Abstract
purpose. Bactericidal/permeability-increasing protein (BPI), an antibacterial
and lipopolysaccharide-neutralizing protein, also has an antiangiogenic
effect. To evaluate the therapeutic role of BPI in ischemic
retinopathies, the antiangiogenic activity of a human recombinant
21-kDa modified N-terminal fragment of BPI (rBPI21), which
has the biological properties of the holoprotein, and a peptidomimetic
(XMP.Z) derived from BPI were examined.
methods. The effects of rBPI21 and XMP.Z on VEGF-induced growth of
bovine retinal microvascular endothelial cells (BRECs) and on
serum-induced growth of bovine retinal pericytes (BRPs) and retinal
pigment epithelial cells (BRPECs) were evaluated by determining total
DNA content. The neonatal mouse model of retinopathy of prematurity
(ROP) was used to study the effect of XMP.Z in vivo. Intraperitoneal
injections of the peptidomimetic (10 mg/kg) were administered every 24
hours for 5 days (postnatal [P]12–P17) during induction of
neovascularization. Retinal neovascularization was evaluated using
flatmounts of fluorescein-dextran–perfused retinas and quantitated by
counting retinal cell nuclei anterior to the internal limiting
membrane.
results. VEGF (25 ng/mL) increased the total DNA per well of BRECs by 120% ±
50% (P < 0.001), which was inhibited by addition
of rBPI21 or XMP.Z, with decreases of 77% ± 15%
(P < 0.05) and 107% ± 19%
(P < 0.01) at maximum effective doses of 75 and 15μ
g/mL rBPI21 and XMP.Z, respectively. In contrast,
rBPI21 at 75 μg/mL enhanced the total DNA per well of BRP
53% ± 14% (P < 0.001) in the presence of 5%
fetal bovine serum (FBS), whereas XMP.Z enhanced BRP growth by 27% ±
7% (P < 0.001) at 5 μg/mL. In the presence of
10% FBS, rBPI21 and XMP.Z increased BRP growth by 91% ±
35% (P < 0.001) and 43% ± 18%
(P < 0.01), respectively. In the oxygen-induced
ROP neonatal mouse model, retinal neovascularization was decreased by
40% ± 16% (n = 5, P < 0.01)
when animals were treated with XMP.Z.
conclusions. Two BPI-derived compounds, rBPI21 and XMP.Z, significantly
suppressed VEGF-induced BREC growth in vitro, while conversely
enhancing the growth of BRPs, even above that induced by 20% FBS. When
tested in animals, XMP.Z also suppressed ischemia-induced retinal
neovascularization in mice. These data suggest that BPI-derived
compounds may have unique therapeutic potential for proliferative
retinal diseases such as diabetic retinopathy, if physiological levels
can be achieved in clinical settings.
Ischemic retinal diseases, such as diabetic
retinopathy, retinopathy of prematurity, and central retinal vein
occlusion, are major causes of blindness in the United
States.
1 Diabetic retinopathy remains the leading cause of
visual loss and new-onset blindness among working-age U.S.
citizens.
2 One of the earliest and most specific
histologic changes in diabetic retinopathy is the loss of
pericytes,
3 4 which leads to capillary loss and retinal
ischemia, finally resulting in extensive retinal neovascularization and
severe visual loss.
5 6 The neovascularization in ischemic
retinal diseases is thought to be mediated primarily by growth factors
such as vascular endothelial growth factor (VEGF),
7 8 9 whose expression is increased by hypoxia.
VEGF is a potent endothelial cell mitogen
10 11 12 and
vasopermeability factor
10 13 14 that mediates its effect
through high-affinity, cell-surface transmembrane receptors such as
fms-like tyrosine kinase (VEGFR1, previously Flt) and fetal liver
kinase 1 (VEGFR2, previously Flk-1).
15 16 17 18 Retinal
endothelial cells possess numerous VEGF receptors,
16 18 and a variety of retinal cells produce VEGF, including retinal pigment
epithelial cells (RPECs), pericytes, endothelial cells, Müller
cells, and astrocytes.
7 19 One of the most potent inducers
of VEGF is hypoxia, which increases VEGF mRNA expression by up to
30-fold.
7 Intraocular VEGF concentrations are increased
during periods of active proliferation,
20 and its
intraocular concentration decreases after successful laser therapy,
which induces regression of neovascularization.
20 Inhibition of VEGF activity accomplished by a variety of methods
prevents ischemia-induced retinal and iris neovascularization in animal
models.
21 22 23
Current therapies designed to control this aberrant angiogenesis, such
as panretinal photocoagulation and cryotherapy, are only partially
effective and are inherently destructive to the
retina.
24 25 VEGF inhibition has the potential to modulate
the neovascular response in a nondestructive manner and therefore may
have significant therapeutic value.
21 22
Bactericidal/permeability-increasing protein (BPI) and its derivative
compounds have antiangiogenic properties.
26 Recent reports
indicate that BPI can inhibit angiogenesis through induction of
apoptosis in human umbilical vein–derived endothelial
cells.
27 BPI is a 55-kDa cationic protein present in the
azurophillic granules of neutrophils,
28 also known as the
cationic antibacterial protein of 57 kDa (CAP57)
29 and the
bactericidal protein of 55-kDa molecular mass (BP55).
30 31 The high-affinity binding of BPI with the structurally conserved lipid
A region
32 of lipopolysaccharide (LPS, or endotoxin, a
glycolipid structural component of the bacterial cell wall) makes it
specifically bactericidal to Gram-negative organisms. Moreover, the
high-affinity interaction of BPI with lipid A also results in
inhibition of LPS-dependent biological responses in vitro and in
vivo.
33
In this study, we examined a recombinant modified 21-kDa N-terminal
fragment of human BPI (rBPI
21), which has
equivalent or greater activity than the holoprotein in bactericidal and
LPS binding assays,
32 34 and a 1.4-kDa peptidomimetic
(XMP.Z)
35 derived from BPI. These studies suggest that
rBPI
21 and XMP.Z can effectively inhibit bovine
retinal endothelial cell (BREC) growth at low doses, leading to
inhibition of angiogenesis both in vitro and in vivo. Further, both
rBPI
21 and XMP.Z appear to exhibit the unusual
and unique property of also stimulating the proliferation of bovine
retinal pericytes (BRPs).
Full-length BPI (55 kDa) and compounds derived from it are known
to have anti-infective and antiangiogenic
properties.
26 28 33 34 More recently, the full-length BPI
has been shown to induce apoptosis in human umbilical vein endothelial
cells and to partially inhibit angiogenesis in a corneal pocket
assay.
27
In the present study, we characterized two compounds derived from BPI
with regard to their actions on retinal vascular and nonvascular cells
and retinal microvessels in vivo. Both of these molecules were derived
from the aminoterminal region of the protein, because this part of the
molecule exhibits high affinity binding to heparin
35 and
inhibits angiogenesis in other systems.
26 One of the
compounds (rBPI
21) is a recombinant 21-kDa
protein, whereas the other (XMP.Z) is a 1.4-kDa peptidomimetic. The
results from the mitogenic assays in retinal endothelial cells shows
that both rBPI
21 and XMP.Z are able to inhibit
VEGF-induced endothelial cell proliferation. The median effective doses
(ED
50) for rBPI
21 and XMP.Z
are similar (between 2 and 5 μM). This is a much higher effective
dose than that for several other inhibitors of angiogenesis such as
thrombospondin-1 (ED
50, 0.5 nM), endostatin
(ED
50, 3 nM),
41 angiostatin
(ED
50, 1 nM),
42 43 and PEDF
(ED
50, 0.4 nM).
43 These other
antiangiogenic factors appear to mediate their actions by binding to
high-affinity cellular receptors, which typically bind in the nanomolar
range. In contrast, the mechanism(s) of antiangiogenic action for the
BPI-derived compounds is not yet fully understood but could involve
binding to growth factors directly or to cellular receptors that have
not been reported.
Antiangiogenic effects of XMP.Z are confirmed in the neonatal hyperoxia
model, which shows a 40% reduction in retinal neovascularization.
These results indicate that XMP.Z has potent antiangiogenic properties.
Because retinal neovascularization in this model may involve angiogenic
factors other than VEGF, including other non–heparin-binding growth
factors such as insulin-like growth factors (IGFs), complete inhibition
of angiogenesis may be difficult. Further studies are therefore needed
to determine whether greater antiangiogenic effects can be achieved
with higher doses of XMP.Z. In addition, correlative studies between
plasma levels of XMP.Z and its retinal antiangiogenic effects are also
needed. Comparative studies between rBPI21 and
XMP.Z may also identify whether there are other active domains within
the BPI protein that have antiangiogenic actions.
The finding that compounds derived from BPI can enhance pericyte growth
while inhibiting VEGF-induced proliferation of BRECs and exhibiting
antiangiogenic activity in vivo is surprising for several reasons. Most
antiangiogenic factors do not have growth-promoting actions on other
types of nontransformed cells. In this case,
rBPI21 and, to a lesser extent, XMP.Z clearly
enhanced pericyte proliferation, even above that observed with 20%
FBS. This is the first growth-promoting or -inhibiting factor that has
been reported to enhance pericyte growth equal to 20% FBS, while also
inhibiting endothelial cell growth. This finding is even more
interesting, because pericytes are very slow-growing cells, with a
doubling time of several days rather than hours.
These stimulatory activities are located in the amino terminal region
of BPI, as illustrated by the results with rBPI21 and, to a lesser extent, the XMP.Z peptidomimetic. However, it is
likely that the region of BPI that is responsible for the mitogenic
actions of pericytes does not coincide exactly with XMP.Z, because at
high concentration and low serum levels, XMP.Z had an inhibiting effect
on the pericyte. This was not observed with
rBPI21 or in the presence of high levels of
serum. Thus, the peptidomimetic with different structures in the same
region must be studied to better define the region of pericyte growth.
The mechanism of rBPI
21’s unexpected stimulatory
actions on pericytes is interesting, because no cellular receptor for
BPI has been identified. BPI has, however, been shown to opsonize
Gram-negative bacteria, suggesting that there may be receptors on
phagocytic cells such as polymorphonuclear cells.
44 The
antiangiogenic activity of BPI and its derivatives could also be the
result of competition between its heparin-binding domain and the many
growth factors that require heparin binding for activity. Some
additional possibilities to explain these results include: (1)
rBPI
21 binds to pericyte growth factors and
enhances their stimulatory actions, (2) rBPI
21 could bind and inactivate inhibitory factors in the serum and thereby
indirectly enhance the actions of serum, and (3)
rBPI
21 may bind to high-affinity sites on the
pericytes and induce stimulatory actions directly.
Irrespective of its mechanism of action, BPI-derived compounds clearly
exhibit antiangiogenic and anti-VEGF properties on retinal endothelial
cells without inhibiting the growth of BRPs and BRPECs. In addition,
BPI itself is stimulatory for the pericyte. Given that retinal
pericytes replicate only slowly if at all in vivo, are preferentially
lost early in the course of diabetic retinopathy, and may act to
suppress retinal endothelial cell growth, the BRP-stimulatory action of
BPI-derived compound could be important in the treatment of diabetic
retinopathy and related disorders.
45 However, the amount
of rBPI
21 needed for the suppression of
endothelial cell growth in the range of 25 to 75 μg/mL is very high
and may be difficult to achieve physiologically. The ideal approach
would be to identify a peptidomimetic in the region of
rBPI
21 that has both inhibitory actions on
endothelial cells and mitogenic actions on pericytes without any
inhibitory actions on nonendothelial cells, even at high
concentrations. Thus, the spectrum of biological properties exhibited
by the BPI-derived compounds represents a potentially ideal combination
for a therapeutic agent directed at diabetic retinopathy. However, this
remains a speculation that requires further study before it can even be
considered as a candidate for clinical trial.
Supported by National Institutes of Health Grant EY5110, Xoma (US) LLC,
Berkeley, California. KS is a recipient of the Mary K. Iacocca
Fellowship, and LPA is a recipient of the Research to Prevent Blindness
Dolly Green Scholarship.
Submitted for publication June 7, 2001; revised September 27, 2001;
accepted October 18, 2001.
Commercial relationships policy: F.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked“
advertisement” in accordance with 18 U.S.C. §1734
solely to indicate this fact.
Corresponding author: George L. King, Research Division, Joslin
Diabetes Center, One Joslin Place, Boston, MA 02215;
[email protected].
The authors thank Edward Feener and Sven-Erik Bursell of the Joslin
Diabetes Center for helpful discussions and Stephen F. Carroll of Xoma
(US) LLC for providing rBPI21 and XMP.Z and reviewing the
manuscript.
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