|Molecular Vision 2003;
Received 21 February 2003 | Accepted 3 April 2003 | Published 9 April 2003
Localization of endothelin-1 mRNA expression and immunoreactivity in the anterior segment of human eye: Expression of ETA and ETB receptors
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
1Unidad de Investigación, Departamento de Medicina Interna III, and 2Departamento de Oftalmología, Hospital Clínico San Carlos, Ciudad Universitaria, Madrid, Spain
Correspondence to: Raquel Fernández-Durango, Unidad de Investigación, Departamento de Medicina Interna III, Hospital Clínico San Carlos, 28040 Madrid, Spain; Phone: 91-3303434; FAX: 91-5445432; email: email@example.com
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.
Endothelin-1 (ET-1) is a very potent endogenous vasoconstrictor, isolated from culture medium of porcine aortic endothelial cells . Three distinct endothelin genes encode the closely related products ET-1, ET-2, and ET-3 . 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 . 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) , human ciliary epithelium cells , retina, and optic nerve . 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 .
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 . 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) . In studies with perfused monkey eyes, ET-1 increases outflow facility, probably by a direct effect on the CM . Tanaguchi, et al.  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 .
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 subjects [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).
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 . 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.
Immunohistochemistry for ET-1
Immunohistochemistry for ET-1 was performed as previously described . 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%.
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 (ΔRn) 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 ΔRn 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: 5'-GCTTCCTGGTTACCACTCATCAA-3', forward and, 5'-TAGTCTGCTGTGGGCAATAGTTG-3', reverse for ETA and 5'-GCCAAGGACCCATCGAGAT-3', forward and,5'-GAAGTGTGGAGTTCCCGATGAT-3' for ETB, reverse. The β-actin primers were 5'-AGATGACCCAGATCATGTTTGAGA-3', forward and, 5'-ATAGGGACATGCGGAGACCG-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.
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.
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 (Figure 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 β-actin, 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 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.
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 . In agreement with our results, Wollensak, et al.  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 . However, in the rabbit iris sphincter, ETA receptors constitute about 72% of the total ET-1 receptor population and they were coupled to phosphoinositide hydrolysis and muscle contraction . ET-1 causes contraction of the rabbit iris dilator muscle . In contrast, ET-1 does not affect the pupil diameter of monkeys . 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 . 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 . Interestingly, we have previously localized, using autoradiography methods, ETA and ETB receptors in the rat ciliary body in a ratio of 35:65 . 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-1-like 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 . 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 . 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 . 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 ET-1 dependent mechanisms . 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 .
Furthermore, from a physiological point of view, there is evidence indicating that endothelins control the IOP . Our findings suggest that ET-1 and its receptors may affect the IOP through changes in the AH formation and in the outflow facility. The expression of ET-1 was observed in the cornea epithelium, which is in agreement with a previous observation . It is known that ET-1 promotes corneal epithelium wound healing in rabbits . 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 . 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.
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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