Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice.
ABSTRACT To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization.
The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad). Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy. The ability of recombinant 16K hPRL expressed in E. coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC).
16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation. Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls. r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs.
Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy. 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells. These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies.
- SourceAvailable from: Joseph Augustin Martial[show abstract] [hide abstract]
ABSTRACT: We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells.Molecular Endocrinology 11/2000; 14(10):1536-49. · 4.75 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The cornea is an avascular organ, where induction of new blood vessels involves the turn-on of proangiogenic factors and/or the turn-off of antiangiogenic regulators. Prolactin (PRL) fragments of 14 kDa and 16 kDa bind to endothelial cell receptors and inhibit angiogenesis. This study was designed to determine whether antiangiogenic PRL-like molecules are involved in cornea avascularity. 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. 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. 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.Investigative Ophthalmology & Visual Science 11/1999; 40(11):2498-505. · 3.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.Molecular Endocrinology 10/2003; 17(9):1815-23. · 4.75 Impact Factor
Molecular Targeting of Antiangiogenic Factor 16K hPRL
Inhibits Oxygen-Induced Retinopathy in Mice
Hao Pan,1Ngoc-Quynh-Nhu Nguyen,2Hiroshi Yoshida,3Frauke Bentzien,3Lynn C. Shaw,1
Francoise Rentier-Delrue,2Joseph A. Martial,2Richard Weiner,3Ingrid Struman,2and
Maria B. Grant1
PURPOSE. To examine the ability and mechanism of the 16 kDa
N-terminal fragment of human prolactin (16K hPRL) in the
inhibition of abnormal retinal neovascularization.
METHODS. The 16K hPRL-encoding sequence was inserted into
an adenoviral vector (16K-Ad). Western blot analysis verified
the expression of 16K hPRL and inhibition of proliferation,
confirming functional activity of the 16K hPRL in virus-infected
adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhib-
ited retinal neovascularization in a mouse model of oxygen-
induced retinopathy. The ability of recombinant 16K hPRL
expressed in E. coli (r16K hPRL) was compared to that of
endostatin in inducing apoptosis of cultured human retinal
endothelial cells (HREC).
RESULTS. 16K was expressed in virus-infected ABAE cells and
resulted in a dose-dependent inhibition of cell proliferation.
Eyes injected with 16K-Ad showed a reduction in preretinal
neovascularization of 82.3 ? 9.3% (P ? 0.00001) when com-
pared to uninjected controls. r16K hPRL was 100 times more
potent than endostatin in inducing apoptosis in HRECs.
CONCLUSIONS. Intravitreal administration of 16K hPRL inhibited
neovascularization in the mouse model of oxygen-induced ret-
inopathy. 16K hPRL stimulated apoptosis in HRECs and inhib-
ited cell proliferation in ABAE cells. These results suggested a
potential therapeutic role for 16K hPRL in the treatment of
proliferative retinopathies. (Invest Ophthalmol Vis Sci. 2004;
(PDR), and “wet” age-related macular degeneration (ARMD),
roliferative retinopathies, which include retinopathy of
prematurity (ROP), proliferative diabetic retinopathy
are the leading causes of blindness in the western world. The
underlying etiology common to these diseases is the prolifera-
tion of aberrant blood vessels.1To date, photocoagulation is
the mainstay of management of patients with PDR. While
effective at reducing severe vision loss, there can be serious
side effects, including diminished night vision, reduced periph-
eral vision, and decreased visual acuity. Moreover, in some
patients the disease progresses despite costly maximal laser
therapy. ROP therapies are limited to laser and cryosurgery of
the avascular zone. Clearly, the development of an affordable
and efficacious drug therapy for the proliferative retinopathies
would be a major breakthrough in medicine.
Vascular endothelial growth factor (VEGF) is currently
viewed as the major effector for retinal neovascularization in all
proliferative retinopathies. One approach in inhibiting neovas-
cularization associated with eye disease is the use of antiangio-
genic peptides. This concept has been extensively tested in
animal models. The antiangiogenic peptides shown to inhibit
retinal neovascularization in rat and mouse models include:
plasminogen kringle 5 fragment,4
bospondin type 1 repeat fragments,5carboxyl-terminal frag-
ment of tryptophanyl-tRNA synthases,6and pigment epithe-
The study of inhibition of retinal vascularization by antian-
giogenic peptides was extended to the antiangiogenic factor
16K hPRL for several reasons. 16K hPRL is a naturally occur-
ring, specific antiangiogenic factor that has been evaluated in
multiple in vitro and in vivo systems.716 K hPRL is endothelial
cell specific and extremely potent. 16K hPRL inhibits VEGF-
and fibroblast growth factor-2 (FGF-2)-induced capillary endo-
thelial cell growth by blocking activation of the mitogen-acti-
vated protein kinase (MAPK) signaling cascade and induces
apoptosis by inhibiting NF-?B activation.8
An adenovirus vector expressing 16K hPRL was developed
to test the possibility that this well-characterized angiogenic
factor could be used to prevent retinal neovascularization.9
Primary cultures of human retinal endothelial cells (HREC)
were used to test the efficacy of r16K hPRL to induce apopto-
sis, a well-characterized action of antiangiogenic factors.10Pri-
mary cultures of ABAE cells were infected with the 16K-Ad to
show that sufficient 16K hPRL was expressed after infection to
inhibit endothelial cell proliferation. In vivo studies were car-
ried out in the mouse model of oxygen-induced retinopathy.11
This model is well established and resembles the human dis-
ease with extra retinal neovascularization. Increases in VEGF
and VEGF receptors are closely correlated with neovasculariza-
tion in this model.1216K-adenoviral infection of the eye dra-
matically inhibited neovascularization in the mouse model of
MATERIALS AND METHODS
Human embryonic kidney (HEK) 293 cells, adenoviral E1-transformed
HEK cells (BD Biosciences, San Diego, CA) were grown in Minimal
From the1Department of Pharmacology and Therapeutics, Uni-
versity of Florida, Gainesville, Florida;2Laboratoire de Biologie Mole ´cu-
laire et de Ge ´nie Ge ´ne ´tique, Universite ´ de Lie `ge, Lie `ge, Belgium; and
3Department of Obstetrics, Gynecology, and Reproductive Sciences,
University of California at San Francisco, San Francisco, California.
Supported by grants from The Juvenile Diabetes Research Foun-
dation International; NIH Grants EY012601 and EY007739 (MBG);
University California CRCC Grant (RW); the Fond pour la Recherche
Industrielle et Agricole (N-Q-NN); the Fond National pour la Recherche
Scientifique (IS); Te ´le ´vie, les Services Fe ´de ´raux des Affaires Scienti-
fiques, Techniques et Culturelles de Belgique (PAI5/35); Fortis Bank
Assurances, l’Universite ´ de Lie `ge (fonds spe ´ciaux); and 4C Biotech,
Submitted for publication September 10, 2003; revised January 23,
2004; accepted February 2, 2004.
Disclosure: H. Pan, None; N.-Q.-N. Nguyen, None; H. Yoshida,
None; F. Bentzien, None; L.C. Shaw, None; F. Rentier-Delrue,
None; J.A. Martial, None; R. Weiner, None; I. Struman, None; M.B.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Maria B. Grant, Department of Pharmacol-
ogy and Therapeutics, University of Florida, Gainesville, FL;
Investigative Ophthalmology & Visual Science, July 2004, Vol. 45, No. 7
Copyright © Association for Research in Vision and Ophthalmology
Essential Medium (MEM) supplemented with 10% fetal calf serum
(FCS), 1% nonessential amino acids and 100 U/mL penicillin/strepto-
mycin and 2.5 ?g/mL fungisome.
ABAE cells were isolated as previously described.13The cells were
grown and serially passaged in low-glucose Dulbecco’s modified Ea-
gle’s medium (DMEM) containing 10% FCS and 100 U/mL penicillin/
streptomycin and 2.5 ?g/mL fungisome (10% FCS/DMEM medium).
Recombinant human FGF-2 (Promega, Madison, WI) was added (1
ng/mL) to the culture every other day. Confluent cells corresponding
to passages 7 to 12 were used in the experiments.
Primary cultures of HRECs were prepared and maintained in
DMEM/Hams F12 with 10% plasma-derived serum (PDS), 5 ?g/mL
transferrin, 2 ?g/mL selenium, 1 ?g/mL insulin, 0.584 mg/mL glu-
tamine, and 15 mg/mL endothelial growth supplement (Sigma, St.
Louis, MO)10and cells in passages 3 to 6 were used in the studies. The
identity of endothelial cells in cultures was validated by demonstrating
endothelial cell incorporation of fluorescence-labeled acetylated LDL
and by flow cytometry analysis, as previously described.14To maintain
purity of HRECs, the cells were grown in PDS, which is free of
platelet-derived growth factor and does not promote the growth of
pericytes (the contaminating cell type in these preparations). r16K
hPRL was produced as previously described.15Endotoxin levels in the
preparations were 100 times lower than amounts needed to mimic the
actions of 16K hPRL. Endostatin was obtained from Sigma.
Construction and Generation of
All recombinant adenovirus was prepared using Adeno-X expression
system purchased from BD Biosciences. The cDNA encoding 16K hPRL
was excised from the pRC/CMV-hPRL 16K vector (Fig. 1A).9This
construct was engineered to produce a secreted peptide consisting of
the first 139 amino acids of the PRL. Codon 140 was replaced by a stop
codon using PCR-directed mutagenesis.16Cysteine 58 (TGG) was re-
placed by a serine (TCC) to avoid non-native disulfide bridges. The
rabbit ?-globin intron was inserted 5? of the 16K hPRL signal peptide.
The restriction sites NheI and KpnI were added respectively to 5? and
3? ends by PCR. The sense primer was 5?-GGGCTAGCGATCCT-
GAGAACTTCAGGGT-3? and the antisense primer, containing a stop
codon (underlined), was 5?-CGGTACC[UNDERLN]TCA[/UNDERLN]-
A replication defective adenoviral vector based on human Ad 5
serotype was used for the study. The 16K hPRL coding sequence was
inserted in the expression cassette into the adenoviral shuttle vector,
pShuttle, at the NheI and KpnI restriction sites and was verified by
sequencing. The cytomegalovirus (CMV) promoter drives the expres-
sion of the expression cassette. The expression cassette was then
excised from pShuttle and inserted to Adeno-X Viral DNA via the I-CeuI
and PI-SceI restriction sites. The recombinant Adeno-X vector (16K-Ad)
was propagated in HEK 293 cells, purified by cesium chloride density
gradient ultracentrifugation, and stored at ?80°C. Adenovirus vector
titer was determined by tissue culture infectious dose 50 (TCID50).
Adenovirus vector carrying an empty expression cassette was used as
Western Blot Analysis
ABAE cells (1.5 ? 104) were plated in 24-well plates in 0.5 mL 10%
FCS/DMEM medium. On the following day the cells were infected with
16K-Ad or Null-Ad at a multiplicity of infection (MOI) of 200 pfu/cell,
and the conditioned media were collected 48 hours postinfection.
Since the cDNA for 16K hPRL contains a signal peptide, 16K hPRL is
secreted into the culture medium. Conditioned media (20 ?L) was
heated in sample buffer containing 1% 2-mercaptoethanol for 5 min-
utes at 95°C and separated by SDS/PAGE 15%/4% before being trans-
ferred to a nitrocellulose membrane (Hybond ECL; Amersham, Piscat-
away, NJ). The membrane was saturated overnight in TBS-8% dry milk,
followed by 1 hour of incubation with a 1/200 dilution of polyclonal
antiserum directed against 16K hPRL (SB30) and 1 hour of incubation
with a 1/5000 dilution peroxidase-conjugated goat antirabbit serum
(Gamma; BioWhittaker, Verviers, Belgium). 16K hPRL detection was
then carried out by chemiluminescence using the ECL Plus kit (Amer-
sham). For the deglycosylation experiments, the N-Glycosidase F de-
glycosylation Kit was used according to the manufacturer’s instruc-
tions (Roche, Palo Alto, CA). Briefly, 20 ?L of conditioned medium
from Ad-16K hPRL-infected ABAE cells were incubated with 10 ?L of
denaturation buffer for 3 minutes at 95°C. Twenty microliter of reac-
tion buffer and 15 ?L of N-glycosidase F (1.2 units/?L) were added and
incubated for 1 hour at 37°C. The deglycosylated product was precip-
itated with two volumes of cold methanol and analyzed by Western
blotting as described above.
All animal procedures used were in agreement with the NIH Guide for
the Care and Use of Laboratory Animals, with the ARVO Statement
for the Use of Animals and with institutional guidelines, and approved
by the University of Florida Institutional Animal Care and Use Commit-
tee. Timed pregnant C57BL/6J mice were purchased from Jackson
Laboratories (Bar Harbor, ME).
Intravitreal Injection into Mouse Model
In the mouse model of oxygen-induced retinopathy,11mice at postna-
tal day (P)7 are placed with their nursing dams in a 75% oxygen
vector: (A) Schematic representation of the 16K hPRL expression
vector, the I-CeuI/PI-SceI restriction fragment cloned in adenovirus5
genome is shown. CMV IE, human cytomegalovirus immediate early
promoter/enhancer; ?-globin intron, rabbit ?-globin intron; 16K hPRL,
16K hPRL coding sequence including the signal peptide; polyA, SV40
polyadenylation signal. (B) Analysis of protein expression by Western
blot analysis from ABAE cells uninfected or infected at an MOI of 200
pfu/cell with 16K-Ad or Null-Ad. Lane 1: E. coli 16K hPRL; lane 2:
protein from cells infected with 16K-Ad; lane 3: protein from cells
infected with Null-Ad. Twenty ?L of conditioned media were sub-
jected to SDS-PAGE and probed with an anti-PRL antibody. The band
corresponding to 16K hPRL (16 kDa) is labeled. Thirty nanograms of
recombinant 16K hPRL produced in E. coli was loaded as a control. (C)
Analysis of glycosylation. Lane 4: E. coli 16K hPRL; lane 5: protein
from cells infected with 16K-Ad; lane 6: deglycosylated protein from
cells infected with 16K-Ad. Twenty microliters of conditioned media
from 16K-Ad-infected ABAE cells were incubated or not with N-glyco-
sidase and subjected to Western blotting as in B. The band correspond-
ing to 16K hPRL (16 kDa) is labeled.
Construction and characterization of 16K hPRL adenovirus
2414Pan et al.
IOVS, July 2004, Vol. 45, No. 7
atmosphere for 5 days. On return to normal air, these mice develop
retinal neovascularization, with peak development occurring 5 days
(P17) after their return to normoxia. Newborn mice (P1) were chilled
on ice for 30 to 45 seconds to reduce their activity. Using a 32G needle
attached to a 10 ?L Hamilton syringe, 0.5 ?L of a mixture containing
the adeno-construct and 0.1% (v/v) fluorescein sodium (Angiofluor;
Alliance Pharmaceuticals, Richmond, TX) was injected into the vitre-
ous OD (Ad-16k ? 9 ? 109pfu/mL) or OS (Ad-Null ? 2.2 ? 1010
pfu/mL); 0.1% fluorescein sodium was used to visualize the injection.
After the fifth day following return to normoxia, the animals were
killed, the eyes removed, fixed in 4% paraformaldehyde, and embed-
ded in paraffin. Three hundred serial sections (6 ?m) were cut sagitally
through the cornea parallel to the optic disc. Every thirtieth section
was placed on a slide and stained with hematoxylin-eosin (H&E). This
resulted in ten sections from each eye being scored in a masked fashion
using light microscopy to count endothelial nuclei extending beyond
the inner limiting membrane into the vitreous as previously de-
scribed.11The efficacy of treatment with a particular plasmid was then
calculated as the percent average nuclei per section in the injected eye
versus the uninjected eye.
For qualitative analysis the retinas from some mice were dissected
and flatmounted, as described by D’Amato et al.17These animals were
perfused with 3 mL of fluorescein-isothiocynate (FITC)-dextran (FD-
2000S; Sigma) in 4% paraformaldehyde. The eyes were enucleated and
the retinas removed and flatmounted for fluorescence microscopic
In Vitro Endothelial Cell Proliferation Assay
On day 1, ABAE cells were plated at a density of 1.5 ? 104cells per
well in a 24-well plate, in 0.5 mL 10% FBS/DMEM medium. On day 2,
cells were infected for 4 hours with increasing MOI of adenovirus
vector. On day 3, the cells were stimulated with FGF-2 (1 ng/mL) for
16 hours. On day 4, the cells were incubated with 5 ? 106cpm of
thymidine for 4 hours,18washed in 5% trichloroacetic acid, solubilized
in NaOH, and counted as previously described.16
DNA Fragmentation ELISA Assay
The levels of mono- and oligonucleosomal DNA released in the cytosol
of apoptotic cells were measured using the Cell Death Detection ELISA
kit (Boehringer Mannheim, Mannheim, Germany). This is a quantitative
sandwich–enzyme-immunoassay using antibodies against DNA and
histones. By isolating nucleosomes in the cytosol with antibodies to
histones, this assay specifically measures apoptosis and not necrosis.
For these studies, 100,000 HREC were plated per well in 12-well plates.
Twenty-four hours later, r16K hPRL, r23K hPRL, endostatin or endo-
toxin were added for 24 hours. The Cell Death Detection assay was
performed on cytosolic fractions prepared using reagents supplied
with the kit according to instructions. Levels of DNA fragmentation
were expressed as an enrichment factor, calculated by dividing the
absorbance of a given sample by the absorbance of the corresponding
10% FCS control.
cell proliferation by direct infection
of 16K hPRL adenovirus vector.
ABAE cells were infected with indi-
cated MOI (pfu/cell) of 16K-Ad or
Null-Ad and treated, 24 hours later,
with FGF-2 (1 ng/mL). Proliferation
was assessed by 3H-thymidine incor-
poration 48 hours postinfection. The
data are expressed as percentages of
the stimulation obtained with FGF-2
alone, 0% being the basal growth
level. Data are the mean of triplicate
wells; bars ? SE. The experiments
were repeated at least three times,
with similar results.
Inhibition of endothelial
oxygen-induced mouse model of retinopathy. The y-axis indicates the
average number of nuclei of preretinal endothelial cells per section.
For each mouse the right eye (OD) was injected with the adeno-
construct and the left eye was uninjected. For the 16K-Ad eyes (n ?
11) and for the Null-Ad eyes (n ? 4). Neovascularization was reduced
by 66 ? 8% (P ? 3 ? 10?8) in the 16K-Ad injected eyes, while there
was no significant reduction in the Null-Ad injected eyes (P ? 0.3).
Results of the injection of the adeno-constructs in the
IOVS, July 2004, Vol. 45, No. 7
eyes from mice that have undergone
the oxygen-induced model of reti-
nopathy. (A) [OS] and (B) [OD]
paired eyes from a mouse that re-
ceived no injection OS and was in-
jected OD with the Ad-Null virus. (C)
[OS] and (D) [OD] paired eyes from a
mouse that received no injection OS
and was injected OD with the Ad-
16K virus. Only the eye injected with
the Ad-16K virus shows significant
reduction in preretinal nuclei (*).
Original magnification, ?5.
Retinal cross-sections of
mice perfused with FITC-dextran.
(A) Retina from an uninjected mouse
eye on postnatal day 17 after the OIR
timecourse; (B) Retina from a mouse
eye injected with the 16K-Ad con-
struct on postnatal day 17 after the
OIR timecourse; (C) Retina from an
uninjected normoxic mouse eye on
postnatal day 17; (D) Retina from a
normoxic eye injected with the
16k-Ad construct on postnatal day
Flatmounted retinas from
2416Pan et al.
IOVS, July 2004, Vol. 45, No. 7
All statistical analysis was done using the student t-test in Microsoft
Excel. P-values ? 0.001 are indicated with an asterisk on the graphs.
16K-Ad Infected ABAE Cells Express and Secrete
Biologically Active 16K hPRL
To extend findings with 16K hPRL to an in vivo setting, a
16K-Ad vector was engineered. To validate the usefulness of
the 16K-Ad vector, ABAE cells were infected to determine that
the infected cells expressed and secreted substantial amounts
of biologically active 16K hPRL. ABAE cells were infected with
a MOI of 200 pfu/cell of 16K-Ad or Null-Ad as a control for viral
infection. ABAE cells infected with the 16K-Ad vector ex-
pressed 16K hPRL (Fig. 1A). Western blot analysis revealed a
band running higher than that seen for recombinant 16K hPRL
produced in Escherichia coli (Fig. 1B), possibly the result of
N-glycosylation of Asn31 within 16K hPRL. After deglycosyla-
tion of the conditioned media with N-Glycosidase F, the band
co-migrated with the r16K hPRL made in E. coli (Fig. 1C).
The amount of 16K hPRL produced in 16K-Ad infected
ABAE cells, as estimated by Western blot analysis, appeared
sufficient to obtain a biologically active concentration to in-
hibit cell proliferation. Consistent with this, FGF-2-induced
ABAE proliferation was inhibited in a dose-dependent manner
with increasing MOI of 16K-Ad while Null-Ad had no effect
Reduced Neovascularization by 16K hPRL in
Mouse Model of Oxygen-Induced Retinopathy
The oxygen-induced model of retinopathy11was used to test
the effect of the 16K hPRL in vivo. Mice, at P1, were injected
in the right eye with the adeno-constructs (Null-Ad or 16K-Ad).
The extent of preretinal neovascularization was scored by
counting the average number of preretinal nuclei per section
of eye (Fig. 3). Injection of the Null-Ad construct exhibited no
significant difference from control (P ? 0.41), as expected.
The 16K-Ad construct showed a reduction in the average
number of preretinal nuclei per section when compared to
control left eyes of 66 ? 8% (P ? 3 ? 10?8). Cross-sections
from representative eyes (Fig. 4) confirm the reduction in
preretinal vessels in mice injected with the16K-Ad construct.
Perfusion of uninjected eyes from mice that underwent the
time course of the OIR model demonstrated the leaky nature of
the preretinal blood vessels that resulted from the hypoxic
stimulus of this model (Fig. 5A). Injection of the 16K-Ad con-
struct significantly reduced the number of leaky vessels in mice
subjected to the OIR model (Fig. 5B). No difference in vascu-
lature was found between uninjected eyes (Fig 5C) and eyes
injected with the 16k-Ad construct (Fig. 5D) from mice that
had not been subjected to the OIR model.
16K hPRL-Induced Apoptosis in HRECs
Increasing concentrations of r16K hPRL progressively in-
creased the level of DNA fragmentation (Fig. 6). Although the
stimulation of DNA fragmentation was similar with 20 nM r16K
hPRL and 10 U of endotoxin, there was ?0.01 U of endotoxin
in the 20 nM r16K hPRL preparation. Endostatin (10 ?g/mL,
500 nM) gave the same response as 5 nM r16K hPRL (80
ng/mL); that is, the potency of r16K hPRL was 100 times
greater. Although intact 23 kDa prolactin (10 nM) contained
similar amounts of endotoxin to the r16K hPRL preparation, it
had no effect on DNA fragmentation, as previously reported in
human umbilical vein endothelial cells (HUVEC) and bovine
brain endothelial (BBE) cells.15
Vascular retinopathies are significant vision-threatening dis-
eases.19Occlusion of capillaries leads to retinal ischemia. At-
tempts to reperfuse areas of retinal ischemia with new, albeit
aberrant, blood vessels lead to further vascular pathology. In all
proliferative retinopathies the state of the nonperfused retina
produces angiogenic growth factors such as VEGF and FGF-2,
which stimulate this new, compensatory, blood vessel growth.
This study explored the effect of the antiangiogenic peptide
16K hPRL in both in vitro and in vivo systems relevant to the
retina. Previously it was demonstrated that 16K hPRL, but not
intact PRL, inhibits VEGF- and FGF-2-induced proliferation of a
variety of capillary endothelial cells in nM concentrations16,20
including endothelial cells isolated from the rat retina.21The
signaling pathways for the antiproliferative action of 16K hPRL
include the inhibition of VEGF- and FGF-2-induced Ras activa-
tion.22This is consistent with the idea that 16K hPRL might be
particularly efficacious in inhibiting VEGF-driven retinopathy.
The inhibition of VEGF- and FGF-2-induced proliferation
appears to be mediated through blocking of the MAPK signal-
ing pathway. Activation and tyrosine phosphorylation of the
Flk1 receptor by VEGF are not affected by the addition of 16K
hPRL23; neither is the association of the receptor with Shc, and
by recombinant 16K hPRL in HREC.
The level of DNA fragmentation (cy-
tosolic mono- and oligonucleosomes)
was measured in HREC treated with
increasing concentrations of recom-
binant 16K hPRL produced in E. coli
(5, 10, and 20 nM), 10 U of endo-
toxin or 10 ?g/mL endostatin.
Activation of apoptosis
IOVS, July 2004, Vol. 45, No. 7
Antiangiogenic Prolactin 2417
Grb2 or the recruitment of Sos.22However, activation of Ras is
inhibited. When activated by VEGF alone, Ras is converted
from the GDP- to the GTP- bound state. This conversion is
blocked when 16K hPRL is added to VEGF. The blockade of
Ras activation is consistent with the downstream inhibition of
the VEGF-induced translocation of Raf-1 to the plasma mem-
brane and the blockade of the activation of MEK and MAPK 42
16K hPRL activates apoptosis in capillary endothelial
cells,15inhibits capillary endothelial cell migration, their orga-
nization into capillaries,16and urokinase activity.24Impor-
tantly, 16K hPRL inhibits neovascularization in the chick cho-
rioallantoic assay,16rat corneal assay,25and in solid tumors in
mice.9The 16 K hPRL fragment is naturally formed in rodents
and in humans by proteolytic processing,26–28and PRL, the
precursor molecule, is expressed by retinal endothelial cells.21
16K hPRL had been used successfully in gene therapy to inhibit
the growth of the HCT116 human colon cancer cell line in
immuno-incompetent mice.916K hPRL has no direct effect on
the growth of HCT116 cells, but acts directly on the tissue
The studies described here support the efficacy of this
peptide in inhibiting growth of the relevant cell type (i.e.,
HREC). Furthermore, the 16 K hPRL fragment is unique in
having endothelial cell specificity. Thus, unlike many other
anti-angiogenic agents considered for possible therapy, the 16
K hPRL would not be expected to affect other cell types
adversely in the retina.
These data demonstrate that 16K hPRL is potent in the nM
range to activate apoptosis in HREC. 16K hPRL has also been
reported to inhibit cell proliferation of rat corneal and retinal
endothelial cells.21,25Clearly, 16K hPRL was considerably
more potent than the preparation of endostatin tested. This is
in close agreement with earlier findings that r16K hPRL stim-
ulates DNA fragmentation in BBE and HUVE cells15and that
activation of 16K hPRL-induced apoptosis is dependent on
activation of the caspase cascade and can be blocked by the
addition of caspase inhibitors. The activity of the r16K hPRL
does not involve contamination with endotoxin. Heat denatur-
ation of 16K hPRL by boiling, digestion with trypsin, or co-
incubation with the endotoxin blocker polymyxin-B has no
effect on the action of 16K hPRL. Furthermore, immuno-neu-
tralization of 16K hPRL with specific antibodies to 16K hPRL
can block its ability to induce DNA fragmentation in BBE cells.
These findings with 16K hPRL were extended to an in vivo
setting by using an engineered 16K-Ad vector. The usefulness
of the 16K-Ad vector was validated by first infecting ABAE cells
and demonstrating that the infected cells secreted substantial
amounts of biologically active 16K hPRL. However, the 16K
hPRL expressed in ABAE cells infected with the 16K-Ad vector
was highly N-glycosylated. This is in agreement with observa-
tions made in HCT116 cells stably transfected with a 16K hPRL
expression vector.9Glycosylated peptide produced in both
cells was biologically active while the 16K hPRL expressed in
E. coli as expected was not glycosylated.
In conclusion, the ability of 16 K hPRL to inhibit retinal
neovascularization was studied because it is a potent and spe-
cific antiangiogenic factor.2016K hPRL is a naturally occurring
fragment found in rodents26and in humans,27,28and the par-
ent protein prolactin is specifically expressed by retinal endo-
thelial cells.21The signaling pathways regulating the inhibition
of endothelial cell proliferation by 16K hPRL include the
unique action of inhibiting Ras activation by both VEGF and
FGF-2.29Previously, 16K hPRL was shown to activate apoptosis
in BBCE.15But the present study demonstrated that r16K hPRL
was 100 times more potent than endostatin in inducing apo-
ptosis in the most relevant cell type, HRECs.
Infection of ABAE cells with increasing amounts of 16K-Ad
inhibited cell proliferation and eyes injected with 16K-Ad
showed a dramatic reduction in preretinal neovascularization
when compared to uninjected controls, while having no effect
on animals exposed to normoxia. This combination of studies
supports 16K hPRL as an endothelial cell-specific candidate
therapeutic agent for the treatment of aberrant neovasculariza-
tion of the retina.
The authors thank Se ´bastien Tabrun for his help in proliferation studies
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