Increased expression of multifunctional serine
protease, HTRA1, in retinal pigment epithelium
induces polypoidal choroidal vasculopathy in mice
Alex Jonesa,1, Sandeep Kumara,1, Ning Zhanga,2, Zongzhong Tonga,3, Jia-Hui Yanga, Carl Watta, James Andersona,
Amritaa, Heather Fillerupa, Manabu McCloskeya, Ling Luoa, Zhenglin Yangb, Balamurali Ambatia, Robert Marca,
Chio Okac, Kang Zhangd,e, and Yingbin Fua,f,4
aDepartment of Ophthalmology and Visual Sciences, andfDepartment of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City,
UT 84132;bThe Key Laboratory for Human Disease Gene Study of Sichuan Province, Sichuan Academy of Medical Sciences and Sichuan Provincial People’s
Hospital, Chengdu 610072, China;cDivision of Gene Function in Animals, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan;dMolecular
Medicine Research Center and Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041, China; andeInstitute 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-
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
brane and up-regulation of VEGF. Our results indicate that in-
creased HTRA1 is sufficient to cause PCV and is a significant risk
factor for CNV.
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 refined 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-specific,
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 (11–13, 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 influence of AMD-associated
polymorphisms on the expression of ARMS2 and HTRA1 have
yielded widely conflicting results (12, 16, 18–24). 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
dvanced age-related macular degeneration (AMD) can be
classified into wet AMD and geographic atrophy (1, 2). Wet
that increased HTRA1 is sufficient to cause PCV and occult
CNV, two types of wet AMD.
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-specific 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 (26–28). Human HTRA1 was specifically
expressed in mouse RPE, as determined by real-time RT-PCR
(Fig. 1A). Because we added a myc-His6tag 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 humanbut not mouse
HTRA1. By comparing with purified (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 C–E). 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 Bruch’s
membrane, and the choroid of hHTRA1+mice, suggesting that
transgenic HTRA1 was secreted from the basal RPE toward
Bruch’s 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 conflict of interest.
This article is a PNAS Direct Submission.
1A.J. and S.K. contributed equally to this work.
2Present address: Department of Pharmacology, School of Medicine, Case Western Re-
serve University, Wood Building, 10900 Euclid Avenue, Cleveland, OH 44106.
3Present address: Department of Oncological Sciences and Medicine, University of Utah,
15 North 2030 East, Salt Lake City, UT 84112.
4To whom correspondence should be addressed. E-mail: Yingbin.firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 30, 2011
| vol. 108
| no. 35www.pnas.org/cgi/doi/10.1073/pnas.1102853108
with the age of onset varying between 3 and 5 wk: (i) numerous
small hyperfluorescent 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 fluores-
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 hyperfluorescence 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 Bruch’s 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, quantification 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, quantification shown at the
bottom of the figure). These features were similar to histopatho-
logic findings on surgically excised human PCV specimens
Relative hHTRA1 mRNA
0.5 1.0 2.0
Recombinant hHTRA1 (ng)
hHTRA1 (ng/15 g lysate)
specifically 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 μg of hHTRA1+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-His6tagged 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 immunos-
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
Bruch’s membrane. The cross reactivity of HTRA1 antibody with the Bruch’s
membrane (SI Appendix, Fig. S11) (negative control with Htra1−/−) made the
detection of HTRA1 in the Bruch’s 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).
Expression of human HTRA1 in mouse RPE. (A) Human HTRA1 was
ICGA, and fundus images of hHTRA1+and WT littermates. The hHTRA1+
mice exhibited hyperfluorescent lesions on ICGA (red arrowheads) and or-
ange-yellow lesions on the fundus photograph (red circles). (B) Higher
magnification shows polypoidal, grape-cluster structure (Upper) or loop
structure (Lower, red arrowhead) of ICGA lesions in hHTRA1+mice. (C) FA of
an 11-mo-old hHTRA1+mouse showed speckled hyperfluorescence 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.
Transgenic hHTRA1+mice developed PCV. (A) Representative FA,
Jones et al. PNAS
| August 30, 2011
| vol. 108
| no. 35
(32, 33). Consistent with histological findings (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 Bruch’s
membrane was severely compromised (64.3% vs. 94.8% in WT;
EL integrity was defined as the total length of EL divided by the
total length of Bruch’s 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 μm in hHTRA1+mice compared with 32.9 μm in
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 (∼9–10 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 Bruch’s membrane allows the invasion of
choroid vessels into the RPE (35). In both the inner and outer
collagenous layers of the Bruch’s 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 fibers 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 Bruch’s membrane,
hHTRA1+-PCV+mice showed degenerative changes in the RPE
and photoreceptors. There were vacuoles in the RPE (Fig. 4B,
Bottom, with quantification), some of which were filled 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 significantly 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 Bruch’s 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). Purified
recombinant human HTRA1 can degrade elastin with a specific
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 Bruch’s 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
Pools of accumulated blood cells
WT: 0 hHTRA1+-PCV+: 9
Quantification of thin-wall vessels
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 (five eyes) and 30 choroidal regions from
three WT (five 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
magnified 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).]
Degradation of the choroidal vessels in hHTRA1+mice (PCV+; 11 mo
| www.pnas.org/cgi/doi/10.1073/pnas.1102853108Jones et al.
Bruch’s membrane and the choroid vessels in PCV+mice (see
more in Discussion).
Increased VEGF in hHTRA1+Mice. VEGFplaysasignificantroleinthe
progression of CNV (37), although its role in PCV is controversial
(32,33,38, 39).Weexamined theexpression ofVEGFin theRPE/
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)increasedHTRA1canleadtoup-regulationof
VEGF in the RPE/choroid of hHTRA1+mice, and (ii) VEGF el-
evation is not sufficient to induce PCV although it is critical for
CNVpathogenesis (seeDiscussion).Our resultsexplainthelimited
effect of anti-VEGF therapy in treating PCV (40–43).
Human HTRA1 Alone Was Sufficient to Cause PCV. Because hHTRA1+
mice expressed both mouse and human HTRA1 in their RPE, we
explored whether overexpression of human HTRA1 alone was
sufficient to cause PCV by breeding hHTRA1+mice into the
Htra1−/−background (SI Appendix, Fig. S8 A–E). Consistent with
findings on CARASIL (cerebral autosomal recessive arteriopathy
with subcortical infarcts and leukoencephalopathy) patients with
reduced or no HTRA1 protein (44), we did not find any PCV or
CNV features in Htra1−/−mice determined by histology, FA,
ICGA, and SD-OCT (SI Appendix, Fig. S8 F 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 significant 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).
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 Bruch’s 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
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 significant
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., Bruch’s 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.
the RPE? One possible explanation is that the Bruch’s membrane
(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 fibers in the
Bruch’s 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 fibers, 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-
flammatory 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 fibers in the Bruch’s 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 significantly 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 Bruch’s membrane between hHTRA1+and WT mice
EL Integrity* (%)
6.76 ± 0.57 (n = 118)
0.35 ± 0.04 (n = 103)
94.8 ± 0.8 (n = 31)
4.79 ± 0.35 (n = 107)
0.58 ± 0.05 (n = 103)
89.7 ± 1.1 (n = 26)
2.13 ± 0.11 (n = 297)
1.09 ± 0.07 (n = 293)
64.3 ± 3.3 (n = 31)
Thirty-one regions of Bruch’s 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. Significant 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
to elucidate the exact mechanism of VEGF regulation in
Because both the integrity and thickness of the EL in the
macula are significantly 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 Bruch’s 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 significantly 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 sufficient to cause PCV and is a significant 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 significantly 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 influence on the phenotype. Currently, there
is no effective pharmacological treatment for PCV. Designing
HTRA1-specific inhibitors and determining HTRA1 modulating
0 10 20
DQ elastin fluorescence (x103)
12 ng elastase
100 ng HTRA1
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 confirmed 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.
Elastase assay of recombinant human HTRA1 (A) and Western
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-
Up-regulation of VEGF in the RPE/chroid of hHTRA1+(PCV+and PCV−)
Quantification of RPE vacuoles
WT: 4 hHTRA1+-PCV+: 45
tion, RPE atrophy, and photoreceptor degeneration. (A) Degradation of the
EL of Bruch’s membrane in hHTRA1+-PCV+mice. EL gaps are bracketed
(Lower). (Scale bar, 1 μm.) (B) Ultrastructure of the choroid, Bruch’s 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 figure because of space
limitation). (Scale bars, 2 μm.)
Transgenic hHTRA1+mice (PCV+; 11 mo old) displayed EL degrada-
| www.pnas.org/cgi/doi/10.1073/pnas.1102853108 Jones et al.
factors will be invaluable in disease prevention and therapeutic Download full-text
intervention for both PCV and CNV.
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-His6 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-His6, and bovine
growth hormone poly(A) were sequence-verified. The transgene construct
was excised by BglII-RsrII digestion, gel-purified, 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
1. Swaroop A, Chew EY, Rickman CB, Abecasis GR (2009) Unraveling a multifactorial
late-onset disease: From genetic susceptibility to disease mechanisms for age-related
macular degeneration. Annu Rev Genomics Hum Genet 10:19–43.
2. Ding X, Patel M, Chan CC (2009) Molecular pathology of age-related macular de-
generation. Prog Retin Eye Res 28(1):1–18.
3. Yannuzzi LA, Sorenson J, Spaide RF, Lipson B (1990) Idiopathic polypoidal choroidal
vasculopathy (IPCV). Retina 10(1):1–8.
4. Spaide RF, Yannuzzi LA, Slakter JS, Sorenson J, Orlach DA (1995) Indocyanine green vid-
eoangiography of idiopathic polypoidal choroidal vasculopathy. Retina 15(2):100–110.
5. Lim TH, Laude A, Tan CS (2010) Polypoidal choroidal vasculopathy: An angiographic
discussion. Eye (Lond) 24:483–490.
6. Majewski J, et al. (2003) Age-related macular degeneration—A genome scan in ex-
tended families. Am J Hum Genet 73:540–550.
7. Fisher SA, et al. (2005) Meta-analysis of genome scans of age-related macular de-
generation. Hum Mol Genet 14:2257–2264.
8. Kondo N, Honda S, Ishibashi K, Tsukahara Y, Negi A (2007) LOC387715/HTRA1 variants
in polypoidal choroidal vasculopathy and age-related macular degeneration in
a Japanese population. Am J Ophthalmol 144:608–612.
9. Lee KY, et al. (2008) Association analysis of CFH, C2, BF, and HTRA1 gene poly-
morphisms in Chinese patients with polypoidal choroidal vasculopathy. Invest Oph-
thalmol Vis Sci 49:2613–2619.
10. Lima LH, et al. (2010) Three major loci involved in age-related macular degeneration
are also associated with polypoidal choroidal vasculopathy. Ophthalmology 117:
11. Dewan A, et al. (2006) HTRA1 promoter polymorphism in wet age-related macular
degeneration. Science 314:989–992.
12. Yang Z, et al. (2006) A variant of the HTRA1 gene increases susceptibility to age-
related macular degeneration. Science 314:992–993.
13. Kanda A, et al. (2007) A variant of mitochondrial protein LOC387715/ARMS2, not
HTRA1, is strongly associated with age-related macular degeneration. Proc Natl Acad
Sci USA 104:16227–16232.
14. Nie GY, Hampton A, Li Y, Findlay JK, Salamonsen LA (2003) Identification and cloning
of two isoforms of human high-temperature requirement factor A3 (HtrA3), char-
acterization of its genomic structure and comparison of its tissue distribution with
HtrA1 and HtrA2. Biochem J 371:39–48.
15. Zurawa-Janicka D, Skorko-Glonek J, Lipinska B (2010) HtrA proteins as targets in
therapy of cancer and other diseases. Expert Opin Ther Targets 14:665–679.
16. Fritsche LG, et al. (2008) Age-related macular degeneration is associated with an
unstable ARMS2 (LOC387715) mRNA. Nat Genet 40:892–896.
17. Kortvely E, et al. (2010) ARMS2 is a constituent of the extracellular matrix providing
a link between familial and sporadic age-related macular degenerations. Invest
Ophthalmol Vis Sci 51:79–88.
18. Friedrich U, et al. (2011) Risk- and non-risk-associated variants at the 10q26 AMD locus
influence ARMS2 mRNA expression but exclude pathogenic effects due to protein
deficiency. Hum Mol Genet 20:1387–1399.
19. Yang Z, et al. (2010) Genetic and functional dissection of HTRA1 and LOC387715 in
age-related macular degeneration. PLoS Genet 6:e1000836.
20. Chan CC, et al. (2007) Human HtrA1 in the archived eyes with age-related macular
degeneration. Trans Am Ophthalmol Soc 105:92–97, discussion 97–98.
degeneration in multiple case-control samples. Ophthalmology 115:1891–1898.
22. Kanda A, et al. (2010) Age-related macular degeneration-associated variants at
chromosome 10q26 do not significantly alter ARMS2 and HTRA1 transcript levels in
the human retina. Mol Vis 16:1317–1323.
23. Wang G, et al. (2010) Analysis of the indel at the ARMS2 3′ UTR in age-related
macular degeneration. Hum Genet 127:595–602.
24. An E, Sen S, Park SK, Gordish-Dressman H, Hathout Y (2010) Identification of novel
substrates for the serine protease HTRA1 in the human RPE secretome. Invest Oph-
thalmol Vis Sci 51:3379–3386.
25. Esumi N, Oshima Y, Li Y, Campochiaro PA, Zack DJ (2004) Analysis of the VMD2
promoter and implication of E-box binding factors in its regulation. J Biol Chem 279:
26. Picanço-Castro V, Russo-Carbolante EM, Fontes AM, Fernandes AC, Covas DT (2008)
An enhancer/promoter combination strengthens the expression of blood-coagulation
factor VIII in non-viral expression vectors. Genet Mol Res 7:314–325.
27. Ong ST, Li F, Du J, Tan YW, Wang S (2005) Hybrid cytomegalovirus enhancer-h1
promoter-based plasmid and baculovirus vectors mediate effective RNA interference.
Hum Gene Ther 16:1404–1412.
28. Barnhart KM, Hartikka J, Manthorpe M, Norman J, Hobart P (1998) Enhancer and
promoter chimeras in plasmids designed for intramuscular injection: A comparative
in vivo and in vitro study. Hum Gene Ther 9:2545–2553.
29. Canfield AE, Hadfield KD, Rock CF, Wylie EC, Wilkinson FL (2007) HtrA1: A novel
regulator of physiological and pathological matrix mineralization? Biochem Soc Trans
30. Clausen T, Southan C, Ehrmann M (2002) The HtrA family of proteases: Implications
for protein composition and cell fate. Mol Cell 10:443–455.
31. Yuzawa M, Mori R, Kawamura A (2005) The origins of polypoidal choroidal vascul-
opathy. Br J Ophthalmol 89:602–607.
32. Terasaki H, Miyake Y, Suzuki T, Nakamura M, Nagasaka T (2002) Polypoidal choroidal
vasculopathy treated with macular translocation: Clinical pathological correlation. Br
J Ophthalmol 86:321–327.
33. Nakashizuka H, et al. (2008) Clinicopathologic findings in polypoidal choroidal vas-
culopathy. Invest Ophthalmol Vis Sci 49:4729–4737.
34. Chong NH, et al. (2005) Decreased thickness and integrity of the macular elastic layer
of Bruch’s membrane correspond to the distribution of lesions associated with age-
related macular degeneration. Am J Pathol 166:241–251.
35. Booij JC, Baas DC, Beisekeeva J, Gorgels TG, Bergen AA (2010) The dynamic nature of
Bruch’s membrane. Prog Retin Eye Res 29(1):1–18.
36. Curcio CA, Millican CL (1999) Basal linear deposit and large drusen are specific for
early age-related maculopathy. Arch Ophthalmol 117:329–339.
37. Witmer AN, Vrensen GF, Van Noorden CJ, Schlingemann RO (2003) Vascular endo-
thelial growth factors and angiogenesis in eye disease. Prog Retin Eye Res 22(1):1–29.
38. Tong JP, et al. (2006) Aqueous humor levels of vascular endothelial growth factor and
pigment epithelium-derived factor in polypoidal choroidal vasculopathy and choroi-
dal neovascularization. Am J Ophthalmol 141:456–462.
39. Matsuoka M, et al. (2004) Expression of pigment epithelium derived factor and vas-
cular endothelial growth factor in choroidal neovascular membranes and polypoidal
choroidal vasculopathy. Br J Ophthalmol 88:809–815.
40. Gomi F, et al. (2008) Efficacy of intravitreal bevacizumab for polypoidal choroidal
vasculopathy. Br J Ophthalmol 92(1):70–73.
41. Lai TY, Chan WM, Liu DT, Luk FO, Lam DS (2008) Intravitreal bevacizumab (Avastin)
with or without photodynamic therapy for the treatment of polypoidal choroidal
vasculopathy. Br J Ophthalmol 92:661–666.
42. Cho M, Barbazetto IA, Freund KB (2009) Refractory neovascular age-related mac-
ular degeneration secondary to polypoidal choroidal vasculopathy. Am J Ophthalmol
43. Stangos AN, et al. (2010) Polypoidal choroidal vasculopathy masquerading as neo-
vascular age-related macular degeneration refractory to ranibizumab. Am J Oph-
44. Hara K, et al. (2009) Association of HTRA1 mutations and familial ischemic cerebral
small-vessel disease. N Engl J Med 360:1729–1739.
45. Runyon ST, et al. (2007) Structural and functional analysis of the PDZ domains of
human HtrA1 and HtrA3. Protein Sci 16:2454–2471.
46. Murwantoko, et al. (2004) Binding of proteins to the PDZ domain regulates pro-
teolytic activity of HtrA1 serine protease. Biochem J 381:895–904.
47. Ng TK, et al. (2011) Interactive expressions of HtrA1 and VEGF in human vitreous
humors and fetal RPE cells. Invest Ophthalmol Vis Sci 52:3706–3712.
48. Sivaprasad S, Chong NV, Bailey TA (2005) Serum elastin-derived peptides in age-
related macular degeneration. Invest Ophthalmol Vis Sci 46:3046–3051.
49. Andreoli MT, et al. (2009) Comprehensive analysis of complement factor H and
LOC387715/ARMS2/HTRA1 variants with respect to phenotype in advanced age-
related macular degeneration. Am J Ophthalmol 148:869–874.
50. Gotoh N, et al. (2008) Correlation between CFH Y402H and HTRA1 rs11200638
genotype to typical exudative age-related macular degeneration and polypoidal
choroidal vasculopathy phenotype in the Japanese population. Clin Experiment
51. Sobrin L, et al. (2011) ARMS2/HTRA1 locus can confer differential susceptibility to
the advanced subtypes of age-related macular degeneration. Am J Ophthalmol 151:
Jones et al. PNAS
| August 30, 2011
| vol. 108
| no. 35