Graefe's Archive for Clinical and Experimental Ophthalmology
https://doi.org/10.1007/s00417-018-4184-5

BASIC SCIENCE

Developmental and age-related changes to the elastic lamina of Bruch’s
membrane in mice
Hidetsugu Mori 1 & Haruhiko Yamada 1 & Keiko Toyama 1 & Kanji Takahashi 1 & Tomoya Akama 2 & Tadashi Inoue 2 &Tomoyuki Nakamura 2
Received: 15 May 2018 / Revised: 10 October 2018 / Accepted: 9 November 2018
# The Author(s) 2018

Abstract
Background Fibrillin-1, tropoelastin, fibulin-5, and latent transforming growth factor beta-binding protein-2 and protein-4(LTBP-2 and LTBP-4) are essential proteins for the elastic lamina (EL). In this study, we analyzed each of these molecules inthe EL of Bruch’s membrane (BM) through development and aging.
Methods C57BL/6 mice (embryonic (E) days E12.5, E15.5, and E18.5; postnatal (P) days P1, P4, and P7 and P3, P6, and P75
weeks of age) were used. To investigate localization, immunohistochemical staining (IH) was performed. Transmission electron
microscopy (TEM) was used to evaluate the formation of microfibrils and tropoelastin. mRNA expression was determined byquantitative real-time PCR (qRT-PCR).
Results All five proteins were expressed in the EL of BM by IH except in embryonic mice. TEM results showed that tropoelastin
co-stained with microfibrils. Between 3 and 6 weeks of age, microfibrils became longer and thicker. It was difficult to evaluate theEL of BM in senile mice at 75 weeks of age because of abundant deposits which correspond to drusen. mRNA levels of eachprotein increased dramatically from E15.5 to P1 days and plateaued by P3 weeks as shown by qRT-PCR.
Conclusions In conclusion, these five proteins are possibly involved in elastic fiber assembly in BM. We define the date of full
assembly of the EL of BM as 3 weeks of age in mice.
Keywords Elastic lamina of Bruch’s membrane . fibrillin1 . Fibulin-5 . Tropoelastin . LTBP-2 . LTBP-4

Introduction
Bruch’s membrane (BM) has a thin and complex structurewith five stratified layers that contain the basement membrane of the retinal pigment epithelium (RPE), the innercollagenous layer, the central elastic layer, the outer collagenous layer, and the basement membrane of thechoriocapillaris endothelium (CCE) [1, 2]. Bruch’s membrane plays a critical role in the eye. Transport of oxygen,glucose, growth factors, and fluids derived from choroidal
vasculature to the retina/RPE and removal of waste* Hidetsugu Mori
morih@hirakata.kmu.ac.jp
1

Department of Ophthalmology, Kansai Medical University, 2-5-1
Shinmachi, Hirakata, Osaka 573-1191, Japan2

Department of Pharmacology, Kansai Medical University,
Osaka, Japan

materials from the RPE through BM supports the function
and health of RPE and photoreceptors [3–5]. Bruch’s membrane also works as a tight barrier to prevent abnormal
angiogenesis [6, 7] as the rupture of BM may induce choroidal neovascularization (CNV) [8–15]. Therefore, maintaining the five-layer structure of BM is essential for retinal
health and normal visual function. Particularly, damage ofelastic lamina and/or mutations in the genes encoding elastic fibers in BM is associated with severe vision loss by
CNV formation according to previous studies [6, 15, 16].
Chong et al. reported that discontinuities within the elastic
lamina of BM are significantly longer in patients with agerelated macular degeneration (AMD) than in age-matched
controls [6]. Sivaprasad et al. reported that serum levels ofelastin-derived peptides in patients with AMD are significantly higher than that in non-AMD patients [16]. Joneset al. reported that mouse RPE overexpression of humanhigh temperature requirement A 1 (HTRA1) induced
polypoidal choroidal vasculopathy (PCV) which is a subtype of AMD [15]. The degeneration of the elastic lamina

Graefes Arch Clin Exp Ophthalmolof BM induced by human HTRA1 as a multifunctional serine
protease that works as elastase activity [17, 18] was the major
factor for the occurrence of PCV.
Discontinuities of the elastic lamina of BM may be predictors for the occurrence of CNVs [6]. Among the five layers in
BM, proper homeostasis and turnover of the elastic lamina of
BM seems to be essential to prevent CNV. Therefore, it isimportant to improve understanding of the elastic lamina of
BM in terms of histology and molecular biology characteristics.
Elastic fibers give connective tissue resilience, deformability,
and recoil abilities without energy input [19, 20]. Elastic fibers
play the principal role in the structural integrity and function ofvarious organs [20]. Knowledge in the field of elastic fiber
assembly-related molecules has spread in recent years owing
to the identification of gene mapping of elastic fibers using
in vivo studies (transgenic mouse models) and in vitro studies(biochemical examinations) [19–48]. Elastic fibers are composed of two major composite biomaterials including an outerlattice of microfibrillar mantle mainly consisting of polymers offibrillin-1 and an inner amorphous core of crossed-linking
tropoelastin (elastin monomers) [20–22]. Several molecules associated with the coacervation and cross-linking between microfibrils and tropoelastin have been implicated in the processof elastic fiber assembly. Recent genetic studies have revealed
that fibulin-5, latent transforming growth factor-β-binding
protein-2 (LTBP-2), and LTBP-4 are involved in elastic fiber
assembly in addition to microfibrils and tropoelastin [34, 38].
Schematic view of elastic fiber assembly involves recruitment
and organized deposition of molecules including fibrillin-1,
tropoelastin, fibulin-5, LTBP-2, and LTBP-4, as shown in
Fig. 1. The complete molecular mechanism in elastic fiber assembly remains unclear. Structural components of these five
molecules (fibrillin-1, fibulin-5, tropoelastin, LTBP-2, and
LTBP-4) differ between organs. For example, ciliary zonules
consist of rich microfibrils and LTBP-2 only [34]. There havebeen no reports about whether these five molecules are
expressed in the elastic lamina of BM. Researching the elastic

Fig. 1 Roles of elastogenic molecules in elastic fiber assembly. LTBP-2
leads each microfibril to the formation of a microfibril bundle. The coacervation of tropoelastin is carried out by fibulin-5. A cluster of
tropoelastin and fibulin-5 is deposited onto microfibril bundles bylamina of BM may lead to elucidating the mechanism of CNV
development.
In this study, we analyzed the relationships between these
five molecules and the elastic lamina of BM through prenatal
and postnatal development and in aging by both qualitativeand quantitative assessments using wild-type mice.

Materials and methods
Animals
In the following descriptions, the day or week of embryonic or postnatal development were used (e.g., E15 to designate the 15th day after fertilization, P6 weeks to indicate
the 6th week after birth). In this study, male and female
C57BL/6 mice (each E12.5, E15.5, and E18.5 days; P1,
P4, and P7 days; and P3, P6, and P75 weeks of age) werepurchased from CLEA Japan (Tokyo, Japan). All animal
experiments were carried out in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic
and Vision Research and the Guideline for Animal
Experimentation at Kansai Medical University. Major ocular structures appear and are able to be differentiated between E10 and E15 days [49]. Therefore, fetal mice werestudied starting at E12.5 days in this study. Animals werehoused with a normal laboratory diet in a temperature andhumidity-controlled environment at 22 °C and 50% filteredair. All mice were kept in pathogen-free plastic cages with
12-h light-dark cycles and had continuous free access towater and food (CLEA Japan, Tokyo, Japan). All plastic
cages, water and bedding feed were purified before use.
Sixteen eyes of eight mice per time point were used foreach examination: immunohistochemistry, transmission electron microscopy (TEM), and quantitative real-time polymerase chain reaction (qRT-PCR).

LTBP-4, which is localized on microfibrils. Then, LOX-like enzyme
forms a cross-linked structure made up of the tropoelastin/fibulin-5 complex on microfibrils, which marks the completion of the elastic fiber
assemblyGraefes Arch Clin Exp Ophthalmol

We defined the embryonic period as three stages: early(E12.5 days), middle (E15.5 days), and late (E18.5 days).

Immunohistochemistry
All mice were killed by cervical dislocation after deep intraperitoneal injection of a mixture containing 7 μl/mg of ketamine hydrochloride (Daiichisankyo, Tokyo, Japan) and0.7 μl/mg of xylazine (Bayer, Leverkusen, Germany). Theeyes were enucleated immediately and frozen using dry ice,followed by embedding in optimal cutting temperature compound (Tissue-Tek OCT; Sakura Finetek, Torrance, CA,
USA). The frozen blocks were preserved overnight at −80 °C. Vertical and 8 μm thin-sliced sections were cut with acryostat (Leica CM3050S®; Leica Biosystems, Nußloch,
Germany). To minimize tissue damage during dissection,
Kawamoto’s films (SECTION-LAB, Hiroshima, Japan) wereused [50]. Each section with Kawamoto’s film was air dried atroom temperature for 20 min. Immunohistochemistry was
performed by the indirect immunofluorescence technique.
Briefly, cryostat sections were fixed with cold methanol at −20 °C for 5 min, washed with phosphate-buffered saline (PBS)
for 5 min, and blocked with 4% blocking agent prepared fromlactoprotein (Sumitomo Dainippon Pharma, Osaka, Japan) for
20 min. The sections were then incubated with the primary
antibodies anti-fibrillin-1 (1:100, original), mouse polyclonalanti-tropoelastin (1:100, Elastin Products Company), rabbit
polyclonal anti-fibulin-5 (1:100, BSYN2473), anti-LTBP-2(1:100, original) and goat polyclonal anti-LTBP-4 (1:100,
R&D Systems) for 1 h at room temperature. After incubation,these sections were rinsed with PBS, and then incubated witha combination of Alexa Fluor®-488, Alexa Fluor®-546(1:200, Thermo Fisher Scientific, Waltham, MA, USA), andDyLight®-650 (1:200, Leinco technologies, Fenton, MO,
USA) secondary antibodies for 1 h at room temperature. The
combinations of primary antibodies and secondary antibodies
are shown in Table 1. After rinsing with PBS, the sections
were mounted with ProLong®Gold mounting media

Table 1 All primary and secondary antibodies used in this study for
immunohistochemistryPrimary antibody

Secondary antibody

Fibrillin-1, tropoelastin, fibulin-5
LTBP-4
LTBP-2Anti-rabbit Alexa Fluor® 488
Anti-goat Alexa Fluor® 546
Anti-chicken DyLight® 650

Anti-fibrillin-1, tropoelastin, and fibulin-5 primary antibodies were
stained with Alexa Fluor⑧ 488 as the secondary antibody. Anti-LTBP-4
primary antibody was stained with Alexa Fluor⑧ 546 as the secondary
antibody. Anti-LTBP-2 primary antibody was stained with Alexa
Fluor⑧ 650 as the secondary antibody(Thermo Fisher Scientific, Waltham, MA, USA) for
preventing fluorescence photobleaching. The sections wereobserved and images were taken with a fluorescence microscope (BZ-X700®; KEYENCE, Osaka, Japan). Negative
control slides were stained by primary non-specific IgG (anti-goat, 144-09531; anti-rabbit, 148-09551) and/or IgY (antichicken, PSC-3707-1) antibodies one at a time to confirm
positive staining of immunohistochemistry.
Triple staining with the immunofluorescent technique was
performed; fibrillin-1 (Alexa Fluor®-488)-LTBP-4 (Alexa
Fluor®-488)-LTBP-2 (DyLight®-650), tropoelastin (Alexa
Fluor®-488)-LTBP-4 (Alexa Fluor®-488)-LTBP-2(DyLight®-650), fibulin-5 (Alexa Fluor®-488)-LTBP-4(Alexa Fluor®-488)-LTBP-2 (DyLight®-650).

TEM
After deep intraperitoneal injection as described above, allmice were killed and pre-fixed with perfusion fixation fromthe heart by 2% glutaraldehyde (Electron Microscopy
Sciences, Hatfield, PA, USA), 1 mM CaCl2 in 0.1 M sodium
cacodylate (Electron Microscopy Sciences, Hatfield, PA,
USA), and 1/15 M phosphate buffer without NaCl (pH 7.4).
Immediately afterwards, each eye was enucleated. We dissected the eyes carefully under a microscope at the corneal limbus
and removed the lens. Posterior eyecups were immersed in
fixation liquid of 2% glutaraldehyde and 1 mM CaCl2 in
0.1 M sodium cacodylate buffer (pH 7.4) and preserved at
4 °C for 2 h. After pre-fixation, samples were washed twice
by 0.2 M sucrose in 0.1 M sodium cacodylate buffer (pH 7.4)
for 10 min. Posterior eyecups were postfixed for 2 h in 1%
osmium tetroxide (Nissin EM, Tokyo, Japan) and 0.15 M sodium cacodylate buffer at 4 °C, dehydrated through a series ofsubsequent high-grade ethanol concentrations, and embedded
in epoxy resin (TAAB Laboratories Equipment Ltd.,
Aldermaston, Berks, England). Semi-thin sections (1 μm
thick) were cut with an ultramicrotome (REICHERT-NISSEIULTRACUT S®; Leica AG, Wien, Austria). These semi-thinsections were stained with toluidine blue for identifying BM
under a light microscope. Then, ultrathin sections were cut at
50 nm by an ultramicrotome with diamond knives (Diatome
AG, Biel, Switzerland) and mounted on 100 mesh copper
grids (Veco B.V., Karel van Gelreweg, Eerbeek, The
Netherland). These sections were stained with uranyl acetate(Cerac, Inc., Milwaukee, WI, USA) for 10 min and with leadcitrate (TAAB Laboratories Equipment Ltd., Aldermaston,Berks, England) for 5 min. Then these sections were stainedwith tannic acid (Nacalai Tesque, Kyoto, Japan) and p-nitrophenol (Wako, Osaka, Japan) for 1 h to facilitate the visualization of tropoelastin as small black dot-like deposits.
Electron micrographs of BM were viewed and taken with
80 kV under TEM (JEM-1400A®; JEOL, Tokyo, Japan).

Graefes Arch Clin Exp Ophthalmol

qRT-PCR
All mice were killed by cervical dislocation after deep intraperitoneal injection as described above. Immediately, after
each whole eye was enucleated, they were stored in
RNAlater® (Thermo Fisher Scientific, Waltham, MA, USA)and homogenized by Biomasher (Nippi Inc., Tokyo, Japan).
Total RNA was isolated using RNeasy Plus Micro® andMini® Kit (Qiagen, Venlo, The Netherlands) according to
the manufacturer’s protocol. Total RNAs were reversetranscribed to generate single-stranded complementary DNA(cDNA) using the SuperScript® VILO™ cDNA Synthesis
Kit (Thermo Fisher Scientific, Waltham, MA, USA). The
reverse-transcriptase cDNAs were then subjected to qRTPCR (Thermal Cycler Dice® Real Time System II;
TAKARA Bio, Otsu, Shiga, Japan). Each qRT-PCR was performed in a 20-μl volume using the SYBR® Premix Ex
TaqII™ PCR Kit (TAKARA Bio, Otsu, Shiga, Japan) protocol as follows: pre-denaturing at 95 °C for 30 s, 5-s denaturing
at 95 °C, and 30-s annealing and extending each at 60 °C.
Denaturing, annealing, and extending were repeated for
40 cycles.
Targeting sequences used in this study were amplified withthe following primers.
Fibrillin-1:&&

Forward, 5′-AATATCTCGGAGCCATTTGC-3′
Reverse, 5′-CAGGTCTACGGCAGTTGTCA-3′
Tropoelastin:&&

Forward, 5′-CTATGGAGGACCCCTTGGAG-3′
Reverse, 5′-CACAGGATTTCCCAAAGCAG-3′
Fibulin-5:&&

Forward, 5′-CCGATACCCTGGTGCCTATT-3′
Reverse, 5′-GCACTGATAGGCCCTGTTTG-3′
LTBP-2:&&

Forward, 5′-AAACCCCTCAGCGACCCGCGGCTGC-3′
Reverse, 5′-TGCTTCTGTGAGGACCGGGTGCTCT-3′
LTBP-4:&&

Forward, 5′-GCACAAATACTAAAGGCTCCTTCCAC-3′
Reverse, 5′-GACACTCGTCAATGTCAAGGCAGGAG-3′
Cyclophilin:&&

Forward, 5′-CAGACGCCACTGTCGCTTT-3′
Reverse, 5′-TGTCTTTGCAACTTTGTCTGCAA-3′

Quantitative mRNA expression levels in each age groupwere normalized to the amount of cyclophilin mRNA as aninternal control. Quantitative mRNA expression levels in eachage group were calculated by relative quantification (standard
curve method). We used the mRNA of the whole eye at P12
weeks as a standard sample. Quantitative mRNA expression
levels in eye tissues was calculated with logarithmic transformation and analyzed by JMP software (SAS institute, Inc.,
Cary, NC, USA). All data were presented as mean ± SD according to normal distribution by Bartlett’s test. Statisticalanalyses were performed by Tukey-Kramer test. P values <0.05 were considered statistically significant.

Results
Immunohistochemistry
Bruch’s membrane was observed as the gap between RPE
pigment and the choroid layer under bright-field light microscope (Fig. 2a). At embryonic stages, BM could not be detected clearly under the light microscope because BM was too
faint and fragile (Fig. 2b). The BM gap was not obvious in
the developing mouse at the embryonic early stage (Fig. 2b,left). However, this gap became visible from the embryonic
middle stage and onwards (Fig. 2b, center and right).
At embryonic early and middle stages, staining of the fiveproteins (fibrillin-1, tropoelastin, fibulin-5, LTBP-2, LTBP-4)
was not detected in BM (data not shown). However, fibrillin-1
showed a strong staining signal in the sclera at the embryonic
early stage (data not shown). Tropoelastin, fibulin-5, LTBP-2,
and LTBP-4 showed a strong staining signal in the sclera at theembryonic middle stage (data not shown). At the embryoniclate stage, positive staining of fibrillin-1 and LTBP-4 in BM
was isolated and weak (data not shown), but tropoelastin,fibulin-5, and LTBP-2 were positive in BM in some slides.
Immunohistochemical staining confirmed the consistent expression of all five matrix proteins in BM except in embryonicstages, which indicated that production and/or deposition of
the matrix proteins were dramatically enhanced at P1 days andonwards (Fig. 3). A summary of immunohistochemical staining in BM in each age group is shown in Fig. 4.
We further analyzed the localization pattern of each protein
in BM. Figure 5 represents a summary of the characteristic
staining pattern of each protein. Fibrillin-1 was detected aslinear continuous long lines at postnatal stages (Fig. 5a).
Tropoelastin was confirmed as a dot-like pattern at postnatal
stages (Fig. 5b). LTBP-2 and LTBP-4 were co-localized withfibrillin-1 (Fig. 5c). Moreover, there was co-localization between tropoelastin and LTBP-2 by merged images (Fig. 5d).
There was no clear difference in staining intensity among
these five proteins with regard to exposing time, fluorescenceintensity, or location (peripheral or center regions).

Graefes Arch Clin Exp OphthalmolFig. 2 Light microscopic appearance of Bruch’s membrane during
development. a Bruch’s membrane of C57/BL6 mice is observed as thewhite layer between the retinal pigment epithelium and the choroid undera bright-field microscope (red arrow). b Three bright-field images show

embryonic early (E12.5 days), middle (E15.5 days), and late (E18.5 days)
stages. A clear white layer confirms that Bruch’s membrane is fully assembled by the embryonic late stage. All images were taken at × 400
magnification

TEM

that developmental formation of BM was completed from
P3 to P6 weeks. The elastic lamina of BM could not be clearly
identified at P75 weeks because abundant dense deposits in
BM localized to the RPE and disturbed the ability to distinguish the structure of BM (Fig. 6i).
The elastic lamina of BM presented discontinuity fromembryo to senile stages in this study. Furthermore, the thickness of BM varied in location (Fig. 7).

At the embryonic early stage (E12.5 days), BM was mostly
observed as an empty space between the RPE and CCE.
Single layer as the basement membrane of RPE was lined
along with RPE. Short and small clusters of fibrous microfibrils were detected. Tropoelastin occasionally overlappedwith clusters of microfibrils (Fig. 6a). At the embryonic middle stage (E15.5 days), another single layer inside of the basement membrane of CCE appeared parallel to CCE. Two layersof collagenous fibrils were observed in the space between thebasement membrane of the RPE and elastic fibers. The basement membrane of the RPE was lined along with the CCE.
Clusters of microfibrils connected by tropoelastin graduallybegan to accumulate in the middle layer of BM (Fig. 6b). At
the embryonic late stage (E18.5 days), short and small clustersof immature elastic fibers began to increase and connect toeach other. Two collagenous fibrils began to form a threedimensional meshwork-like helix structure (Fig. 6c). At P1
day, extended microfibrils attached by tropoelastin grew andconnected to form as cross-linked structure in BM (Fig. 6d).
From P4 to P7 days, microfibrils with tropoelastin became
thick central bundles, indicating that it became a layer of relatively uniform structure (Fig. 6e, f). From P3 to P6 weeks, amostly consecutive single structure of microfibrils attached bytropoelastin became longer and thicker (Fig. 6g, h). Two collagenous layers became thick and formed a tight meshwork.
Bruch’s membrane was able to be identified as five discrete
layers during P3 to P6 weeks. Their morphology indicated

qRT-PCR
Fibrillin-1 mRNA
The mean mRNA level of fibrillin-1 (Fig. 8a) at E12.5 days
was low (926.3 ± 249.1). Significant elevation of mRNAlevels were observed at E15.5 days (13,954.4 ± 4371.5) compared with those of other time points (P < 0.01). mRNA expression levels were maintained until P7 days even thoughmRNA expression level was reduced between E15.5 and P7
days. mRNA expression levels were reduced back to early
embryonic levels by P3 weeks. There was no significant difference between E12.5 days and P6 weeks or between E12.5
days and P75 weeks.
Tropoelastin mRNA
The mean mRNA level of tropoelastin (Fig. 8b) at E12.5 days
was relatively low (540.5 ± 258.6). Significant elevations of
tropoelastin mRNA expression levels were observed at E15.5

Graefes Arch Clin Exp OphthalmolFig. 3 Immunohistopathological findings in Bruch’s membrane. Figure 3
is an image from a 6-week-old mouse. From postnatal stages and onwards, all proteins in Bruch’s membrane clearly showed positive

expression by staining. In each image, Bruch’s membrane is shown as ayellow arrow. All images were taken at × 400 magnification

days (23,843.1 ± 8030.1) compared with E12.5 days
(P < 0.01). The highest mRNA levels were observed at
P1 day (35,266.3 ± 8700.0). High mRNA levels were maintained between E15.5 and P7 days, and there were no significant differences in expression level between these time
points. At P3 weeks, tropoelastin mRNA levels were reduced.
No significant difference was detected between P75 weeks
and E12.5 days.

Fibulin-5 mRNA

Fig. 4 Summary of
immunohistochemistry results in
Bruch’s membrane from each age
group. The five proteins were
found to be expressed in Bruch’s
membrane byimmunohistochemistry inpostnatal mice; B+^ positive
staining, B−^ negative staining,
B±^ between negative andpositive

The mean mRNA level of fibulin-5 (Fig. 8c) were lowest at
E12.5 days (150.8 ± 68.6). At E15.5 days, mRNA levels were
elevated (2635.7 ± 689.1) and at their highest compared withthose of other time points (P < 0.01). Fibulin-5 mRNA levels
were significantly reduced at E18.5 days compared with
E15.5 days (P < 0.01) and were maintained at high values

Graefes Arch Clin Exp OphthalmolFig. 5 Patterns of protein expression in Bruch’s membrane. a Fibrillin-1,
b tropoelastin, c fibrillin-1, and LTBP-2 (left image) and fibrillin-1 and
LTBP-2 (middle image); d tropoelastin and LTBP-2. Fibrillin-1 proteinsshow relatively long, linear staining (red bracket). Tropoelastin proteinsshow dot-like staining (yellow, round). Co-localization of fibrillin-1 and

LTBP-2, fibrillin-1 and LTBP-2, and tropoelastin and LTBP-2 is observed. LTBP-2 and LTBP-4 staining overlaps with fibrillin-1.
Tropoelastin and LTBP-2 punctate staining overlapped. All images weretaken at × 400 magnification

between E18.5 days and P7 days (E18.5 days, 1406.7 ± 230.3;
P1 day, 1751.8 ± 408.9; P4 days, 1341.2 ± 312.3; P7 days,
1653.9 ± 417.7) (no significant difference among time points).
By P3 weeks, fibulin-5 mRNA levels showed significant reduction compared to that of P7 days (P < 0.01).

LTBP-4 mRNALTBP-2 mRNA
The mean mRNA level of LTBP-2 (Fig. 8d) was at its lowest
at E12.5 days (40.0 ± 83.0) compared with those of other time
points (P < 0.01). Significant elevations of mRNA levels wereobserved at E15.5 days (1859.9 ± 1206.9, P < 0.01). The
highest mRNA expression level was observed at E18.5 days
(3639.6 ± 1442.7). High mRNA levels were maintained fromE15.5 to P1 days. mRNA levels at P4 days were reduced
significantly compared with P1 day (P < 0.01). By P4 days,
mRNA expression levels plateaued.

The mean mRNA (Fig. 8e) level at E12.5 days was lowest(136.1 ± 41.7) compared with those of other time points(P < 0.01). Elevations of mRNA levels were observed at
E15.5 days (1433.4 ± 707.8). By E15.5 days, mRNA levels
plateaued.

Discussion
The number of AMD patients is increasing for many reasons
such as change of lifestyle (e.g., a western diet), an increase in
the senior population, and genetic factors [51–55]. Clinically,
AMD is divided into three types: typical AMD, PCV, and retinal angiomatous proliferation (RAP) [51–55]. Moreover, thedevelopment of CNV in AMD is divided into two types [52]:Graefes Arch Clin Exp OphthalmolFig. 6 Ultrastructural pre- and postnatal changes of Bruch’s membrane inwild-type mice. Transmission electron micrographs of the elastic lamina
of Bruch’s membrane at embryonic 12.5 days (a), 15.5 days (b), 18.5 days(c), P1 day (d ), P4 days (e), P7 days (f), P3 weeks (g), P6 weeks (h), andP74 weeks (i). Tropoelastin is stained as black dots by tannic acid and pnitrophenol. A microfibril/tropoelastin (red arrow) complex appeared atembryonic early stage (a), increased in expression in the embryonic middle stage (b), and formed connections with one other by the embryoniclate stage (c, d ). Extended microfibrils grew longer and thicker between
P4 days and P3 weeks (e–g). Mature structure of the elastic lamina of
Bruch’s membrane revealed relatively continuous and thick fibers by P3
to 6 weeks (f, g). At P74 weeks (i), basal linear deposits prevented a clear
elastic layer from being detected. Magnification—a × 28,700, b ×
90,400, c × 59,200, d × 74,800, e × 28,700, f × 28,700, g × 21,400, h ×
14,200, and i × 221,400

type 1 CNV arises from choroid to reach the sub-RPE space,
and type 2 CNV arises from choroid to reach the sub-retinal
space. Typical AMD has type 1 and/or type 2 CNV separately[51, 52]. PCV has only type 1 CNV [53]. RAP has types 1 and2 CNVs in addition to chorioretinal anastomosis [55]. Amongthe three subtypes of AMD, the number of PCV patients in Asia
is larger than that in Western countries [51, 53]. PCV can cause
severe visual loss due to massive hemorrhage in the eye by
ruptured polypoid lesions [54]. There have been many reports
about animal models of CNVs [8–15, 56]. Laser and surgicallyinduced CNVs by penetrating BM have been widely used as
experimental CNV models [8–11]. Recently, transgenic animalmodels have also been used for the investigation of CNVs
[12–14]. Most of CNVs in these animal models belong to type2 [8–14]. Conversely, only a few animal models of type 1 CNV
have been produced [15, 56]. Moreover, there has been only 1report about an animal model of PCV by Jones et al. This study
found that the degeneration of the elastic lamina of BM is afundamental factor for the occurrence of PCV [15]. There havebeen many reports characterizing the elastic lamina of BM;
however, most previous reports assessed BM by only histological evaluation. There have been no reports about the relationship between essential molecules (fibrillin-1, tropoelatin,
fibulin-5, LTBP-2 and LTBP-4) for elastic fiber assembly inthe elastic lamina of BM. In this study, we found that fibrillin1, tropoelastin, fibulin-5, LTBP-2 and LTBP-4 proteins wereexpressed constitutively in BM at all postnatal stages byimmunohistochemistry.
Fibrillins account for the majority of the microfibril complex [22–28]. Fibrillin monomers are long flexible molecules[24] encoded by three kinds of genes (fibrillin-1, fibrillin-2
and fibrillin-3) [27–29]. Each fibrillin-1 and fibrillin-2 plays
a role in elastic fiber assembly. Fibrillin-1 and fibrillin-2 double knockout mice showed hypoplasia of elastic fibers, andmice deficient of either fibrillin gene resulted in elastogenesis[28, 31]. The molecular organization of fibrillin-1 andfibrillin-2 is unclear. They have been found in the cornea[32], crystalline lens [33] and ciliary zonule [34, 35].

Graefes Arch Clin Exp OphthalmolFig. 7 Maturation of the elastic
lamina of Bruch’s membrane atpostnatal week 6. Transmission
electron microscopy findings inBruch’s membrane of C57/BL miceat P6 weeks showed a relatively
continuous elastic layer, with small
gaps between long microfibrils(yellow asterisks). RPE, retinal pigment epithelium; BM, Bruch’s
membrane; CC, choriocapillaris.
Magnification, × 7140

Mutations in the fibrillin-1 gene cause Marfan syndrome,
which is associated with ectopia lentis [35]. Whether
fibrillin-3 is involved in elastic fiber assembly or not remains
unknown [29]. Within the three isoforms of the fibrillin family, fibrillin-1 is mainly responsible for forming microfibrils[25]. Thus, we studied fibrillin-1. Fibrillin microfibrils werefirst described as small-diameter fibrils located close to thebasement membranes and also at the periphery of elastic fibers
under the electron microscope [23]. In this study, fibrilllin-1rich microfibrils were seen as long bundles in BM at all postnatal stages under TEM. This result corresponded with theappearance in immunohistochemistry, which indicated thatfibrillin-1 protein presents as a long linear pattern in BM at
all postnatal stages.
Tropoelastin is the major component of elastic fiber, andfibrillin-1 forms microfibrils that act as a scaffold fortropoelastin. Tropoelastin presents as the non-cross-linked soluble precursor of elastin at less than 20 °C. Tropoelastin represents two stages in elastic fiber assembly; coacervation andcross-linking. The aqueous solutions aggregate at 37 °C byinteractions between hydrophobic domains. This processtermed coacervation is reversible and thermodynamically controlled. The coacervate becomes steady and insoluble bycross-linking via lysyl oxidase (LOX)-like enzyme in an irreversible manner. LOX-like enzyme aligns tropoelastin molecules prior to cross-linking [19, 36, 37]. Thus, themicroaggregation of tropoelastin deposited onto microfibrils
and the cross-linked structure of tropoelastin by LOX-likeenzyme are the major processes that form mature elastic fibers. In this study, the microaggregation of tropoelastin wasobserved as aggregation of black dots on the microfibril bundle using tannic acid and p-nitrophenol under TEM. This result corresponded with the appearance of tropoelastin in immunohistochemistry as well, in which tropoelastin protein
stained as a dot-like pattern.
LTBPs are also integral components in elastic fiber assembly. There are four isoforms within the LTBP family (LTBP-1,
LTBP-2, LTBP-3, and LTBP-4). The LTBP family regulates
transforming growth factor-β (TGF-β) bioavailability bybinding and sequestering TGF-β [42]. Among the four isoforms of the LTBP family, LTBP-1 and LTBP-3 are not required for the elastic fiber assembly reported by studies on
mice and humans that are deficient in these genes [43].
LTBP-2, however, does not bind to TGF-β [44]. Mutations
in LTBP2 occur in ocular diseases including primary or secondary glaucoma, megalocornea, microspherophakia andectopia lentis [45–47]. LTBP-2 facilitates the formation of
thick microfibrils by binding fibrillin microfibrils into bundles
in ciliary zonules [34].
In the absence of LTBP-4, the elastin/fibulin-5 complex isunable to linearly deposit on microfibrils, and misplaced aggregation of elastin/fibulin-5 forms globular structures [48]. In
previous reports, interactions between fibrillin-1 and LTBP-2
or LTBP-4 indicated that LTBP-2 and LTBP-4 play a role information and maturation of microfibrils [34, 38]. In thisstudy, we confirmed interaction between fibrillin-1 and
LTBP-2 or LTBP-4 by localization in immunohistochemistryin BM. Moreover, we found a novel co-localization of LTBP2 and tropoelastin. We could not confirm co-localization offibulin-5 and tropoelastin or LTBP-4 by immunohistochemistry. Unfortunately, we did not have evidence for how LTBP-2
and tropoelastin interact with each other in BM.
LTBP-4 localized on microfibril bundles is essential for
linear assembly of elastin through interaction with fibulin-5[48]. LTBP-4 and fibulin-5 play a role in proper accumulationof tropoelastin to the microfibril scaffold [48]. Nakamura et al.
reported that the elastic fiber assembly process requires
fibulin-5 using fibulin-5-deficient mice. These knockout mice
resulted in loose skin, tortuous vessels, and emphysematous
lungs [38, 39]. Yanagisawa reported that fibulin-5 can strongly interact with tropoelastin [40]. Hirai reported that fibulin-5
not only binds with tropoelastin but also promotes coacervation of tropoelastin in cell culture [41]. In human donor eyes,
the fibulin-5 protein was localized in BM using immunohistochemistry [57]. In this study, fibulin-5 protein was expressed
in BM in postnatal mice similar to human BM.
We have shown that the elastic fibers in BM were divided
into microfibrils and tropoelastin separately using TEM. Our

Graefes Arch Clin Exp Ophthalmol
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Fig. 8 mRNA abundance of each elastic fiber assembly-related protein inwhole eye tissue. mRNA levels of fibrillin-1 (a), tropoelastin (b), fibulin5 (c), LTBP-2 (d ), LTBP-4 (e), and total mRNA (f) were evaluated at preand postnatal stages with quantitative real-time PCR. Total mRNA expression levels were upregulated before and after birth. From postnatal

weeks 3 to 6, mRNA expression plateaued at low levels, which indicatesthat production of each protein is finished in adult mice. The bars represent mean ± SD. Statistics were analyzed by Tukey-Kramer (n = 16
mice/group, P < 0.05)

TEM showed that there were slightly negligible clusters of
microfibrils in the hollow space of BM at the embryonic earlystage, which was consistent with the negative staining in immunohistochemistry and low level of each mRNA in qRTPCR at this same stage. TEM indicated that microfibril expression levels began to increase and tropoelastin began todeposit onto the cluster of microfibrils in the embryonic middle stage, which was consistent with the results of qRT-PCR.
The expression levels of these five molecules (fibrillin-1,
tropoelastin, fibulin-5, LTBP-2, and LTBP-4) were elevatedin the embryonic middle stage.
Connections of each microfibril by tropoelastin between
embryonic to postnatal stages allowed the elastic lamina of
BM to gradually become mature fibers as shown under
TEM. This TEM result coincides with immunohistochemistry,
which shows positive staining of the five proteins by postnatal
stages in immunohistochemistry.
Hirabayashi et al. defined the completion of the development of the elastic lamina of BM in normal mice as P6 weeks
by the continuity of elastic fibers using TEM [2]. However,
the elastic lamina of BM does not have full continuity fromembryo to senile stages. In this study, we showed the maturation of the elastic lamina of BM using qualitative (TEM and
immunohistochemistry) and quantitative (qRT-PCR) examinations. Relatively thick and continuous microfibrils withtropoelastin were observed from P3 to P6 weeks under
TEM. Immunohistochemistry of all five proteins showed positive staining starting at postnatal stages. The mRNA levels offibrillin-1, tropoelastin, and fibulin-5 remained high until P7days and decreased by P3 weeks as shown by qRT-PCR. The
mRNA levels of LTBP-2 and LTBP-4 remained high until P4days and decreased by P7 days as shown by qRT-PCR. We
also found that mRNA levels of the five proteins involved inelastic fiber assembly decreased starting at either P7 days or
P3 weeks. According to the results of TEM, immunohistochemistry, and qRT-PCR, we define the maturation of elastic
lamina of BM at P3 to P6 weeks.
With age, BM loses normal function and leads to degenerative changes [6, 58–65]. Discontinuities of elastic fiber of
BM were observed in senescence accelerated mice (SAM),
aging mice, and elderly people according to numerous studies[6, 16–23]. Increased length of discontinuities of the elastic
lamina of BM may be the predilection for the occurrence of
CNVs [6]. Furthermore, lipid deposits and other debris which
are likely to play a role in the pathogenesis of AMD accumulated in BM in aging eyes [66, 67]. In this study, we showedabundant dense deposits in BM of senile mice (P75 weeks).
These deposits aligned sub-RPE and BM in a similar mannerto the basal linear deposits of human AMD. It was difficult to
detect the elastic lamina of BM clearly in senile mice because

Graefes Arch Clin Exp Ophthalmolof the abundant dense deposits located between the basement
membrane of the RPE and the elastic lamina. Stone et al.
hypothesized that fibulin-5 may be involved in basal deposit
formation from the result that human patients with AMD havemissense mutations in fibulin-5 [60]. The mechanism of how
fibulin-5 mutations are related with AMD is not understoodcompletely and must be addressed.
There are some limitations of this study. Further studies are needed to elucidate the elastic fiber assembly,
age-related changes, and the formation of deposits in
BM in the context of these five molecules. In this study,
mRNA expression levels by qRT-PCR were measured
from the whole eye. The eye itself contains various
elastic fiber tissues such as the sclera, ciliary zonules,
and BM. To strictly examine mRNA levels of the elastic
lamina of BM, we would need to perform qRT-PCR
using laser capture microdissection microscopy. Instead,
we qualitatively assessed our proteins by using immunohistochemistry. To compare the quantitative protein
expression levels, using Western blot or ELISA is recommended. In this study, we performed qRT-PCR on
total ocular tissue because to collect proteins from sucha small tissue as Bruch’s membrane would yield a levelof protein that is too small for analysis. In the future,
we will have to study how each molecule interacts byusing an immunoelectron microscope to fully understandhow these proteins are assembled. Further studies including fibrillin-1, tropoelastin, fibulin-5, LTBP-2, and
LTBP-4 transgenic mice may lead to understanding theelastic fiber formation in BM, which furthermore maylead to understanding the pathology of types 1 and 2
CNVs.
In summary, we studied immunohistochemical localization, ultrastructural changes, and mRNA levels of five proteins (fibrillin-1, tropoelastin, fibulin-5, LTBP-2, and LTBP4) in the EL of BM. We conclude that the EL became maturefrom P3 to P6 weeks as formerly stated. The structure of EL
remained discontinuous throughout the age groups. Elastic
fibers are still poorly understood, particularly in the BM, because they are complex and difficult to illustrate. Degradationof elastic fibers in BM may cause age-related disease such as
AMD. Future therapeutic interventions which regenerate elastic fibers may be used instead of, or alongside, intravitreal
anti-vascular endothelial growth factor (VEGF), and/or placental growth factor (PlGF) for the treatment of CNV.
Acknowledgements The authors thank Dr. Tomoya Akama and Dr.
Tomoyuki Nakamura (Department of Pharmacology, Kansai Medical
University, Osaka, Japan) for helpful discussion and for the gift of antibodies against fibrillin-1 and LTBP-2.
Author contributions Research design was performed by Hidetsugu
Mori, Keiko Toyama, Tomoya Akama, Tadashi Inoue, and Tomoyuki
Nakamura. Assays were performed by Hidetsugu Mori and Keiko

Toyama. Data analysis was performed by Hidetsugu Mori. Hidetsugu
Mori wrote the paper with support of Haruhiko Yamada, Kanji
Takahashi, Tomoya Akama, and Tomoyuki Nakamura.
Funding information None.

Compliance with ethical standards
All animal experiments were carried out in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research andthe Guideline for Animal Experimentation at Kansai Medical University.
Conflict of interest The authors declare that they have no conflict ofinterest.
Abbreviations LTBP, Latent transforming growth factor beta-binding
protein; EL, Elastic lamina; BM, Bruch’s membrane; IH,
Immunohistochemical staining; TEM, Transmission electron microscopy; qRT-PCR, Quantitative real-time PCR; RPE, Retinal pigment epithelium; CCE, Choriocapillaris endothelium; CNV, Choroidal neovascularization; AMD, Age-related macular degeneration; HTRA1, Human hightemperature requirement A 1; PCV, Polypoidal choroidal vasculopathy;
PBS, Phosphate-buffered saline; LOX, Lysyl oxidase; TGF-β,
Transforming growth factor-β; VEGF, Vascular endothelial growth factor; PlGF, Placental growth factor
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.

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