v9a16-fernandez-durango pgmkr

Molecular Vision 2003; 9:103-9 <http://www.molvis.org/molvis/v9/a16>
Received 21 February 2003 | Accepted 3 April 2003 | Published 9 April 2003
© 2003 Molecular Vision
Localization of endothelin-1 mRNA expression and immunoreactivity in the anterior segment of human eye: Expression of ETA
and ETB receptors
Raquel Fernández-Durango,1 Raquel Rollín,1 Aránzazu Mediero,1 Manuela Roldán-Pallares,2 Julián García
Feijo,2 Julián García Sánchez,2 Arturo Fernández-Cruz,1 Ainhoa Rípodas1
1
Unidad de Investigación, Departamento de Medicina Interna III, and 2Departamento de Oftalmología, Hospital Clínico San Carlos,
Ciudad Universitaria, Madrid, Spain
Purpose: Endothelin-1 (ET-1), a potent vasoactive peptide, is an important regulator of intraocular pressure. Actually,
there is evidence of a role for ET-1 in the pathogenesis of glaucoma. However, the expression pattern of ET-1 and its
receptors, ETA and ETB, in the anterior segment of human eye are not known. In the current study, we have examined the
expression and distribution of ET-1 as well as the expression profile of ETA and ETB genes in the iris, ciliary muscle, and
ciliary processes of human eyes.
Methods: Six normal human eyes with no history of eye diseases were fixed, embedded in paraffin and sectioned. Cellular localization of ET-1 was identified by in situ hybridization and immunohistochemistry. Iris, ciliary processes, and
ciliary muscles were dissected from six normal human eyes and quantitative real time RT-PCR was used to quantify the
expression of ETA and ETB.
Results: In situ hybridization revealed the presence of ET-1 transcripts in the iris, nonpigmented epithelial ciliary cells,
and ciliary muscle. Immunohistochemical studies showed that ET-1-like immmunoreactivity appeared in the same regions where ET-1 mRNA was expressed as well as in trabecular cells, inner and outer endothelial cells lining Schlemm’s
canal, corneal epithelial, and limbus cells. Quantitative real time RT-PCR demonstrated that the expression of ETA and
ETB receptors is greatest in the iris, followed by ciliary muscle and ciliary processes.
Conclusions: ET-1 and its receptors ETA and ETB are constitutively expressed in the anterior segment of human eye.
These results indicate that ET-1 may play a physiological role in the regulation of intraocular pressure through its ETA and
ETB receptors in human eye. In addition, ET-1 present in corneal epithelium and limbus may function in regulating cell
proliferation and/or differentiation.
lium cells [13], retina, and optic nerve [14]. ET-1 mRNA expression has been localized in human retina and optic nerve
and ETA and ETB gene expression have been detected in human retina [14].
There is much evidence suggesting that ET-1 plays a role
in the local regulation of intraocular pressure (IOP). In vivo,
intracameral and intravitreous injections of ET-1 into rabbit
eyes produced a prolonged reduction of IOP [6]. The mechanisms contributing to ET-1-induced ocular hypotension could
be a reduction in AH formation and/or an increase in the outflow facility. In fact, ET-1 induced contraction in bovine ciliary muscle (CM) and trabecular meshwork (TM) [15]. In studies with perfused monkey eyes, ET-1 increases outflow facility, probably by a direct effect on the CM [16]. Tanaguchi, et
al. [17] also observed that intravitreous injection of ET-1 in
the rabbit eye produced an increase of outflow facility and a
decrease of AH formation. The inhibition of Na+/K+-ATPase
produced by ET-1 in human nonpigmented ciliary epithelial
(NPCE) cells could explain the reduction in AH production
[18].
Furthermore, IR-ET-1 concentrations in the AH of patients with primary open angle glaucoma are significantly
higher than in those matched normal non-glaucomatous sub-
Endothelin-1 (ET-1) is a very potent endogenous vasoconstrictor, isolated from culture medium of porcine aortic
endothelial cells [1]. Three distinct endothelin genes encode
the closely related products ET-1, ET-2, and ET-3 [2]. These
three isoforms mediate their biological actions by two receptor subtypes, ETA and ETB [3,4]. The ETA receptor, which is
located on vascular smooth muscle cells, mediates potent vasoconstrictor actions and binds preferably the ET-1 isoform [5].
The ETB receptor is located on vascular endothelial cells and
binds equipotently all three ET isoforms. The ETB receptor is
thought to mediate vasodilatation through the release of nitric
oxide and prostaglandins [2,5].
In the eye, the presence of ET system has been intensely
investigated. Immunoreactive ET-1 (IR-ET-1) and IR-ET-3 as
well as their mRNAs have been detected in the iris-ciliary
body, choroid, and retina of the rat [6-9]. ETA and ETB receptors have been located in the ciliary body and in the vascular
and neural retina of the rat [7,10,11]. In human, IR-ET-1 is
present in aqueous humor (AH) [12], human ciliary epitheCorrespondence to: Raquel Fernández-Durango, Unidad de
Investigación, Departamento de Medicina Interna III, Hospital Clínico
San Carlos, 28040 Madrid, Spain; Phone: 91-3303434; FAX: 915445432; email: [email protected]
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© 2003 Molecular Vision
jects [19,20]. A significant increase in the plasma levels of
IR-ET-1 of normotensive glaucoma patients were found compared to plasma of normal subjects [21,22].
All these data suggest that ET-1 has an important role as
a modulator of IOP and that might be involved in the development of glaucoma. However, as far as we know, in the anterior segment of human eye the localization of IR-ET-1 is not
well defined and the distribution of ET-1 mRNA expression is
yet unknown.
Therefore, the present study investigates in the human
anterior segment the distribution of ET-1 mRNA expression
by in situ hybridization, the localization of IR-ET-1 by immunohistochemistry, and the expression of ETA and ETB by quantitative real time polymerase chain reaction (PCR).
Immunohistochemistry for ET-1: Immunohistochemistry for ET-1 was performed as previously described [14].
Briefly, deparaffinized and hydrated sections were incubated
with 5% bovine serum albumin (BSA) in 0.1 M phosphate
buffer saline (PBS), for 10 min at room temperature to reduce
non-specific binding. A rabbit anti-ET-1 polyclonal antibody
(Peninsula Laboratories, Inc. Belmont, CA) was used at 1:400
dilution. Immunostaining was developed using the “super sensitive immunodetection system” kit (Biogenex, San Ramon,
CA). Sections were incubated for 20 min with diluted
biotinylated anti-rabbit immunoglobulins as directed by the
supplier and, after washing, incubated with the alkaline phosphatase conjugated streptavidin diluted in PBS for 20 min at
room temperature. The alkaline phosphatase activity was visualized with naphthol phosphate and Fast Red chromogen
(Biogenex), which resulted in red staining. The sections were
lightly counterstained with Mayer’s haematoxylin. For control, tissue sections were incubated either with primary antibody preabsorbed with 10 nM ET-1 or with normal rabbit serum instead of primary antibody. The commercially available
polyclonal anti-ET-1 serum has cross reactivity with ET-2 of
91% and with ET-3 of 0.05%.
RNA extraction: Anterior segments of the enucleated eyes
were removed, placed in a dish with the inner side up, and the
iris, ciliary process (CP), and CM were microdissected. Tissues were immediately frozen in liquid nitrogen and stored at
-70 °C until use. Total mRNA was isolated using the RNeasy
Mini kit (Qiagen, Santa Clara, CA). Extraction procedure was
performed according to the manufacturer’s instructions.
Real time quantitative RT-PCR: First strand cDNA was
synthesized using 2.5 µM random hexamers and 1.25 U/µl
MultiScribe Reverse Transcriptase (Applied Biosystems. Foster City, CA) according to the manufacturer’s recommendations.
Real time quantitative RT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR
Green dye on a ABI Prism® 7700 Sequence Detection System (Applied Biosystems). The normalized fluorescent intensity of the reporter dye (∆Rn) is plotted against cycle number
to derive a graphical representation of the PCR reaction. The
threshold cycle Ct is defined as the cycle number at which the
∆Rn passes the statistically significant level of 10 times the
standard deviations of the baseline emission during the fist 10
cycles of the PCR The Ct is linearly proportional to the logarithm of the input copy number and the slope of the best fit
line is a measure for the reaction efficiency E=10-(1/slope) according to the manual instructions. The quantity of cDNA was
calculated by normalizing the Ct of genes of interest (target)
to the Ct of the housekeeping β-actin(endogenous reference)
of the same sample, according to the standard curve method
(Bulletin number 2 ABI PRISM® 7700 Sequence Detection
System). All reactions were performed at least in duplicated
and controlled by standards (nontemplate controls and standard positive control).
Primers were designed with the Primer Express software
package that accompanies the Applied Biosystems Prism®
7700 Sequence Detector. The sequences of these primers were:
METHODS
Tissue specimens: Donor human eyes, provided by the Tissue
Bank (Hospital Clínico San Carlos, Madrid, Spain) were removed after death and enucleated within 2-3 h post-mortem.
The eyes were from twelve subjects (7 women and 5 men; age
range, 65-75 years old) without diabetes or ocular diseases as
determined by their clinical history. All research procedures
involving humans were in accordance with institutional guidelines on the Declaration of Helsinki.
Six eyes were fixed in phosphate buffered 4% paraformaldehyde (pH 7.4) for 2 days, dehydrated with a graded series
of ethanol, hemisected horizontally and embedded in paraffin
wax. Serial transverse sections of 4 µm thickness were mounted
on either polylysine- or 2% 3-aminopropyl triethoxysilane in
acetone (APES)-treated slides, for immunohistochemistry and
in situ hybridization, respectively. The other six eyes were
used for real time PCR.
In situ hybridization for ET-1: In situ hybridization for
ET-1 was performed as previously described [14]. Briefly, template DNA was a 600 bp cDNA (537-1162 bp) encoding human ET-1, which was subcloned in both orientations into the
EcoR I site of Bluescript M13 KS+ vector. Those cDNA clones
were generously donated by Dr. Derek Nunez (Cambridge,
UK). The plasmids were linearised with EcoR V prior to transcription. Single strand sense and antisense digoxigenin labelled RNA probes were generated by in vitro transcription of
the DNA with T7 RNA polymerase using the DIG RNA labeling Kit (Boerhinger-Mannhein, Bedford,MA).
Prehybridisation was performed for 30 min at 55 °C. Hybridization was done with 10 ng/µl denatured digoxigenin-11-UTP
labeled riboprobes in hybridization buffer overnight at 45 °C
in a moist chamber. After washing at room temperature to a
final stringency of 0.2X SSC, the slides were digested with 50
µg/ml RNase A at room temperature for 5 min. Data acquisition for of digoxigenin was carried out using a Detection Kit
(Boerhinger Mannheim) according to the manufacturer’s instructions. The slides were developed in NBT/BCIP (nitroblue
tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate).
Control sections were taken as serial sections from the
same tissue block and included; (1) hybridization with sense
probe, (2) RNase treatment before hybridization with antisense
probe, and (3) the omission of the RNA probe.
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© 2003 Molecular Vision
5'-GCT TCC TGG TTA CCA CTC ATC AA-3', forward and,
5'-TAG TCT GCT GTG GGC AAT AGT TG-3', reverse for
ETA and 5'-GCCAAGGACCCATCGAGAT-3', forward
and,5'-GAAGTGTGGAGTTCCCGATGAT-3' for ETB, reverse. The β-actin primers were 5'-AGA TGA CCC AGA TCA
TGT TTG AGA-3', forward and, 5'-ATA GGG ACA TGC GGA
GAC CG-3' reverse. PCR was performed using a kit (Applied
Biosystems) The PCR mixture consisted of 12.5 µl 2X SYBR
Green PCR Master Mix, 5µl (0.15 ng) of RT product and 300
nM primers in a total volume of 25µl. Standard amplification
parameters were used and were as follow: 50 °C for 2 min for
AmpErase, 95 °C for 10 min to inactivate the AmpErase and
to activate the Ampli Taq Gold DNA polymerase, followed by
45 cycles, each of which comprised melting step at 95 °C for
15 s and annealing extension at 60 °C for 1 min.
Data analysis: To compare the relative abundance of ETA
and ETB mRNAs, standard curves of ETA, ETB, and β-actin
were generated using cDNA synthesis from serial 1:5 dilutions of a RNA sample, prepared by pooling a fraction of the
RNAs of all individual samples included in this study. For
each sample, the amount of ETA, ETB, and β-actin was determined from those standard curves. The resulting ETA and ETB
amounts were divided by the β-actin amount to obtain a normalized value.
RESULTS
Localization of ET-1 mRNA by in situ hybridization: In situ
hybridization revealed the presence of ET-1 mRNA transcript
in the iris, ciliary body, and CM (Figure 1). In the iris, strong
hybridization signals were obtained in sphincter muscles,
stroma and blood vessels (Figure 1A). Circular, radial and longitudinal fibers of the CM showed intense hybridization signal (Figure 1C). Also, strong ET-1 mRNA expression was seen
in the human NPCE cells and stroma of the ciliary body (Fig-
Figure 1. ET-1 expression in the
anterior segment of human eye.
Expression of ET-1 mRNA in human iris, ciliary processes and ciliary muscle by in situ hybridization
with DIG labeled antisense probe
(A, C, E). Positive ET-1 mRNA
signals (blue) were observed in the
iris (A), in the CM (C), and in the
NPCE and stroma of the CP (C, E).
No substantial staining was seen
when hybridized with the sense
probe (B, D). Abbreviations are
used in the figure for iris (I), ciliary process (CP), and ciliary
muscle (CM).
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© 2003 Molecular Vision
ure 1C,E).Sections incubated with the sense probe for ET-1
mRNA showed no positive signal (Figure 1B,D). Similarly,
RNase treatment before hybridization or the omission of RNA
probe did not labeled any anterior segment structures.
Localization of ET-1 immunoreactivity: In the iris, specific and intense immunolabelling for ET-1 was obtained on
the cytoplasm of fibroblasts and clump cells as well as on
endothelial cells within blood vessels (Figure 2A). The adventicia of the iris vessels was strongly positive. The sphincter muscles showed positive signal. The cells from the anterior, circular and longitudinal portion of the CM reacted similarly to the anti-ET-1 antibody (Figure 2C,E). The nonpigmented epithelium and the stroma of CP were strongly stained
(Figure 2E).
In the cornea, positive immunostaining for ET-1 was
present in all cell layers (the surface layer, and in the cytoplasm of the superficial, wing and basal cells). All the endothelial cells exhibited positive staining (data not shown). However, in the limbus only wing cells showed strong positive
staining (Figure 2F). ET-1 immunoreactivity was also located
on trabecular cells in the uveal and corneoscleral meshwork
as well as on endothelial cells lining the Schlemm’s canal (Figure 2H). Sections of human eyes incubated with normal serum or anti-ET-1 preincubated with ET-1 showed no specific
labeling (Figure 2B,D,G,I).
Quantification of ETA and ETB mRNAs in the iris, CP,
and CM: The reaction efficiencies (E) values derived for βactin, ETA and ETB primers were close to 2, indicating near
optimum PCR amplifications. Moreover, the real time detection of dsDNA allows construction of a dissociation curve at
the end of the PCR run by ramping the temperature of the
sample from 60 °C to 95 °C while continuously collecting
fluorescence data. The curves of the melting profiles of ETA
and ETB receptors and housekeeping gene did not reveal an
accumulation of primer dimers (data not shown). Figure 3
shows the electrophoresis of the specimens after real time PCR
for ETA and ETB in iris, CP, and CM. The obtained bands
were of the expected 84 bp and 98 bp sizes, respectively.
No effects were found of either enucleation or postmortem interval on β-actin, ETA and ETB genes. No correlation
with age was found for the β-actin, ETA and ETB encoding
Figure 2. Localization of ET-1 immunoreactivity in the anterior segment of the human eye. Immunolocalization of ET-1 (A, C, E, F,
H). Positive ET-1 immunoreactivities (red) were shown in the iris
(A), the CM (C), the stroma and NPCE cells of the CP (C, E), the
epithelial cells of limbus (F), the trabecular cells and in the outer and
inner walls of Schlemm’s canal (H). Negative controls were obtained
with anti-ET-1 preabsorbed with 10 nM ET-1 (B, D, G, I). All images were magnified 66 times. Abbreviations are used in the figure
for iris (I), ciliary process (CP), ciliary muscle (CM), limbus (L),
trabecular meshwork (TM), and Schlemm’s canal (SC).
Figure 3. Detection of ETA and ETB mRNAs in human anterior segment. mRNA was extracted from human iris (I), ciliary muscle (CM)
and ciliary processes (CP), reversed transcribed and amplified by
real time PCR. The products were electrophoresed in 2.5% agarose
gel and visualized by staining with ethidium bromide. The lanes labeled “MW” and “BL” are a molecular weight marker and
nontemplate control, respectively.
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iris sphincter, ETA receptors constitute about 72% of the total
ET-1 receptor population and they were coupled to
phosphoinositide hydrolysis and muscle contraction [25]. ET1 causes contraction of the rabbit iris dilator muscle [26]. In
contrast, ET-1 does not affect the pupil diameter of monkeys
[16]. These discrepancies could be due to species differences.
In the human CP, both ET-1 mRNA and ET-1 immunoreactivity signals were demonstrated in the NPCE cells and in the
stroma. IR-ET-1 has been detected in the iris-ciliary body of
rat and rabbit [6,7] and in cultured human NPCE cells [13].
Our real time PCR data shows that in the human CP, the gene
expression of ETA was similar to that of ETB receptor. In
contrast, ETA receptors predominate in primary and transformed human NPCE cells [27]. Interestingly, we have previously localized, using autoradiography methods, ETA and ETB
receptors in the rat ciliary body in a ratio of 35:65 [10]. These
discrepancies could be due to species differences. All these
results indicate that ET-1, synthesized and released by human
NPCE cells, may act in an autocrine manner to regulate AH
formation.
We also identified both ET-1 gene expression and ET-1like immunoreactivity in the TM, in the outer and inner walls
of Schlemm’s canal, in the anterior circular, radial and longitudinal CM, as well as in the connective tissues of the human
ciliary body. Our quantitative real-time PCR demonstrates that
ETA gene expression in human CM is similar to that of ETB
receptor. However, using RT-PCR techniques, only the ETA
receptor was expressed in human CM and TM cells [27]. This
discrepancy could be explained either because the sensitivity
of our real time RT-PCR is greater than that of the normal
PCR or because in culture the cells suffer a variation of the
proportion of ETB receptor. In fact, it has been suggested that
culture conditions may induce species dependent changes in
ET receptor subtype proportions [28]. Furthermore, ET-1 has
been shown to induce contraction of isolated human CM strips
and cultured human CM cells through ETA receptors [12,29].
In isolated bovine CM strips, ET-1 caused contraction through
the ETA receptor and relaxation as a result of ETB receptor
activation [30]. Recently, there was evidence that Unoprostone
isopropyl (Rescula; Novartis Ophthalmic AG, Basel, Switzerland), a new docosanoid, may lower IOP in patients with glaucoma by affecting aqueous outflow through inhibition of ET1 dependent mechanisms [31]. Thus, the synthesis and secretion of ET-1 by CM and TM could regulate their contraction
or relaxation and ultimately affect AH outflow either by
paracrine or autocrine pathways.
Our results demonstrate that ET-1 is expressed in most of
the cells that are in contact with the AH, corneal cells, endothelial cells, NPCE cells, TM cells, and endothelial cells lining
Schlemm’s canal. This suggests that these cells could contribute to the secretion of ET-1 into AH where the ET-1 concentration levels are 2-3 higher than the corresponding plasma
levels [15].
Furthermore, from a physiological point of view, there is
evidence indicating that endothelins control the IOP [6]. Our
findings suggest that ET-1 and its receptors may affect the
IOP through changes in the AH formation and in the outflow
genes in the range included in our study (65-75 years). The
expression profile for ETA and ETB mRNAs levels relative
to β-actin in ocular tissues is shown in Figure 4A and B, respectively. The results, presented in order of abundance, show
higher levels of ETA gene expression in the iris, followed by
the CM; the CP showed the lower expression levels (Figure
4A). Similarly, the ETB expression levels were more abundant in the iris followed by the CM and CP (Figure 4B). In the
iris, the ETB mRNA levels were 2 fold higher than those of
ETA mRNA levels. However, in CM and CP the ETA mRNA
levels were similar to those of ETB mRNA levels.
DISCUSSION
In this study, we have described for the first time the expression of the ET-1 gene in the anterior segment of the human
eye. In the iris, we demonstrated that ET-1 mRNA as well as
ET-1 peptide appear in the stroma, vessels, and in the sphincter and dilator muscles. Previously, ET-1 mRNA has been
shown in the rat iris [23]. In agreement with our results,
Wollensak, et al. [24] have localized ET-1 immunoreactivity
signals in the stroma and in the adventitia of the vessels. Our
results using quantitative real time PCR demonstrated that in
human iris, ETB expression was 2 times that of the ETA. The
expression of both receptors was higher in the iris than in the
CP and CM. It was determined that ETA and ETB receptor
subtypes exist in the bovine iris sphincter, and that ETB receptors represent 80% of the total ET-1 receptors and were
linked to increases in cyclic AMP formation and not to muscle
contraction, in agreement with [25]. However, in the rabbit
Figure 4. ETA and ETB gene expression in the human anterior segment. Quantification by real time RT-PCR of ETA and ETB expression in human iris, ciliary muscle (CM) and ciliary processes (CP).
A: ETA expression in iris, CM and CP was normalized to β-actin
expression levels in the same cDNA samples. B: ETA expression in
iris, CM and CP was normalized to β-actin expression levels in the
same cDNA samples. The bars represent the mean; the error bars
represent the standard deviation.
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9. de Juan JA, Moya FJ, Fernandez-Cruz A, Fernandez-Durango R.
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facility. The expression of ET-1 was observed in the cornea
epithelium, which is in agreement with a previous observation [24]. It is known that ET-1 promotes corneal epithelium
wound healing in rabbits [32]. Interestingly, ET-1 was expressed in the basal, wing and superficial cells of peripheral
cornea and in the wing cells of the limbus. These epithelial
cells are formed by differentiation and migration of multipotent
stem cells in the limbal basal layer to produce the stratified
squamous epithelium [33]. It is thus of interest to study the
possible involvement of ET-1’s action on the migration of
epithelial cells during wound healing in the cornea and limbal
conjunctiva.
In conclusion, in human eye ET-1 is synthesized and secreted in the iris, NPCE cells, CM, trabecular cells, endothelial cells lining the Schlemm’s canal, corneal epithelial cells,
and limbus cells. All those cells are in contact with the AH,
thus ET-1 may regulate the IOP in the eye through ETA and
ETB receptor subtypes either in a paracrine or autocrine way.
Moreover, the ET-1 found in corneal epithelial and limbus cells
may function in regulating cell proliferation and/or differentiation. Further studies will be required to elucidate the possible involvement of ET-1 in glaucoma and in ocular inflammation.
ACKNOWLEDGEMENTS
This work was supported by Fondo de Investigaciones
Sanitarias, grant FIS 01/266. Raquel Rollín is a fellow from
Fondo de Investigaciones Sanitarias, grant BEFI 00/9140.
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The print version of this article was created on 10 Apr 2003. This reflects all typographical corrections and errata to the article through that
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