Increased expression of multifunctional serine protease, HTRA1, in retinal pigment epithelium induces polypoidal choroidal vasculopathy in mice

Article (PDF Available)inProceedings of the National Academy of Sciences 108(35):14578-83 · August 2011with33 Reads
DOI: 10.1073/pnas.1102853108 · Source: PubMed
Abstract
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly. Wet AMD includes typical choroidal neovascularization (CNV) and polypoidal choroidal vasculopathy (PCV). The etiology and pathogenesis of CNV and PCV are not well understood. Genome-wide association studies have linked a multifunctional serine protease, HTRA1, to AMD. However, the precise role of HTRA1 in AMD remains elusive. By transgenically expressing human HTRA1 in mouse retinal pigment epithelium, we showed that increased HTRA1 induced cardinal features of PCV, including branching networks of choroidal vessels, polypoidal lesions, severe degeneration of the elastic laminae, and tunica media of choroidal vessels. In addition, HTRA1 mice displayed retinal pigment epithelium atrophy and photoreceptor degeneration. Senescent HTRA1 mice developed occult CNV, which likely resulted from the degradation of the elastic lamina of Bruch's membrane and up-regulation of VEGF. Our results indicate that increased HTRA1 is sufficient to cause PCV and is a significant risk factor for CNV.
Increased expression of multifunctional serine
protease, HTRA1, in retinal pigment epithelium
induces polypoidal choroidal vasculopathy in mice
Alex Jones
a,1
, Sandeep Kumar
a,1
, Ning Zhang
a,2
, Zongzhong Tong
a,3
, Jia-Hui Yang
a
, Carl Watt
a
, James Anderson
a
,
Amrita
a
, Heather Fillerup
a
, Manabu McCloskey
a
, Ling Luo
a
, Zhenglin Yang
b
, Balamurali Ambati
a
, Robert Marc
a
,
Chio Oka
c
, Kang Zhang
d,e
, and Yingbin Fu
a,f,4
a
Department of Ophthalmology and Visual Sciences, and
f
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City,
UT 84132;
b
The Key Laboratory for Human Disease Gene Study of Sichuan Province, Sich uan Academy of Medical Sciences and Sichuan Provincial Peoples
Hospital, Chengdu 610072, China;
c
Division of Gene Function in Animals, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan;
d
Molecular
Medicine Research Center and Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041, China; and
e
Institute for Genomic
Medicine and Shiley Eye Center, University of California at San Diego, La Jolla, CA 92037
Edited by Jeremy Nathans, The Johns Hopkins University, Baltimore, MD, and approved July 27, 2011 (received for review February 19, 2011)
Age-related macular degeneration (AMD) is the leading cause of
irreversible blindness in the elderly. Wet AMD includes typical
choroidal neovascularization (CNV) and polypoidal choroidal vas-
culopathy (PCV). The etiology and pathogenesis of CNV and PCV are
not well understood. Genome-wide association studies have linked
a multifunctional serine protease, HTRA1, to AMD. However, the
precise role of HTRA1 in AMD remains elusive. By transgenically
expressing human HTRA1 in mouse retinal pigment epithelium,
we showed that increased HTRA1 induced cardinal features of
PCV, including branching networks of choroidal vessels, polypoidal
lesions, severe degeneration of the elastic laminae, and tunica
media of choroidal vessels. In addition, HTRA1 mice displayed
retinal pigment epithelium atrophy and photoreceptor degenera-
tion. Senescent HTRA1 mice developed occult CNV, which likely
resulted from the degradation of the elastic lamina of Bruchs mem-
brane and up-regulation of VEGF. Our results indicate that in-
creased HTRA1 is sufcient to cause PCV and is a signicant risk
factor for CNV.
A
dvanced age-related macular degeneration (AMD) can be
classied into wet AMD and geographic atrophy (1, 2). Wet
AMD includes the typical choroidal neovascularization (CNV)
and polypoidal choroidal vasculopathy (PCV). CNV is caused by
the growth of new blood vessels from the choroid into the sub-
retinal pigment epithelium (RPE) and subretinal spaces, whereas
PCV is caused by inner choroidal vessel abnormalities (3). PCV
has two key features on indocyanine green angiography (ICGA):
polypoidal vascular dilations and a network of branching ab-
normal choroid vessels (4). Both CNV and PCV can lead to
recurrent serous exudation and hemorrhages (5). The etiology
and pathogenesis of CNV and PCV are largely unknown.
Numerous genetic association studies have shown that chro-
mosome 10q26 is a major candidate region associated with the
susceptibility of several types of AMD (6, 7), including PCV (8
10). The linkage peak was rened to two neighboring genes,
HTRA1 (11, 12) and ARMS2 (or LOC387715) (13). HTRA1 is
a multifunctional serine protease that is ubiquitously expressed
in mammalian tissues (14, 15) but ARMS2 is primate-specic,
with a proposed function in mitochondria (13, 16), extracellular
matrix (17), or as a noncoding RNA (18). Variants in this region
are in strong linkage disequilibrium (1113, 16). There are three
major competing hypotheses attributing increased risk of AMD
to (i) increased HTRA1 (11, 12), (ii) decreased ARMS2 (16), or
(iii) both increased HTRA1 and decreased ARMS2 (19). How-
ever, a series of studies on the inuence of AMD-associated
polymorphisms on the expression of ARMS2 and HTRA1 have
yielded widely conicting results (12, 16, 1824). As a result, the
functional involvement of either HTRA1 or ARMS2 in AMD
remains uncertain, despite strong genetic evidence (18, 22). To
clarify the role of HTRA1 in AMD pathogenesis, we trans-
genically expressed human HTRA1 in mouse RPE. We showed
that increased HTRA1 is sufcient to cause PCV and occult
CNV, two types of wet AMD.
Results
Expression of Human HTRA1 in Mouse RPE. We generated a mouse
line (hHTRA1
+
) overexpressing human HTRA1 (hHTRA1)in
mouse RPE. The transgene was driven by a hybrid promoter
consisting of the human cytomegalovirus immediate-early en-
hancer/promoter (CMV-IE) and the RPE-specic human vitel-
liform macular dystrophy 2 (VMD2) promoter (25). When used
with a cellular promoter, the CMV-IE was shown to enhance the
expression of transgenes (2628). Human HTRA1 was specically
expressed in mouse RPE, as determined by real-time RT-PCR
(Fig. 1A). Because we added a myc-His
6
tag at the C terminal of
human HTRA1, we detected the expression of transgenic
HTRA1 in hHTRA1
+
RPE by Western blotting with anti-myc
antibody 9E10 (Fig. 1B, Upper). The absolute levels of human
HTRA1 in transgenic hHTRA1
+
and normal human (ages be-
tween 50 and 60 y old) RPE were measured by Western blotting
with a monoclonal antibody that recognizes human but not mouse
HTRA1. By comparing with puried (His)
6
-tagged recombinant
human HTRA1 standards, the human HTRA1 level was de-
termined to be 2.96 ± 0.56 ng/15 μg lysate in hHTRA1
+
RPE,
which was 5.3-times that of human RPE (0.56 ± 0.09 ng/15 μg
lysate) (Fig. 1 CE). By immunohistochemistry, human HTRA1
was located in the RPE of hHTRA1
+
mouse (Fig. 1F, Lower,
green signals). Consistent with its role as a secreted protein
functioning in the extracellular matrix (29, 30), we observed sig-
nals at the basal side of RPE (Fig. 1F, Lower, arrows), the Bruchs
membrane, and the choroid of hHTRA1
+
mice, suggesting that
transgenic HTRA1 was secreted from the basal RPE toward
Bruchs membrane/choroid. This pattern is similar to endogenous
HTRA1 expression in human eyes (SI Appendix, Fig. S1).
Transgenic hHTRA1
+
Mice Developed PCV and Occult CNV. On ICGA,
hHTRA1
+
mice exhibited cardinal features of PCV bilaterally,
Author contributions: Y.F. designed research; A.J., S.K., N.Z., Z.T., J.-H.Y., C.W., J.A., A.,
H.F., M.M., Z.Y., R.M., C.O., K.Z., and Y.F. performed research; A.J., S.K., N.Z., L.L., B.A.,
C.O., and Y.F. analyzed data; and Y.F. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
1
A.J. and S.K. contributed equally to this work.
2
Present address: Department of Pharmacology, School of Medicine, Case Western Re-
serve University, Wood Building, 10900 Euclid Avenue, Cleveland, OH 44106.
3
Present address: Department of Oncological Sciences and Medicine, University of Utah,
15 North 2030 East, Salt Lake City, UT 84112.
4
To whom correspondence should be addressed. E-mail: Yingbin.fu@hsc.utah.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1102853108/-/DCSupplemental.
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with the age of onset varying between 3 and 5 wk: (i) numerous
small hyper uorescent dots (diameter less than 0.25 mm) con-
sistent with microaneurysmal dilations (Fig. 2 A and D); (ii) large
polypoidal lesions (diameter more than 0.45 mm) resembling
grape clusters (Fig. 2B, Upper); and (iii) a network of branching
abnormal vessels (i.e., loop or coil-like structures) (Fig. 2B,
Lower, red arrowhead) with terminal dilations (31). None of the
above features were observed in WT littermates.
Of the 114 hHTRA1
+
mice we examined, 67 (59%) showed
PCV phenotypes (PCV
+
) but the other 47 did not (PCV
), al-
though the level of transgenic HTRA1 was similar in their RPE
(Fig. 1B). The genetic background of hHTRA1
+
mice appeared
to have an impact on phenotype progression because 78% of
progenies from PCV
+
parents developed PCV, in contrast to
28% from PCV
parents. In the PCV
+
mice, there was a broad
phenotypic spectrum ranging from weak to severe (SI Appendix,
Fig. S2A). Similar to human PCV (5), some branching vascular
networks and lesions remained quiescent (SI Appendix, Fig.
S2B), whereas others developed new lesions after 2 to 5 mo (SI
Appendix, Fig. S2C). The lesions were rarely visible on uores-
cein angiography (FA), which is more suitable to image retinal
vasculature, and were best seen on ICGA, which is more suitable
to image posterior choroidal vasculature because of its infrared
spectrum (Fig. 2 A, B, and D). The ICGA lesions were located in
the choroid by spectral domain optical coherence tomography
(SD-OCT) (SI Appendix, Fig. S3). On ocular fundus examina-
tion, hHTRA1
+
mice showed prominent orange-yellow lesions
(Fig. 2A, red circles), many of which appeared to have the same
locations as ICGA lesions. In senescent hHTRA1
+
mice (older
than 11 mo), we could see speckled hyperuorescence with
poorly demarcated leakage in late-phase FA, which resembles
occult CNV, in four of eight PCV
+
mice (Fig. 2C, circles). The
same was not observed in either the PCV
(n = 3) or WT lit-
termates (n = 2). The occurrence of occult CNV was not cor-
related with the severity of PCV (SI Appendix, Table S1),
suggesting different factors are involved in controlling the pro-
gression of the two types of wet AMD.
Degradation of the Elastic Lamina of the Bruchs Membrane and
Choroidal Vessels in hHTRA1
+
Mice. The PCV lesions in hHTRA1
+
mice likely resulted from the exudates of compromised choroidal
vessels (Fig. 3). Indeed, pools of blood cells in the sub-RPE space
were frequently present in the hHTRA1
+
mice (Fig. 3A,yellow
arrows, quantication shown at the bottom of Fig. 3), apparently
from hemorrhagic choroidal vessels. In contrast to the normal
choroidal architecture of WT mice, the hHTRA1
+
mice (11 mo
old) contained clusters of abnormally dilated, thin-wall vessels
beneath the RPE (Fig. 3B, arrows, quantication shown at the
bottom of the gure). These features were similar to histopatho-
logic ndings on surgically excised human PCV specimens
AB
RPE
Retina
Liver
Heart
Skin
0.0
0.4
0.8
1.2
Relative
hHTRA1 mRNA
/
Gapdh
mRNA
42
kDa
F
WT
hHTRA1
+
Cho
RPE
Cho
RPE
55
C
recombinant hHTRA1
Human RPE
0.5 1.0 2.0
55
50
kDa
human RPE
(15
g)
hHTRA1
+
RPE
(4
g)
recombinant
hHTRA1 (ng)
0.0 0.5 1.0 1.5 2.0
0
25
50
75
100
125
AU
Recombinant hHTRA1 (ng)
hHTRA1
+
-
RPE
52
D
Human
hHTRA1
+
***
n=6
0.0
1.0
2.0
3.0
hHTRA1 (ng/15 g lysate)
E
PCV+
hHTRA1
+
PCV-
WT
µ
µ
µ
Fig. 1. Expression of human HTRA1 in mouse RPE. (A) Human HTRA1 was
specically expressed in the RPE of hHTRA1
+
mice as determined by real-time
RT-PCR. The levels of human HTRA1 mRNA in different tissues were nor-
malized to mouse Gapdh mRNA levels. n = 5 for RPE and retina samples; n =
4 for liver, heart, and skin. (B) Western blot analysis of human HTRA1 ex-
pression in the RPE/choroid of hHTRA1
+
(PCV
+
and PCV
) mice. Human
HTRA1 protein was detected with an anti-myc monoclonal antibody, 9E10
(Upper). Equal loading was indicated by the β-actin level (Lower). (C)A
representative Western blot showing relative levels of human HTRA1 signal
from 4 μgofhHTRA1
+
and 15 μg of human RPE extracts derived from three
mice and two normal human donors, respectively. (His)
6
-tagged recombi-
nant human HTRA1 protein (52 kDa) was used as standards (in nanograms).
The myc-His
6
tagged transgenic HTRA1, (His)
6
-tagged recombinant HTRA1,
and native HTRA1 (from human RPE) ran at 55, 52, and 50 kDa, respectively.
(D) The pixel values of the recombinant protein bands in C in arbitrary units
(AU) were plotted against the protein amounts to construct a standard
curve. The expression levels of human HTRA1 in hHTRA1
+
and human RPE
were determined according to their AU and the standard curve. (E) Com-
parison of human HTRA1 protein levels in hHTRA1
+
and human RPE (n =6).
***P < 0.001. (F) Retinal sections from WT and hHTRA1
+
mice immun os-
tained with a mouse anti-human HTRA1 antibody (green). Transgenic HTRA1
was detected in the basal side of RPE (red arrows), the choroid, and the
Bruchs membran e. The cross reactivity of HTRA1 antibody with the Bruchs
membrane (SI Appendix, Fig. S11) (negative control with Htra1
/
) made the
detection of HTRA1 in the Bruchs membrane challenging, although the
signal intensity was increased in hHTRA1
+
in comparison with WT and
Htra1
/
. RPE, retinal pigment epithelium; Cho, choroid. (Scale bar, 5 μm.)
Data are presented as mean ± SEM (A and E).
A
WT hHTRA1
FAICGAFundus
D
C
B
+
hHTRA1
+
FA
ICGA
hHTRA1
+
ICGA
ICGA
ICGA
WT hHTRA1
+
Fig. 2. Transgenic hHTRA1
+
mice developed PCV. (A) Representative FA,
ICGA, and fundus images of hHTRA1
+
and WT littermates. The hHTRA1
+
mice exhibited hyperuorescent lesions on ICGA (red arrowheads) and or-
ange-yellow lesions on the fundus photograph (red circles ). (B) Higher
magnication shows polypoidal, grape-cluster structure (Upper) or loop
structure (Lower, red arrowhead) of ICGA lesions in hHTR A1
+
mice. (C)FAof
an 11-mo-old hHTRA1
+
mouse showed speckled hyperuorescence at the
same site of an ICGA lesion (red circles), resembling occult CNV. (D) Com-
posite ICGA image shows the distribution of lesions in hHTRA1
+
mice.
Jones et al. PNAS
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14579
GENETICS
(32, 33). Consistent with histological ndings (Fig. 3 A and B),
ultrastructural analysis of 11-mo-old hHTRA1
+
mice showed
marked attenuation of the choroidal vessels (Fig. 3C, Right). The
choroidal arteries in atrophic regions completely lacked both the
elastic interna and elastic externa. The tunica media was severely
degenerated or missing. In sharp contrast, the neighboring retinal
vessels were normal in hHTRA1
+
mice (SI Appendix,Fig.S4),
which is consistent with the normal FA (Fig. 2A), and the im-
munohistochemical results showing HTRA1 was secreted from
the basal RPE toward the choroid side (Fig. 1F).
Another prominent feature of the hHTRA1
+
-PCV
+
mice was
that the integrity of the elastic lamina (EL) of the Bruchs
membrane was severely compromised (64.3% vs. 94.8% in WT;
EL integrity was dened as the total length of EL divided by the
total length of Bruchs membrane in a given region) (Table 1).
The EL of the PCV
+
mice was fragmented, and interrupted by
gaps of varying sizes (Fig. 4A, Lower, brackets). The degradation
of EL shares close similarity to macular EL disruption associated
with AMD lesions (34). The length of the longest uninterrupted
EL was only 9.9 μminhHTRA1
+
mice compared with 32.9 μmin
WT (Table 1). The combined gap length was nine-times larger
in PCV
+
mice than that in WT (318.55 vs. 36.32 μm). It is in-
teresting that the largest gap length in PCV
+
mice, 8 μm, was
close to the average gap length (910 μm) in the macula of
AMD patients (34). Not surprisingly, we observed choroidal
endothelial processes inserting into the EL gaps of PCV
+
mice
(Fig. 4B, Middle Right, red arrowheads), supporting the notion
that the breakdown of Bruchs membrane allows the invasion of
choroid vessels into the RPE (35). In both the inner and outer
collagenous layers of the Bruchs membrane, there were mem-
brane-bound basal linear deposits (Fig. 4B, Bottom Right, red
arrows), which were linked to early AMD (36). Despite the ex-
tensive degeneration of the EL, the collagen bers in both the
inner collagenous layer and outer collagenous layer, and the
basement membranes of both the RPE and the choriocapillaris,
appear to be normal in most regions (SI Appendix, Fig. S5). RPE
cells from PCV
+
mice had normal polygonal morphology, as
revealed by Alexa 488-phalloidin staining (SI Appendix, Fig. S6).
Besides lesions in the choroid and Bruchs membrane,
hHTRA1
+
-PCV
+
mice showed degenerative changes in the RPE
and photoreceptors. There were vacuoles in the RPE (Fig. 4B,
Bottom, with quantication), some of which were lled with ve-
sicular materials (Fig. 4B, Bottom Left, yellow arrows) but others
were empty (Fig. 4B, Bottom Right , yellow arrowheads). In some
areas, RPE was devoid of basal infoldings (Fig. 4B, Middle Left,
red brackets). Areas of RPE hypopigmentation (Fig. 3A, black
arrows) and hyperpigmentation (Fig. 4B, Middle) were evident in
hHTRA1
+
-PCV
+
mice. Both the outer and inner segments of
hHTRA1
+
-PCV
+
mice were disorganized, with marked vacuo-
lization in the inner segment (Fig. 4C, Right, red arrows and
arrowheads indicate vacuoles between and within the inner
segment, respectively). In contrast to PCV
+
mice, hHTRA1
+
-
PCV
mice had signicantly better EL integrity, normal struc-
tures of choroid vessels, RPE, and photoreceptors (Table 1, and
SI Appendix, Fig. S7).
HTRA1 Exhibits Elastase Activity. The most parsimonious explana-
tion for the elastin degradation in both the Bruchs membrane
and the choroid vessels in the hHTRA1
+
mice is that the pro-
tease activity of overexpressed HTRA1, which is secreted from
RPE, caused the degeneration. Because HTRA1 was not known
to have elastase activity, we performed an in vitro elastin deg-
radation assay using DQ elastin, a soluble elastin labeled with
quenched BODIPY FL dye, as a substrate (Fig. 5A). Puried
recombinant human HTRA1 can degrade elastin with a specic
activity of 4.4 ± 0.8 μ /mg (n = 3). This activity was 30-times less
than that of porcine pancreas elastase (135 ± 5.5 μ/mg, n = 4),
suggesting that the basal elastase activity of HTRA1 was low.
To eliminate the possibility that the degeneration of the elastic
layers of the choroidal vessels and Bruchs membrane was the
result of down-regulation of elastin expression, we analyzed the
protein level of soluble elastin in the RPE/choroid of hHTRA1
+
and WT mice by Western blot. The level of tropoelastin, the
soluble precursor of elastin, was similar in both the hHTRA1
+
mice and WT (Fig. 5B), suggesting that the biosynthesis of
elastin is not altered by HTRA1 overexpression. However, there
is an increase of degraded elastin products (Fig. 5B, bracket) in
hHTRA1
+
-PCV
+
mice in comparison with the WT and PCV
mice, suggesting that elastin degradation rather than elastin
down-regulation is likely the cause for the observed lesions in the
A
B
WT
hHTRA1
+
-PCV+
Cho
RPE
OS
IS
ONL
OPL
INL
IPL
GC
IS/OS
Choroid
IS/OS
Choroid
C
endothelium
elastica interna
tunica media
elastica externa
lumen lumen
adventitia
Pools of accumulated blood cells
WT: 0
hHTRA1
+
-PCV+: 9
Quantification of thin-wall vessels
WT: 2
hHTRA1
+
-PCV+: 23
Cho
RPE
Cho
RPE
Fig. 3. Degradation of the choroidal vessels in hHTRA1
+
mice (PCV
+
;11mo
old). (A) H&E staining of retinal sections of an hHTRA1
+
mouse and a WT
littermate. Large numbers of red blood cells are accumulated in the cavity
between RPE and choroid (yellow arrows). There were nine pools of blood
cells in four eyes of hHTRA1
+
-PCV
+
mice and no blood-cell accumulation in
four eyes of WT littermates (one eye per mouse). Pools of blood cells were
counted from one retinal/RPE/choroid section per eye. Black arrows point to
RPE hypopigmentation. OS, outer segment; IS, inner segment; ONL, outer
nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner
plexiform layer; GC, ganglion cell layer. (Scale bars, 20 μm.) (B) Comparison
of choroid architecture between hHTRA1
+
and WT mice. Dilated, thin-wall
vessels in hHTRA1
+
mice are indicated (red arrows). There were 23 thin-wall
vessels in hHTRA1
+
-PCV
+
mice and only 2 in WT. Thirty-one choroidal regions
from three hHTRA1
+
-PCV
+
mice (ve eyes) and 30 choroidal regions from
three WT (ve eyes) were compared (0.75 ± 0.14 per region in PCV
+
vs. 0.07 ±
0.05 in WT, P < 0.001). Thin plastic sections (200 nm) were stained with rabbit
anti
L-aspartate IgG. (Scale bars, 50 μm.) (C) Ultrastructural analysis showed
severe degeneration of the elastic laminae (internal and external) and the
tunica media of a choroid artery in hHTRA1
+
-PCV+ mice. Lower panels show
magnied views of the artery wall of a WT and a PCV
+
mouse in the Upper
panels (red boxes). [Scale bars, 2 μm(Upper) and 1 μm(Lower).]
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Bruchs membrane and the choroid vessels in PCV
+
mice (see
more in Discussion).
Increased VEGF in hHTRA1
+
Mice. VEGF plays a signicant rolein the
progression of CNV (37), although its role in PCV is controversial
(32, 33, 38, 39). We examined the expression of VEGF in the RPE/
choroid of hHTRA1
+
mice by Western blotting (Fig. 6A) and im-
munohistochemistry (Fig. 6B). Compared with WT, the VEGF
level was increased in both PCV
+
and PCV
mice (Fig. 6A). Our
data suggest that (i) increased HTRA1 can lead to up-regulation of
VEGF in the RPE/choroid of hHTRA1
+
mice, and (ii)VEGFel-
evation is not sufcient to induce PCV although it is critical for
CNV pathogenesis (see Discussion). Our results explain the limited
effect of anti-VEGF therapy in treating PCV (4043).
Human HTRA1 Alone Was Sufcient to Cause PCV. Because hHTRA1
+
mice expressed both mouse and human HTRA1 in their RPE, we
explored whether overexpression of human HTRA1 alone was
sufcient to cause PCV by breeding hHTRA1
+
mice into the
Htra1
/
background (SI Appendix, Fig. S8 AE). Consistent with
ndings on CARASIL (cerebral autosomal recessive arteriopathy
with subcortical infarcts and leukoencephalopathy) patients with
reduced or no HTRA1 protein (44), we did not nd any PCV or
CNV features in Htra1
/
mice determined by histology, FA,
ICGA, and SD-OCT (SI Appendix,Fig.S8F and G). However,
overexpression of human HTRA1 in the RPE of Htra1
/
mice
resulted in PCV features similar to those in hHTRA1
+
-PCV
+
mice (SI Appendix,Fig.S9,Right). This result is signicant in terms
of designing new therapeutic interventions for AMD: it suggests
that clinicians can safely target HTRA1 in the eye by local delivery
of reagents (e.g., shRNA/siRNA).
Discussion
By overexpressing human HTRA1 in mouse RPE, we showed
that increased HTRA1 leads to PCV and occult CNV in mice.
The lesions in both the Bruchs membrane and the choroidal
vessels were likely caused by the elastase activity of overex-
pressed HTRA1 (Fig. 5A). The low basal elastase activity of
HTRA1 is consistent with biochemical studies showing that both
the N-terminal Kazal-type serine-protease inhibitor domain and
the C-terminal PDZ domain regulate HTRA1 activity (45, 46). It
suggests that extracellular matrix proteins are likely involved in
activating HTRA1 and producing the PCV phenotype, probably
by interacting with the PDZ domain and the inhibitor domain. It
was shown that the removal of the inhibitor domain or binding of
PDZ domain activates HTRA1 protease activity threefold
(with a combined effect of >ninefold) (46). This result may ex-
plain the wide phenotypic variations in hHTRA1
+
mice. How-
ever, it should be mentioned that HTRA1 is a complex protein
containing a homology domain to Mac25 and insulin-like growth-
factor binding proteins, a Kazal-type inhibitor motif, the protease
domain, and the PDZ domain, all of which may contribute to the
phenotypes we observed. Future work in generating transgenic
mice expressing the protease-inactive form of HTRA1, as well as
HTRA1 lacking various domains, should help clarify the bio-
chemical mechanism on how increased HTRA1 leads to PCV and
occult CNV.
To estimate the percentage of HTRA1 secreted from RPE, we
compared the endogenous HTRA1 level in the supernatant versus
the lysate of the human RPE cell line, ARPE-19. Approximately
43% of HTRA1 was secreted into the supernatant (SI Appendix,
Fig. S10 and SI Materials and Methods). It is likely that a signicant
amount of human HTRA1 remains inside the RPE of hHTRA1
+
mice. The exact role of intracellular HTRA1 is unclear because
hHTRA1
+
mice exhibit most prominent phenotypes in the extra-
cellular space of RPE (i.e., Bruchs membrane and choroid). It is
worth mentioning that the endogenous HTRA1 is ubiquitously
expressed in vertebrates (14, 15). Overexpressing HTRA1 in
mouse RPE may have expedited the development of PCV phe-
notype. However, it may also cause unintended damage to the
target tissue (e.g., RPE lesions), although hHTRA1
+
mice exhibit
phenotypes that closely resemble human PCV.
Why is the degeneration of the choroid vessels much more severe
than that of the Bruchs membrane (Figs. 3C vs. 4A)thatiscloserto
the RPE? One possible explanation is that the Bruchs membrane
acts as a physical barrier for cells mediating inammatory responses
(i.e., leukocytes, lymphocytes, and macrophages), therefore miti-
gating the possible damaging effect by secondary immune respon-
ses (35). Indeed, we found that the basement membranes of both
the RPE and the choriocapillaris and the collagen bers in the
Bruchs membrane were normal in most regions, despite the EL
degeneration (SI Appendix, Fig. S5). In contrast, the tunica ad-
ventitia, which contains large amount of collagen bers, and the
tunica media, which is composed predominantly of smooth muscle,
were severely degenerated in choroid arteries in addition to the
degradation of the elastic lamina (Fig. 3C), suggesting that in-
ammatory processes (involving T cells, macrophages, matrix
metalloproteinases, neutrophil elastase, and so forth) likely par-
ticipated in the degradation of choroid vessel walls following the
initial assault by HTRA1.
As in human PCV patients, we did not observe drusen in
hHTRA1
+
mice. Although hHTRA1
+
mice exhibit robust fea-
tures of PCV, there is no classic CNV formation, suggesting that
additional factors, such as oxidative stress, complement activa-
tion (2), may be required to develop typical CNV. The other
possible explanation is the relative intactness of the basement
membranes and collagenous bers in the Bruchs membrane.
Nevertheless, increased HTRA1 led to the degradation of the
EL of Bruch s membrane (Fig. 4A and Table 1) and up-regula-
tion of VEGF (Fig. 6), both of which are risk factors for CNV
(34, 37). Our result is consistent with a recent study showing that
the vitreous level of HtrA1 was signicantly associated with that
of VEGF in patients with eye diseases (ocular vascular diseases,
retinal detachment, idiopathic macular hole, and traumatic in-
jury) (47). Both HTRA1 and VEGF were up-regulated in human
fetal RPE cells under stress conditions (47). In HTRA1 mice,
VEGF may be up-regulated in response to the RPE stress in-
duced by HTRA1 overexpression. Future studies are necessary
Table 1. Comparison of EL integrity of the Bruchs membrane between hHTRA1
+
and WT mice
WT hHTRA1
+
-PCV
hHTRA1
+
-PCV
+
Uninterrupted EL* 6.76 ± 0.57 ( n = 118) 4.79 ± 0.35 (n = 107) 2.13 ± 0.11 (n = 297)
GAP* 0.35 ± 0.04 (n = 103) 0.58 ± 0.05 (n = 103) 1.09 ± 0.07 (n = 293)
Maximal EL 32.90 23.04 9.87
Maximal GAP 2.19 2.74 7.70
Combined GAP 36.32 59.78 318.55
EL Integrity* (%) 94.8 ± 0.8 (n = 31) 89.7 ± 1.1 (n = 26) 64.3 ± 3.3 (n = 31)
Thirty-one regions of Bruchs membrane from three WT and three hHTRA1
+
-PCV
+
mice and 26 regions from
two hHTRA1
+
-PCV- mice were compared. The mouse ages were between 11 and 12 mo.
*Values are given as mean ± SEM. Signicant differences in uninterrupted EL, GAP, and EL integrity are noted
between WT, hHTRA1
+
-PCV
, and hHTRA1
+
-PCV
+
(all P < 0.001, except that P = 0.04 between WT and hHTRA1
+
-
PCV
in uninterrupted EL).
Jones et al. PNAS
|
August 30, 2011
|
vol. 108
|
no. 35
|
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GENETICS
to elucidate the exact mechanism of VEGF regulation in
hHTRA1
+
transgenic mice.
Because both the integrity and thickness of the EL in the
macula are signicantly decreased compared with the extra-
macular region (34), HTRA1 may be a key molecule predis-
posing the human macula to wet AMD. The degradation of the
elastic lamina in both the Bruchs membrane and the choroid
vessels of HTRA1 mice is particularly intriguing considering
multiple lines of evidence suggesting a relationship between in-
creased elastin degradation and the progression of wet AMD.
For example, serum levels of elastin-derived peptides in patients
with exudative AMD are signicantly higher than those in non-
exudative AMD patients and control patients (48). Decreased
integrity of the macular EL is strongly correlated with early
AMD and CNV (34). Collectively, our data show that increased
HTRA1 is sufcient to cause PCV and is a signicant risk factor
for CNV. It suggests that ARMS2 is unlikely to play a major role
in PCV pathogenesis. This is likely to be the case for CNV
pathogenesis as well because PCV and CNV are genetically
similar in the 10q26 loci (10). The role of HTRA1 in wet AMD
is supported by recent studies showing that genetic variants at
HTRA1-ARMS2 loci are signicantly associated with lesion sizes
in both PCV and CNV patients (49, 50), and confer a higher risk
of CNV than geographic atrophy in a well-powered sample (51).
In contrast, there is no functional evidence to date that ARMS2
plays any angiogenic role related to CNV or PCV.
Finally, we found that the genetic background of hHTRA1
+
mice had a strong inuence on the phenotype. Currently, there
is no effective pharmacological treatment for PCV. Designing
HTRA1-specic inhibitors and determining HTRA1 modulating
01020304050
0
5
10
15
20
25
30
DQ elastin fluorescence (x10
3
)
Time (min)
12 ng elastase
100 ng HTRA1
PCV+
hHTRA1
+
WT
PCV-
B
-actin
tropoelastin
170
kDa
130
100
72
55
40
33
24
degraded
elastin
A
Fig. 5. Elastase assay of recombinant human HTRA1 (A) and Western
blotting analysis of soluble elastin in the RPE/choroid of hHTRA1
+
(PCV
+
and PCV
) mice and WT littermates (B). Data represent the mean activities
of 100-ng HTRA1 (n = 3) and 12-ng porcine pancreatic elastase (n =4)in
A. Equal loading was conrmed by the β-actin level in B. A bracket indicates
the increase of degraded low molecular-mass elastin ranging from 45 to
55 kDa in PCV
+
mice.
A
B
WT
anti- -actin
anti-VEGF
PCV+
PCV-
hHTRA1
+
hHTRA1
+
PCV+
WT
PCV-
Fig. 6. Up-regulation of VEGF in the RPE/chroid of hHTRA1
+
(PCV
+
and PCV
)
mice. Western-blot (A) and immunohistochemistry (B) analysis of VEGF ex-
pression in the RPE/choroid of hHTRA1
+
(PCV
+
and PCV
) mice and WT litter-
mates. (Scale bar, 10 μm.) Equal loading was conrmed by the β-actin level in A.
WT
Cho
RPE
BM
EL
Cho
RPE
BM
EL
Cho
RPE
BM
EL
hHTRA1
+
-PCV+
A
Cho
RPE
EL
Cho
RPE
EL
basal
infolding
B
WT
hHTRA1
+
-PCV+
OS
IS
OS
IS
C
WT hHTRA1
+
-PCV+
Quantification of RPE vacuoles
WT:
4
hHTRA1
+
-PCV+: 45
Fig. 4. Transgenic hHTRA1
+
mice (PCV
+
; 11 mo old) displayed EL degrada-
tion, RPE atrophy, and photoreceptor degeneration. ( A) Degradation of the
EL of Bruchs membrane in hHTRA1
+
-PCV
+
mice. EL gaps are bracketed
(Lower). (Scale bar, 1 μm.) (B) Ultrastructure of the choroid, Bruchs mem-
brane, and RPE of hHTRA1
+
-PCV
+
mice. Red brackets show RPE regions de-
void of basal infoldings (Middle Left). Processes of an endothelial cell have
inserted into EL gaps of hHTRA1
+
mice (Middle Right, red arrowheads).
Red and yellow arrows (or arrowheads) show basal linear deposits and
RPE vacuolization, respectively (Bottom). There were 45 RPE vacuoles in
hHTRA1
+
-PCV
+
vs. 4 in WT. Twenty-six RPE regions from three WT and 24 RPE
regions from three hHTRA1
+
-PCV
+
mice (one eye per mouse) were compared
(1.88 ± 0.28 per RPE region in PCV
+
vs. 0.15 ± 0.07 in WT, P < 0.001). (Scale
bars, 1 μm.) (C) Photoreceptor degeneration in hHTRA1
+
-PCV
+
mice. Red
arrows and arrowheads indicate vacuolization between and within the inner
segment of PCV
+
mice, respectively. In both WT and hHTRA1
+
-PCV
+
, RPE is
horizontal on the top (RPE was not included in the gure because of space
limitation). (Scale bars, 2 μm.)
14582
|
www.pnas.org/cgi/doi/10.1073/pnas.1102853108 Jones et al.
factors will be invaluable in disease prevention and therapeutic
intervention for both PCV and CNV.
Methods
Mice. To generate hHTRA1
+
mice, a 1.5-kb human HTRA1 cDNA (TAG stop
condon was removed) was inserted as a BamHI-XhoI fragment into
pcDNA3.1/myc-His vector (Invitrogen) so that HTRA1 cDNA was in-frame
with myc-His
6
coding region. Subsequently, the human VMD2 promoter
(585 to +38 bp) (25) was inserted as a KpnI-BamHI fragment between the
CMV enhancer/promoter and human HTRA1 cDNA. The transgene regions
containing the CMV-VMD2 hybrid promoter, HTRA1-myc-His
6
, and bovine
growth hormone poly(A) were sequence-veried. The transgene construct
was excised by BglII-RsrII digestion, gel-puried, and injected into C57BL/6 ×
CBA embryos at the University of Utah Transgenic/Gene Targeting Core
Facility. Animal experiments were conducted according to protocols ap-
proved by the Institutional Animal Care and Use Committees. Additional
materials and methods are in the SI Appendix.
ACKNOWLEDGMENTS. We thank N. Esumi for providing the VMD2 pro-
moter, G. Hageman for assistance in H&E staining, W. D. Ferrell for assistance
in histology imaging, and M. E. Hartnett and P. S. Bernstein for discussions
and comments on the manuscript. K.Z. was supported by grants from the
National Eye Institute/National Institutes of Health, National Basic Research
Program of China (973 Program, 2011CB510200), Chinese National 985 Pro-
ject to Sichuan University and West China Hospital, and a Veterans Admin-
istration Merit Award; C.O. was supported by research grants from the Nara
Institute of Science and Technology and the Inamori Foundation, and by
Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and
Technology, Japan; Y.F. was supported by the Career Development Award
(Research to Prevent Blindness), a Research to Prevent Blindness departmen-
tal unrestricted grant, and the Karl Kirchgessner Foundation Award for
Vision Research.
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Jones et al. PNAS
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August 30, 2011
|
vol. 108
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no. 35
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14583
GENETICS
    • "MEFs were isolated from E13.5 and E14.5 embryos derived from four different breedings. Htra1 −/− mice were described previously [8]. SW480 and HCT116 cells were obtained from ATCC. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. Methods: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. Results: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). Conclusions: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.
    Full-text · Article · Dec 2016
    • "Histopathological and animal studies have found pathology in the Bruch's membrane and the choroidal vasculature that may have resulted from abnormal ECM metabolism (Chong et al., 2005; Jones et al., 2011; Nakashizuka et al., 2008 ). In particular, the arteriosclerotic and aneurysmal changes seen in PCV strongly suggest derangements in ECM remodeling (Jones et al., 2011; Nakashizuka et al., 2008). Some studies have found associations of serum levels of MMPs with AMD. "
    [Show abstract] [Hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people globally. It is estimated that there will be more Asians with AMD than the rest of the world combined by 2050. In Asian populations, polypoidal choroidal vasculopathy (PCV) is a common subtype of exudative AMD, while choroidal neovascularization secondary to AMD (CNV-AMD) is the typical subtype in Western populations. The two subtypes share many common clinical features and risk factors, but also have different epidemiological and clinical characteristics, natural history and treatment outcomes that point to distinct pathophysiological processes. Recent research in the fields of genetics, proteomics and imaging has provided further clarification of differences between PCV and CNV-AMD. Importantly, these differences have manifested in disparity in response to intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) treatment between PCV and CNV-AMD, emphasizing the need for accurate diagnosis of PCV and in distinguishing PCV from CNV-AMD, particularly in Asian patients. Current clinical trials of PCV of intravitreal anti-VEGF therapy and photodynamic therapy will provide clearer perspectives of evidence-based management of PCV and may lead to paradigm shifts in therapeutic strategies away from those currently employed in the treatment of CNV-AMD. Further research is needed to clarify the relative contribution of specific pathways in inflammation, complement activation, extracellular matrix dysregulation, lipid metabolism and angiogenesis to the pathogenesis of PCV. Findings from this research, together with improved diagnostic technology and new therapeutics, will allow more optimal management of Asian AMD.
    Full-text · Article · Apr 2016
    • "In PCV and CNV-AMD, histopathological and animal studies have shown that abnormal ECM metabolism may underlie the pathology seen in the bruch's membrane and the choroidal vasculature [66,68,69]. In particular, the arteriosclerotic and aneurysmal changes seen in PCV strongly suggest derangements in ECM remodeling [66,69]. Chau et al., found increased plasma levels of MMP-9 in subjects with early AMD and neovascular AMD, but did not differentiate between PCV and CNV-AMD [70]. "
    [Show abstract] [Hide abstract] ABSTRACT: Age related macular degeneration (AMD) in Asians has been suggested to differ from their Western counterparts in terms of epidemiology, pathogenesis, clinical presentation and treatment. In particular, polypoidal choroidal vasculopathy (PCV) appears to be the predominant subtype of exudative AMD in Asian populations, in contrast to choroidal neovascularization secondary to AMD (CNV-AMD) in Western populations. Epidemiological data on PCV has been largely limited to hospital-based studies and there are currently no data on the incidence of PCV. Similarities and differences in risk factor profile between PCV and CNV-AMD point to some shared pathogenic mechanisms but also differential underlying mechanisms leading to the development of each phenotype. Serum biomarkers such as CRP, homocysteine and matrix metalloproteinases suggest underlying inflammation, atherosclerosis and deranged extracellular matrix metabolism as possible pathogenic mechanisms. In addition, recent advances in genome sequencing have revealed differences in genetic determinants of each subtype. While the standard of care for CNV-AMD is anti-vascular endothelial growth factor (VEGF) therapy, photodynamic therapy (PDT) has been the mainstay of treatment for PCV, although long-term visual prognosis remains unsatisfactory. The optimal treatment for PCV requires further clarification, particularly with different types of anti-VEGF agents and possible benefits of reduced fluence PDT.
    Full-text · Article · Apr 2015
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