Brain Research 912 (2001) 137–143www.elsevier.com/ locate /bres

Research report

Localisation of endothelin-1 mRNA expression and immunoreactivityin the retina and optic nerve from human and porcine eye.

Evidence for endothelin-1 expression in astrocytesa a b a´ ´Ainhoa Ripodas , Jose Angel de Juan , Manuela Roldan-Pallares , Rosa Bernal ,

c c a´ ´ ´Jose Moya, Montserrat Chao , Araceli Lopez , Arturo Fernandez-Cruz ,a ,*´Raquel Fernandez-Durango

a ´ ´Dpto. Medicina Interna III, Hospital Clınico San Carlos, Ciudad Universitaria, C /Martın Lagos s /n, 28040 Madrid, Spainb ´ ´Dpto. Oftalmologıa, Hospital Clınico San Carlos, Ciudad Universitaria, 28040 Madrid, Spain

c ´ ´ ´Dpto. Anatomıa Patologica, Hospital Clınico San Carlos, Ciudad Universitaria, 28040 Madrid, Spain

Accepted 22 June 2001

Abstract

We have investigated the localisation of endothelin-1 (ET-1) mRNA and ET-1-like immunoreactivity in retina and anterior portion ofoptic nerve from human and porcine eyes. In situ hybridisation method revealed expression of ET-1 mRNA mainly in the innermostlayers of the retinas, in the retinal pigment epithelium cells as well as in the astrocytes of the optic nerve. Immunohistochemical studiesshowed that ET-1-like immunoreactivity appeared in the same regions where ET-1 mRNA was expressed as well as in the inner nuclearlayer and in the inner segments of photoreceptors. In the nerve fibre and ganglion cell layers, astrocytes expressed both glial fibrillaryacidic protein and ET-1 proteins suggesting that these cells may secrete ET-1. Expression of ETA and ETB receptors in human retinawere demonstrated by reverse transcription-polymerase chain reaction. Our results demonstrated expression of ET-1 in glial, neural andvascular components of retina and optic nerve from human and porcine eyes. 2001 Elsevier Science B.V. All rights reserved.

Theme: Sensory systems

Topic: Retina and photoreceptors

Keywords: Endothelin; ETA and ETB receptors; Astrocyte; Human and porcine retina and optic nerve

1. Introduction acting through the ETA receptors, located on the vascularsmooth muscle cells [34]. The ETB receptors, located on

Endothelin-1 (ET-1), a potent vasoconstrictor, belongs vascular endothelial cells, give rise to vasodilatation by theto a family of 2.5 kDa isopeptides: ET-1, ET-2 and ET-3 release of endothelium-derived nitric oxide and pros-encoded by distinct genes at separate chromosomial loci tacyclin [40]. ET-1 also evoked mitogenic action on[42,19]. The biological actions of ET-1 are primarily vascular smooth muscle cells [2].mediated by two receptor subtypes, termed ETA and ETB, The presence of the ET system in the eye is wellcloned and sequenced in mammals [1,33]. The ETA established. Immunoreactive ET-1 (IR-ET-1), IR-ET-3 asreceptors have selective affinity for ET-1 whereas the ETB well as their mRNAs have been detected in the iris-ciliaryreceptors have equal affinity for all three isoforms. ET-1, body and choroid of the rat [21], and ETA and ETBproduced by endothelial cells, induced vasoconstriction receptors have also been located in the rat ciliary body

[32]. In the rat, IR-ET-1 and IR-ET-3 have been demon-strated in retinal extracts [21,7,8] and ET-1 and ET-3*Corresponding author. Tel.: 134-91-3303-434; fax: 134-91-5445-mRNAs expression have been detected in the whole rat432.

´E-mail address: rafedu@terra.es (R. Fernandez-Durango). retina [4]. We have previously reported the presence of

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02731-7

138 A. Ripodas et al. / Brain Research 912 (2001) 137 –143

ETA and ETB receptors in neural retinal rat membranes series of ethanol, hemisected horizontally and embedded in[9]. On the other hand, ETA and ETB gene expression paraffin wax. Serial transverse sections of 4 mm thicknesshave been detected in the rat retina [4]. were mounted on either polylysine or 2% 3-aminopropyl

In humans, Wollensak et al. [41], using immunohisto- triethoxysilane in acetone (APES) treated slides, for im-chemical techniques, have localised ET-1 immunoreactivi- munohistochemistry and in situ hybridisation, respectively,ty in the retinal blood vessels and optic nerve but not in theneural retina. However, Sitt et al. [36] have described the 2.2. In situ hybridisationpresence of ET-1 and ET-3 immunoreactivities in the outerplexiform layer and in the photoreceptor inner segments of Template DNA was a 600-base pair (bp) cDNA (537–the retina. Autoradiographic studies have shown that ETA 1162 bp) encoding human ET-1 which was subcloned inreceptors are the main ET-receptor binding sites in human both orientations into the EcoRI site of the Bluescript M13and rabbit retinal blood vessels, while ETB receptors are KS1 vector. Those cDNA clones were generously donatedpresent in neurones and glial cells of the neural retina [22]. by Dr. Derek Nunez (Cambridge, UK). Large-scaled

Several studies have been shown that ET-1 is involved preparations of plasmid DNAs were performed using Ultrain the regulation of retinal blood flow [25,5]. In rats, Clean Kit (Mo Bio Lab, Solana Beach, USA). Theincreases in endogenous ET-1 production and resistance to plasmids were linearised with EcoRV prior to transcription.the vasoconstriction action of the peptide have been Single-strand sense and antisense digoxigenin-labelledimplicated in retinal blood flow alteration in diabetes RNA probes were generated by in vitro transcription of the[3,38]. Recently, we have reported that the ET system is DNA with T7 RNA polymerase using the DIG RNAaltered in both vascular and neuronal component of the rat labelling kit (Boehringer Mannheim, Bedford, MA, USA)retina in the early stages of diabetes and that insulin following the protocol from the manufacturer. Tissuetreatment up-regulated the retinal ETA receptors [9]. sections were deparaffined, rehydrated and fixed with 4%Furthermore, the application of ET-1 in the perineural paraformaldehyde in phosphate-buffered saline (PBS).region of the anterior optic nerve produced ischemia [28]. Digestion was carried out with a solution of proteinase K

All these data suggest that the ET system has an (20 mg/ml) dissolved in 0.2 M Tris–HCl containing 50important role as a modulator of ocular vascular tone and mM EDTA for 15 min at 378C (pH 8.0). Sections werethat might be involved in the development of diabetic then refixed in 4% paraformaldehyde, dehydrated and airretinopathy and glaucoma. However, in human retina the dried. Prehybridisation was performed with the hybridisa-localisation of ET-1 immunoreactivity is not well defined tion buffer (50% formamide, 23 SSC, 100 mg/ml dena-and the distribution of ET-1 mRNA expression is yet tured salmon sperm DNA, 10% dextran sulfate, 100 mMunknown. Therefore, in the present study we have investi- Tris–HCl, 50 mM EDTA, and 10 mM dithiothrietol) forgated in human retina and in anterior optic nerve: (1) the 30 min at 558C. Hybridisation was done with 10 ng/mldistribution of the expression of ET-1 mRNA by in situ denatured digoxigenin-11-UTP-labelled riboprobes in hy-hybridisation, (2) the localisation of ET-1 by immuno- bridisation buffer overnight at 458C in a moist chamber.histochemistry, and (3) the expression of ETA and ETB After washing at room temperature to a final stringency ofmRNA by reverse transcription polymerase chain reaction 0.23 SSC, the slides were digested with 50 mg/ml RNase(RT-PCR). We also study the latter three points in pig A dissolved in 10 mM Tris–HCl, 0.5 M NaCl, 1 mMretina in order to set up the best conditions for the study of EDTA for 30 min at 378C and then rinsed two times in 23

post-mortem eyes. SSC at room temperature for 5 min. Data acquisition fordigoxigenin was carried out using a detection kit (Boeh-ringer Mannheim) according to the manufacturer’s instruc-

2. Materials and methods tions. In brief, the tissues were incubated with anti-digoxi-genin antibody conjugated with alkaline phosphatase

2.1. Tissue specimens (1:5000) for 30 min at 378C. Finally, the slides weredeveloped in NBT–BCIP (nitroblue tetrazolium salt–5-

Donor human eyes, provided by the Tissue Bank bromo-4-chloro-3-indolyl phosphate) mixture in dark con-´(Hospital Clınico San Carlos, Madrid, Spain) were re- ditions, overnight for human eyes and during 4 h for

moved after death and enucleated within 2–3 h post- porcine eyes. The colour reaction was stopped by washingmortem. Porcine eyes were obtained within 1 h post- with 10 mM Tris–HCl and 1 mM EDTA (pH 8).mortem. The eyes were from five subjects (three women Control sections were taken as serial sections from theand two men; age range, 50–70 years old) with no history same tissue block and included: (1) hybridisation withof diabetes or ocular disease as determined by their clinical sense probe, (2) RNase treatment before hybridisation withhistory. All research procedures involving humans were in antisense probe, and (3) the omission of the RNA probe.accordance with institutional guidelines on the Declarationof Helsinki. 2.3. Immunohistochemistry

The eyes were fixed in phosphate-buffered 4% parafor-maldehyde (pH 7.4) for 2 days, dehydrated with a graded Deparaffinised and hydrated sections were incubated

A. Ripodas et al. / Brain Research 912 (2001) 137 –143 139

with 5% bovine serum albumin (BSA) in 0.1 M PBS, for 2.5. Image analysis10 min at room temperature to reduce non-specific bind-ing. After a brief rinse with 0.1% BSA in PBS (washing Images were captured using Leica Qwin image process-solution), the sections were incubated for 2 h at room ing and analysis software (Leica microscopy system,temperature, in a humidified chamber, with either a rabbit Heerbrugg, Switzerland) on a personal computer linked toanti-ET-1 polyclonal antibody (Peninsula Laboratories, a high resolution video camera (Leica DC100) mounted onBelmont, USA) at 1:400 dilution, a rabbit anti-human von a microscope. Results described in the article were derivedWillebrand Factor (vWf) polyclonal antibody (Sigma, St. from examination of at least three sections from all theLouis, MO, USA) at 1:800 dilution or a rabbit anti-human studied eyes.glial fibrillary acidic protein (GFAP) polyclonal antibody(Sigma) at 1:100 dilution.

In all sections, immunostaining was developed using 3. Results‘super sensitive immunodetection system’ kit (Biogenex,San Ramon, USA). Sections were incubated for 20 min 3.1. Localisation of ET-1 mRNA by in situ hybridisationwith diluted biotinylated anti-rabbit immunoglobulins as in retina and anterior portion of the optic nerve fromdirected by the supplier and, after washing, incubated with human and porcine eyesthe alkaline-phosphatase conjugated streptavidin diluted inPBS for 20 min at room temperature. The alkaline-phos- In the human retina, expression of ET-1 mRNA wasphatase activity was visualised with naphthol phosphate detected mostly as a dense band in the region of the innerand Fast Red chromogen (Biogenex) which resulted in red plexiform layer (Fig. 1B). A patchy hybridisation signalstaining. The sections were lightly counterstained with was also observed in the nerve fibre and ganglion cellMayer’s hematoxilin. For control, tissue sections were layers. Some sparse cells of the inner nuclear layer and ofincubated either with primary antibody preabsorbed with the innermost part of the outer nuclear layer were weakly10 nM ET-1 or with normal rabbit serum instead of stained. Strong ET-1 mRNA expression was seen through-primary antibody. The commercially available polyclonal out the choroid. Endothelial cells of the choroid vesselsanti-ET-1 serum has a cross-reactivity with ET-2 of 91% were intensively stained. Retinal pigment epitheliumand with ET-3 of 0.05%. (RPE) cells showed weak signal (Fig. 1D).

Similar results were observed in porcine retina (Fig. 1F).2.4. RT-PCR The strong dense band signal was also localised in the

innermost region of the inner plexiform layer, which isTotal RNA was extracted from the dissected human and wider than the human one. The punctuated and scattered

porcine retinas using a modified guanidinium isothio- distribution pattern of signal in the nerve fibre layer iscyanate method [6]. Total RNA was reverse transcribed consistent with retinal astrocytes. Ganglion cells and someusing oligo(dt) as primer and murine Malone leukaemia sparse cells of the inner nuclear layer were positively16

virus reverse transcriptase (Pharmacia, Piscataway, NJ, stained. In the choroid, dense punctuated signals alienatedUSA) as described previously [13]. PCR primers were the vessels signalling the endothelial cells. Sections incu-selected from published ETA [18] and ETB [29] as bated with the sense probe for ET-1 mRNA showed nodescribed previously [30]. All 59 primers covered splice positive signal neither in human (Fig. 1C) nor in porcinejunctions, thus excluding amplification of genomic DNA. retinas (Fig. 1G). Similarly, RNase treatment beforeThe human-ETA PCR primers selected were 59- hybridisation or the omission of RNA probe did notCCTTTTGATCACAATGACTTT-39 (sense) and 59- labelled any retinal structures.TTTGATGTGGCATTGAGCATACAG-39 (antisense); and In the optic nerve, nerve bundles were positivelythe primers for human-ETB were 59-ACTGGCC- staining and strong focal accumulation of hybridisationATTTGGAGCTGAGAT-39 (sense) and 59-CTGCA- signal was localised in the astrocytes within the axonTGCCACTTTTCTTTCTCAA-39 (antisense). PCR was bundles and in the astroglial layer that separated theperformed in a 50-ml reaction volume containing 1 mM of vessels and connective tissue from the axons (Fig. 1H).each primer, 2 mM of dNTP and 1.25 U Taq DNApolymerase. The reaction mixture was overlaid with two 3.2. Localisation ET-1 and GFAP-likedrops of mineral oil. PCR parameters consist of an initial immunoreactivities in retina and anterior portion ofdenaturation at 948C for 5 min to heat activate the DNA optic nerve from human and porcine eyespolymerase. This was follow by 30 cycles of denaturationat 948C for 30 s, annealing at 608C for 30 s, and extension In human retina, specific and intense immunolabellingat 728C for 30 s with a final extension at 728C for 10 min. for ET-1 was observed in the innermost layers (internalThen PCR-amplified products were electrophoresed on a limiting membrane, nerve fibre layer, ganglion cell layer,2% agarose gel and stained with ethidium bromide. The inner plexiform layer and internal nuclear layer) (Fig. 1I,J)identity of the human ETA and ETB products was verified which are the most vascularised since the vWf signalsby sequencing. extend through all those layers (data not shown). The most

140 A. Ripodas et al. / Brain Research 912 (2001) 137 –143

A. Ripodas et al. / Brain Research 912 (2001) 137 –143 141

intense labelling was observed in the inner plexiform layeras a dense band. The cytoplasm of ganglion cells werelabelled. The immunostaining in the inner nuclear layercould correspond to some cells and to a minority ofcapillaries crossing the outer part of this layer. Severalcells of the outer nuclear layer were also positively stained.The inner segment layers of the photoreceptors werelightly positive. The cells of the RPE were stronglystained. Strong ET-1 immunostaining was showedthroughout the choroid. GFAP positive astrocytes, mostlylocalised in the nerve fibre layer and ganglion layer, madecontact with the adventicia of the large vessels throughtheir processes (Fig. 1L, M). ET-1 immunoreactivity was

Fig. 2. mRNA expression of ETA and ETB receptors. mRNA wasalso located, in an adjacent section, on the cell body of the extracted from human retinas, reversed transcribed and amplified by PCR.astrocytes and their processes enveloping the vessel (Fig. PCR products were electrophoresed in agarose gel and visualised by1J, K). Moderate ET-1 like immunoreactivity was seen in staining with ethidium bromide. Lane description: MW, molecular

marker.the endothelial cells of the arteries and arterioles. Similarresults were obtained in porcine retina. ET-1 immuno-reactive material was localised in the inner retina (Fig. 1P,Q) where both GFAP (Fig. 1S, T) and ET-1 immuno- their sequence determined, and verified to share 100% withstaining (Fig. 1Q, R) exist within the astrocytes and their their respective cDNA sequences.processes surrounding the vessels.

Sections of human and porcine retinas incubated withnormal serum or anti-ET-1 preincubated with ET-1 showed 4. Discussionno specific labelling (Fig. 1N and U, respectively).

In both human (Fig. 1O) and porcine (Fig. 1V) anterior The present study has been designed to investigate theportion of optic nerves, strong ET-1 immunoreactivity was cellular distribution of ET-1 mRNA in human retina andidentified in neural bundles. Adjacent sections incubated optic nerve and to clarify the localisation of the maturewith normal serum or anti-ET-1, preincubated with ET-1, peptide on the different layers of the retina. We have usedshowed no specific labelling (data not shown). the in situ hybridisation technique to identify the ET-1

gene expression and immunohistochemistry to detect the3.3. Immunohistochemistry for von Willebrand factor presence of mature peptide.

To the best of our knowledge, the current findings areVessels positive for vWf (a marker of vessels with viable the first to describe the expression of ET-1 gene in human

endothelial cells) reflected the normal anatomic pattern of retina. This study demonstrates that ET-1 mRNA as wellthe retinal vasculature and were found in the innermost as ET-1 peptide appear in the innermost layer of thepart of the retina (data not shown). The number of viable human and porcine retinas, principally in the internalvessels was highest in the central retina. plexiform layer. The presence of vWf antigen signal

corroborates that those layers are the most vascularised3.4. RT-PCR ones. However, the dense band of ET-1 mRNA in the

inner plexiform layer does not only account for the numberThe amplified 299-bp ETA and 428-bp ETB PCR cDNA of vessels that we observed in this layer. Considering that

fragments were of predicted molecular size (Fig. 2). The this layer is a synapse-enriched zone and that certainPCR products amplified for ETA and ETB were purified, mRNAs have been detected in dendrites of mature brain

Fig. 1. Localisation of ET-1 mRNA (A–H) in human (A–D) and porcine (E–H) eyes by in situ hybridisation. The signal was visualised with nitrobluetetrazolium as the substrate. Sections of human (A) and porcine (E) retinas were stained with hematoxylin-eosin. Positive ET-1 mRNA signals wereobserved mainly in the choroids and innermost layers of human (B) and porcine (F) retinas. RPE cells (filled arrow) showed lightly signal (D). In theanterior portion of the optic nerve neural bundles and astrocytes (filled arrows) were positively labelled (H). Sections hybridised with the sense riboprobeshowed no signal neither in human (C) nor in porcine (G) retinas. Localisation of ET-1 and GFAP immunoreactivities (I–V) in human (I–O) and pig(P–V) eyes. The immunostaining is seen in red. Positive ET-1 immunoreactivity was showed in the choroids and in the innermost layers of human (I, J)and porcine (P, Q) retinas (nuclear counterstained with hematoxylin). RPE cells showed also intense signal in both retinas. Existence of ET-1 and GFAPimmunoreactivities were shown in consecutive sections on astrocytes (filled arrows) of human (J, L, respectively) and porcine (Q, S, respectively) retinas.Higher magnification of the inserts on the squares are shown in K, M, R and T. Strong labelling of ET-1 appeared in neural bundles of human (O) andporcine (V) anterior portion of optic nerves. Negative controls were obtained with anti-ET-1 preabsorbed with 10 nM ET-1 (N, U). Ch, Choroid; RCL,rods and cones layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cellslayer; NFL, nerve fiber layer. All images magnification 3160 except D, K, M, R, T 3400.

142 A. Ripodas et al. / Brain Research 912 (2001) 137 –143

neurones [37], the ET-1 mRNA could be targeted for bound intracellular stores [31]. Therefore, since RPE cellsdelivery into dendrites of ganglion and/or amacrine cells. have been involved in the formation of epiretinal mem-Interestingly, many of the mRNAs localised in dendrites of branes in proliferative vitreoretinopathy (PVR), ET-1 maybrain neurones encode proteins that play a role in synaptic potentially participate in the formation and contraction ofsignalling [39]. Future research will further illuminate the those membranes. In fact, we have recently reportedsignificance of possible dendritic translation of ET-1 increased ET-1 levels in the subretinal fluid of patientsmRNA in human retina. with PVR in comparison with those of patients with

Demonstration of existence of both ET-1 and GFAP uncomplicated retinal detachment [14].immunoreactivities within astrocytes in human and porcine With regard to expression of ET-1 ligand, previousretinas indicate that ET-1 may also be formed and stored studies have yielded conflicting results in human retina, forwithin retinal glial cells thus playing a role in glial instance, Wollensak et al. [41] did not found ET-1 in thefunction. In agreement, primary astrocytes in culture neural retina meanwhile Stitt et al. [36] found ET-1 in theexpress ET-1 and ET-3 [11,23], the ETA and ETB outer plexiform layer. The reasons for the different find-receptors [17,24] as well as the ET-1-converting enzyme ings of those groups remain uncertain but may reflectactivity [10]. However, in human brain in vivo, expression differences between the methodologies. An importantof ET-1 and ET-3 in astrocytes were only detected in low difference between our studies and those of the above citedamounts in the ‘resting state’ [15,20]. The physiological groups is that in the latter the eyes were frozen or fixed insignificance of ET-1 expression in retinal glial elements is the laboratory an average of 24 h post-mortem whereas thefor the moment unknown. Astrocytic proliferation together eyes used in our study were fixed 2 h after death.with an excessive secretion of ET-1 have been reported in In summary, we demonstrated the gene expression ofvivo, such as in cerebral focal ischemia, and subarachnoid ET-1, ETA and ETB receptors in retina and optic nervehaemorrhage [27,43]. Moreover, Sasaki et al. [35] demon- from human and porcine eyes. We also obtained evidencestrated that ET-1 specifically stimulated the efflux of for the expression of ET-1 in retinal neural cells andglutamate via ETB receptors from cultured of rat as- astrocytes. The expression of ET-1 in neural, glial as welltrocytes suggesting that ET-1 may exacerbate neurode- as vascular elements of the normal adult retina suggest thatgeneration. On the other hand, ET-1 acts as a growth factor ET-1 may have a role in the maintenance of both neuralfor astrocytes, inducing DNA synthesis and proliferation and vascular integrity in the mature retina. Further studies[23]. Interestingly, an increase of GFAP expression, that will be required to elucidate the possible involvement ofmay reflects a state of glial hypertrophy, was observed in ET-1 in ocular diseases.human diabetic retinopathy [26]. As ET-1 is increased inthe innermost layers of retinas of experimental diabetic rats[9], a role of ET-1 increased secretion by glial cells in the Acknowledgementsdevelopment of diabetic retinopathy will require furtherinvestigation. In agreement with our results in humans, we ´ ´We thank Dr. J. Alvarez Rodrıguez, Transplant Coor-have reported that ET-1 immunoreactivity was mainly ´dinator (Tissue Bank, Hospital Clınico San Carlos, Mad-localised on the ganglion cell, on the internal plexiform rid, Spain) for his cooperation in collecting human eyesand on the inner nuclear layers of the rat retina [9]. from donors. This work is supported by the Ministerio de

In this study we have found ET-1-like immunoreactivity ´Educacion y Cultura, grant DGES 97/0028.in the photoreceptor inner segments of human and porcineretinas. Similar results were observed by Stitt et al. [36] inrat and human retina. However, we did not found positive ReferencesET-1-like immunoreactivity signal in the rod and conelayer of rat retina [9]. Those results suggest that ET-1 in

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