 
Secretion of bioactive PRL by rat RRCECs. (A) Proliferation of Nb2 cells in response to PRL (NIH standard) alone or together with anti-PRL antibodies. (B) Proliferation of Nb2 cells in response to RRCEC-conditioned medium (CM) was blocked by anti-PRL antibodies to levels similar to those induced by nonconditioned medium (NCM). CM and NCM were concentrated 10-fold. Proliferation was determined by a colorimetric assay, followed by optical density measurement at 595 nm. Data are mean ± SEM of triplicate determinations. *P < 0.05 versus cells without PRL or CM.
Figure 6.
 
Modulation of RRCEC proliferation by 16-kDa but not by 23-kDa PRL. (A) Effect of 16-kDa and 23-kDa PRL on bFGF-induced proliferation of RRCECs. RRCECs were cultured in the absence (▵) or presence (○) of bFGF alone or together with increasing concentrations of rat 16-kDa PRL (•) and rat 23-kDa PRL (▴). (B) Absence of effect of increasing concentrations of rat 23-kDa PRL (▴), human 23-kDa PRL (▪), and human growth hormone (♦) on RRCEC basal proliferation. Data are mean ± SEM of triplicate determinations.* P < 0.05 versus bFGF alone.
Figure 6.
 
Modulation of RRCEC proliferation by 16-kDa but not by 23-kDa PRL. (A) Effect of 16-kDa and 23-kDa PRL on bFGF-induced proliferation of RRCECs. RRCECs were cultured in the absence (▵) or presence (○) of bFGF alone or together with increasing concentrations of rat 16-kDa PRL (•) and rat 23-kDa PRL (▴). (B) Absence of effect of increasing concentrations of rat 23-kDa PRL (▴), human 23-kDa PRL (▪), and human growth hormone (♦) on RRCEC basal proliferation. Data are mean ± SEM of triplicate determinations.* P < 0.05 versus bFGF alone.
Figure 7.
 
Expression of PRL receptor mRNA in whole rat retinas. Southern blot analysis of PCR products using primers complementary to the three forms of the rat PRL receptor cDNA. A 588-bp transcript was amplified from reverse-transcribed total RNA from four different rat retinas (lanes 3–6) but not from rat retinal capillary endothelial cells (lane 1). The 588-bp transcript was also amplified from Nb2 cells (lane 2), rat hypothalamus (lane 8), and PRL receptor cDNA (lane 9) as positive controls. Negative control was without RNA (lane 7).
Figure 7.
 
Expression of PRL receptor mRNA in whole rat retinas. Southern blot analysis of PCR products using primers complementary to the three forms of the rat PRL receptor cDNA. A 588-bp transcript was amplified from reverse-transcribed total RNA from four different rat retinas (lanes 3–6) but not from rat retinal capillary endothelial cells (lane 1). The 588-bp transcript was also amplified from Nb2 cells (lane 2), rat hypothalamus (lane 8), and PRL receptor cDNA (lane 9) as positive controls. Negative control was without RNA (lane 7).
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