|Molecular Vision 2005;
Received 23 July 2004 | Accepted 26 April 2005 | Published 7 July 2005
Immunoreactive ET-1 in the vitreous humor and epiretinal membranes of patients with proliferative vitreoretinopathy
Roldán-Pallarés,1 Raquel Rollín,2 Aránzazu
Mediero,2 Juan Carlos Martínez-Montero,3 Arturo
Fernández-Cruz,2 Carmen Bravo-Llata,4 Raquel
1Departamento de Oftalmología and 2Unidad de Investigación, Departamento de Medicina Interna III, Hospital Clínico San Carlos, Universidad Complutense de Madrid, Madrid, Spain; 3Departamento de Anatomía Patológica, Instituto Oftálmico, Hospital Gregorio Marañón, Madrid, Spain; 4Departamento de Matemáticas, Universidad Complutense de Madrid, Madrid, Spain
Correspondence to: Manuela Roldán Pallares, Rey Francisco, 11, 28008, Madrid, Spain; Phone: 34-91-5477885; FAX: 34-91-3303939; email: firstname.lastname@example.org
Purpose: Endothelin one (ET-1) is a vasomodulator peptide that plays a role on ocular blood flow, glial proliferation, and collagen matrix contraction by retinal pigmented epithelial (RPE) cells. Both glial and RPE cells have been involved in the formation of epiretinal membranes (ERMs). This investigation was conducted to determine whether ET-1 may be associated with ERMs, either idiopathic (IERMs) or from proliferative vitreoretinopathy (PVR).
Methods: Plasma and vitreous samples were collected from patients classified by the presence of PVR membranes, retinal detachment (RD), and other ocular conditions, such as IERMs, that made the patients candidates for vitrectomy. Immunoreactive endothelin one (IR-ET-1) was tested in plasma and vitreous by radioimmunoassay. Immunoreactive-ET-1 was localized in IERMs and PVR membranes immunohistochemically. Expression of endothelin receptors A (ETA) and B (ETB) was confirmed by reverse transcription-polymerase chain reaction.
Results: IR-ET-1 levels in plasma and vitreous were higher in patients with PVR and in patients with RD than in those of the control group. Eyes with IERMs also showed higher IR-ET-1 levels than the control group cases. IR-ET-1 levels in eyes with PVR were higher than those in eyes with IERMs. IR-ET-1 levels in eyes with RD were also higher than those of eyes with IERMs. Immunoreactive ET-1 was localized in the cellular and stromal components of both IERMs and PVR membranes. Furthermore, ETA and ETB receptors were expressed in both IERMs and PVR membranes.
Conclusions: IR-ET-1 in human vitreous is elevated in PVR, RD, and IERMs. ET-1 and its receptors ETA and ETB are present in epiretinal tissue of both idiopathic and PVR membranes. These data suggest an involvement of ET-1 in retinal disease.
Proliferative vitreoretinopathy (PVR) is a major cause of retinal detachment (RD) surgery failure . It is characterized by the formation of fibrous epiretinal membranes (ERMs) at the vitreoretinal interface in the vitreous cavity. These membranes result from inappropriate proliferation, migration, and differentiation of several cell types . The formation of these membranes also occur in a variety of ocular disorders such as proliferative diabetic retinopathy, trauma to the posterior segment of the eye, and chronic intraocular inflammation [3,4]. Gradual contraction of ERMs may result in complex retinal detachments [1,5]. After successful vitreoretinal surgery, the functional prognosis remains poor in RDs complicated with PVR. Therefore, it is important that the pathogenesis of this disorder can be clearly understood in order to devise preventive strategies.
Endothelin-1 (ET-1) is a peptide produced by endothelial cells [6,7] that induces vasoconstriction when it interacts with ETA receptors on the vascular smooth muscle cells. Conversely, it induces vasodilatation by interacting with the ETB receptors on vascular endothelial cells, resulting in the release of endothelium derived nitric oxide and prostacyclin . ET-1 also stimulates mitogenic action on vascular smooth muscle cells . It has been shown that hypoxia induces the ET-1 gene expression in endothelial cells and in certain tumors [10,11]. Furthermore, ET-1 is involved in the autocrine growth of tumors .
The presence of an ET-1 system in the eye is well established. Immunoreactive ET-1 (IR-ET-1) and ET-1 mRNA have been predominately localized in the innermost layers of human retina and ET-1 expression has been reported in glial, neural, and vascular cells of the retina and in the optic nerve of human and porcine eyes . The ET system plays an important role as a modulator of retinal , optic nerve head, and choroidal blood flow [15,16]. An excessive ET-1 secretion associated with astrocytic proliferation in cerebral focal ischemia [17,18] and ET-1 synthesis and its secretion have been recently identified in human retinal pigment epithelial cells (RPE) . There is evidence that RPE is a source for ET-1 in the retina and that its release increases during a breakdown of the blood-retinal barrier (after TNF-μ treatment) . RPE cells have been involved in the elaboration of extracellular matrix and it has been suggested that ET-1 may influence gene expression of extracellular matrix protein . Moreover, using an in vitro contraction assay, it has also been reported that collagen matrix contraction by transdifferentiated RPE calls is stimulated by ET-1 and that this contraction promoted by ET-1 was completely protein synthesis dependent . Also, in cultured bovine RPE cells ET-1 produced the release of free/bound Ca2+ from bound intracellular stores . Considering that both RPE and glial cells have been involved in the formation of ERMs, ET-1 may participate in the formation and contraction of those membranes. Moreover, blood flow slowdown, retinal perfusion defects, and tissular hypoxia (all ET-1 related) have been observed in the detached retina [22,23]. Also, capillary dilatation and permeability changes have been found around the areas of vascular occlusion or in the retinal folds associated with PVR .
Several endogenous growth factors and cytokines appear to participate in development of epiretinal angiogenesis and fibrosis [24-27]. Criteria to implicate these factors in retinal disease have included their presence or upregulation in vitreous humor from patients with proliferative retinopathies and their detection in these patient's pathologic ocular tissue [24-27]. This paragraph treat to explain the steps we have followed to evaluate our hypothesis, that is in the next paragraph.
Recently, we have reported higher levels of IR-ET-1 in the subretinal fluid of patients with RD complicated with PVR than in cases with RD uncomplicated with PVR . These findings led us to speculate that ET-1 plays a role in the formation or contraction of ERMs. As an initial step to evaluate this hypothesis, this study assessed the presence of IR-ET-1 in the vitreous, whether IR-ET-1 vitreous levels correlate with the existence of PVR, the localization of ET-1 by immunohistochemistry in ERMs (either, idiopathic or PVR), and expression of ETA and ETB receptors in ERMs by reverse transcription-polymerase chain reaction.
Selection of patients
All research involving human subjects adhered to the Declaration of Helsinki. Institutional review and approval were obtained, and informed consent was obtained from all patients enrolled in the study. Patients were characterized by age, gender, and associated ocular conditions (Table 1). They had to be candidates for vitrectomy as a surgical procedure in order to obtain the vitreous samples. Based on ocular diagnosis they were divided into three categories. The PVR group consisted of patients with primary or recurrent PVR (grade>=C3)  who were candidates for a first vitrectomy. The RD group included patients with RD and vitreous organization but without associated PVR who were candidates for a first vitrectomy (excluding those patients with clinically evident vitreous hemorrhage and/or early PVR detected during vitrectomy). The control group included patients with neither PVR nor RD but who were candidates for a first vitrectomy. In the control group, cases with IERMs were included as long as the patients did not have any history of other ocular or systemic disease. Patients were excluded if they reported some hematologic, cardiologic, or metabolic disorder, age twenty years and below, history of aphakia, pseudophakia and/or current systemic or topical eye treatment. Selection of the patients was done between January 1998 to November 2003. During this period of time, a total of 1687 RD cases arrived at our Hospital either as emergencies or referrals. Surgery of the included patients was performed under general anesthesia using a similar vitrectomy and membrane peeling technique.
Plasma, vitreous, and epiretinal membranes samples
Blood samples were obtained at the time of vitreoretinal surgery, prior the anesthetic procedure. Undiluted vitreous samples were obtained at the time of the surgery with a syringe attached to an automated vitrector. Both blood and vitreous samples were collected in chilled tubes containing protease inhibitors (10 μM phenyl methyl sulphonyl fluoride (PMSF), 5 μM pepstatin A, and 10 μM trasylol). Blood samples were centrifuged at 3,000 rpm for 20 min at 4 °C "plasma". Vitreous samples were centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatants were then stored at -70 °C for less than three months before testing. In selected patients (n=24), ERMs were removed from the eye, fixed in formalin for 24 h, embedded in paraffin, and sectioned at 4 μm.
Extraction and radioimmunoassay of ET-1
The ET-1 extraction with Sep-Pack C18 cartridge (Waters Associates, Milford, MA) and the measurement of IR-ET-1 were carried out in plasma and vitreous samples as previously published . Briefly, the Sep-Pack C18 cartridges were activated by washing with 8 ml (pure) acetonitrile and 8 ml ammonium acetate (0.2%, pH 4.0). The samples were then applied on the cartridges, washed with 5 ml ammonium acetate and the absorbed ET-1 was eluted with 3 ml acetonitrile (60%) in ammonium acetate. The organic solvent was evaporated under nitrogen stream followed by lyophilization. The residue was taken up in an RIA buffer (0.1 M phosphate, pH 7.4 containing 0.3% NaCl, 0.1% BSA, and 0.1% Triton X-100). IR-ET-1 was assayed by RIA as previously published , using polyclonal antibody against synthetic ET-1 (Peninsula Laboratories, Merseyside, UK) at a 1:90,000 final dilution. The antibody fully reacts with ET-1 (100%) and it also cross-reacts with ET-2 (7%), with ET-3 (7%), with porcine big-endothelin (35%) and with human big-endothelin (17%). However, it did not show any cross-reactivity with somatostatin, b-endorphin, angiotensin I, II, and III, vasopresin and atrial natriuretic factor (ANF). The sensitivity of the RIA was 1 pg/ml. The 50% intercept was 50 pg/ml. The interassay variation was 13% and the intra-assay variation was 10%. Recoveries of 1.5 and 3 pg of ET-1 added to pooled vitreous was 78±3.2% (n=10).
The protein content of the vitreous samples was determined by the method of Lowry et al. .
The statistical evaluation consisted of correlation coefficients and t-tests corrected for multiple comparisons. Two methods of correcting for multiple comparisons were used: the Duncan test using SAS (version 8.2, SAS Institute Inc., Cary, NC) and the Bonferroni test SPSS (version 12, SPSS Inc., Chicago, IL). Results are presented as means±standard error of the mean (SEM).
Epiretinal membranes: Immunohistochemistry for ET-1, GFAP, and cytokeratin
Epiretinal proliferative tissue was obtained in 24 cases during vitrectomy surgery (Table 1). Seven IERMs and nine ERMs from PVR were used for immunohistochemistry; these membranes were fixed in buffered formalin (Sigma-Aldrich, St. Louis, MO) for 24 h, paraffin embedded, sectioned at 4 mm and dried on snowcoat X-tra slides. For light microscopy immunohistochemistry we used a polyclonal antibody to human ET-1 (Peninsula Laboratories, Belmont, CA), a GFAP, clone 6F2 mouse monoclonal antibody to glial fibrillary acidic protein (DAKO, Carpinteria, CA), and an AE1/AE3 mouse monoclonal anti-human cytokeratin antibody (DAKO). Briefly, the deparaffinized and hydrate sections were incubated in a TBT Tris Base Saline (TBS) 0.5 M, blocking solution, pH 7.4, containing 3% (w/v) of BSA and 0.05% (v/v) of Triton X-100, for 30 min at room temperature to reduce nonspecific binding. For antibody cytokeratin, prior to incubation with the primary antibodies, it was necessary a previous step of heat induced antigen retrieval technique with a pressure cooker heating in a 0.01 M sodium citrate solution. The sections were incubated over night with a 1:400 diluted polyclonal ET-1 antibody at 4 °C in a humedifier chamber. Then, the slides were washed for 5 min in TBS. Immunodetection was performed with biotinylated antimouse immunoglobulins followed by streptavidin alkaline phosphatase conjugated (LSAB2 kit supplied by DAKO Corp.) and with naphthol phosphate and Fast Red chromogen (Sigma-Aldrich, St. Louis, MO) which resulted in red staining. After that, the sections were lightly counterstained with Mayer's hematoxylin. Final mounting was done in the Glycergel water soluble media (DAKO).
As controls, tissue sections were incubated either with primary antibody preabsorbed with 10 nM ET-1 or with normal rabbit serum instead of the primary antibody. In addition, antibody to GFAP and antibody to cytokeratin, both at 1:50 dilution overnight at 4 °C, were used to detect glial and RPE cells, respectively.
Images were captured using Leica Qwin image processing and analysis software (Leica microscopy system, Heerbrugg, Switzerland) on a personal computer, linked to a high resolution video camera (Leica DC 100) mounted on a microscope (Zeiss, Oberkochen, Germany). Results presented on the article were worked out from the examination of at least three sections from all the studied ERMs.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from IERMs (four cases) and from PVR membranes (four cases) was extracted using a RNeasy kit, (Qiagen, Santa Clara, CA) according to manufacturer's guidelines. The RNA concentration was spectrophotometrically determined. Following DNAse treatment, first-strand cDNA was synthesized using 2.5 μM random hexamers and 1.25 U/μl Multiscribe reverse transcriptase (RT, Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. Real-time RT-PCR analysis was performed using an automated sequence detection instrument (Prism 7700 Sequence detector; Applied Biosystems) for the real-time monitoring of nucleic acid green dye fluorescence (SYBR Green I; Applied Biosystems).
A series of PCRs was performed on ERMs cDNAs using primer pairs for ETA and ETB receptors, and the housekeeping gene control β-actin. Primer pairs were designed with the assistance of Prism 7700 sequence detection software (Primer Express, Applied Biosystems). Details of the primers and the GenBank accession numbers are given in Table 2. Specificity was checked in a BLAST search (BLASTnr) on 21 February 2002, which compared to all available databases. The primers shared 100% homology with the target sequences but no significant homology with any other sequences.
PCR was performed using a kit (Applied Biosystems) as previously described . Briefly, the PCR mixture contained 12.5 μl 2X SYBR Green PCR Master Mix, 5 μl (10 ng) of RT product, and 300 nM of the primers in a total volume of 25 μl. PCR amplification was performed according to the temperature profile: 2 min at 50 °C for AmpErase followed by 10 min at 95 °C to inactivate the AmpErase and activate the AmpliTaq Gold DNA polymerase. The cycling conditions were: a 15 s melting step at 95 °C followed by 40 1-min cycles of annealing-extension at 60 °C. All reactions were performed in duplicate. For each primer pair, nontemplate controls were included to check for significant levels of contaminants. Agarose gel electrophoretic analysis was used to check whether the amplified products corresponded to the estimated size for cDNA fragments of β-actin, ETA, and ETB receptors.
Description of the patient population
Seventy-five patients having a mean age of 58.46±1.54 years were included in this study. Details of patients are given in Table 1 and Table 3. In the PVR group (n=25) patients were operated on for primary or recurrent PVR with vitrectomy and membrane peeling, membrane peeling, epiretinal tissue was obtained in 13 of these cases. In the RD group (n=25) patients were operated on (first surgical procedure) with vitrectomy for rhegmatogenous RD associated with other ocular conditions (Table 1). In the control group (no RD, n=25) patients were operated on (first surgical procedure) with vitrectomy; membrane peeling was done in eleven cases of this group with idiopathic epiretinal membrane (IERM). None of the patients included in the study reported either an hematologic, cardiologic or metabolic disease or some treatment for these problems.
There were no statistically significant (χ2 test, Fisher's exact test) sex (gender) differences among the three groups studied. Similarly, there were no statistically significant (ANOVA) age differences among the three groups: controls (62.92±1.93), RD (56.72±3.71), and PVR (55.76±2.17).
IR-ET-1 concentrations in human plasma and IR-ET-1 and proteins in the vitreous
A clear parallelism was observed between various dilutions of the extracts of vitreous and the standard curve, indicating that IR-ET-1 present in the vitreous is indistinguishable from the peptide used in the preparation of the standard curve.
Plasma IR-ET-1 levels were similar (p=0.89) in females (2.71±0.074 pg/ml, n=36) and males (2.70±0.072 pg/ml, n=39). Plasma IR-ET-1 levels were not related to age (58.46±13.81, range 30-83 years, n=75; Pearson correlation: r=-0.1951, p=0.09). Plasma IR-ET-1 levels (Figure 1) were the highest in the PVR group (3.24±0.021 pg/ml), intermediate in the RD group (2.71±0.015 pg/ml), and lowest in the control group (2.17±0.018 pg/ml). These groups were all significantly different from each other (PVR compared to RD: p<0.0003; PVR compared to control group: p<0.0003; RD compared to control group: p<0.0001; Table 3). Plasma IR-ET-1 levels of 3.40 pg/ml and higher were only detected in the PVR group. When we compare plasma IR-ET-1 concentrations between cases of the control group with IERMs (2.30±0.01 pg/ml; n=11) and cases of the PVR group with primary (3.21±0.017 pg/ml) or recurrent ERMs (3.44±0.012 pg/ml), the differences were statistically significant (both comparisons significant with p<0.0003).
Vitreous IR-ET-1 levels (Figure 2) were also higher in the PVR group than in the RD and control groups (PVR: 19.35±1.07 pg/ml; RD: 3.56±0.04 pg/ml; control: 2.93±0.05 pg/ml; p<0.0003 between PVR and control and between PVR and RD, while no differences were found between RD and control; Table 3). We calculated the ratio of vitreous IR-ET-1 concentration to the plasmas' one for each patient. The ratio was approximately fourth times higher in the PVR group than in the RD and control groups (PVR: 5.98±0.28 pg/ml; RD: 1.31±0.01 pg/ml; control: 1.34±0.01 pg/ml). The highest vitreous IR-ET-1 concentrations were reported in patients with recurrent PVR (29.19±1.09 pg/ml) and primary PVR (18.22±0.88 pg/ml), whereas they were intermediate in those with RD (3.56±0.04 pg/ml) and in the cases with IERMs (n=11) of the control group (3.28±0.01 pg/ml) and finally, the lowest in the rest of the control group (n=14, 2.80±0.04 pg/ml). Vitreous IR-ET-1 levels of males (8.79±1.35 pg/ml) were similar to those of females (8.55±1.37 pg/ml). It was also found that the ratio of vitreous IR-ET-1 concentration to plasma IR-ET-1 concentration in males (2.92±0.38) was similar to those in females (2.83±0.39).
To assess the effect of RD on vitreous IR-ET-1 levels, we considered three groups of patients. In the control group, patients without RD, candidates of primary vitrectomy were included, they may have IERMs. In the RD group, patients must have rhegmatogenous RD but without associated PVR or ERMs, they must also be candidates of primary vitrectomy (vitreous opacity). In the PVR group, patients with primary or recurrent PVR (PVR must be grade greater than or equal to C3 in order to need vitrectomy) were considered in order to evaluate IR-ET-1 vitreous concentrations when epiretinal tissue proliferation is associated to retinal detachment. The PVR group had a significantly higher level (p<0.001) of vitreous IR-ET-1 than the RD and the control groups. Plasma IR-ET-1 levels were different in the three groups (as tested by ANOVA p<0.001). Plasma and vitreous IR-ET-1 levels were, respectively: in the PVR group: 3.24±0.021 pg/ml, 19.53±1.07 pg/ml; and in the RD group: 2.71±0.01 pg/ml, 3.56±0.04 pg/ml (Figure 1 and Figure 2). If we compare the IERM cases (control group) with the three groups considered in the study (control, RD, and PVR), plasma levels were different in the four groups (p<0.0001). All vitreous measurements: IR-ET-1 (19.53±1.07 pg/ml), the ratio of vitreous IR-ET-1 concentration to the plasma's one (5.98±0.28 pg/ml), the protein vitreous concentration (2.51±0.05 mg/ml), and the ratio of IR-ET-1 to proteins (7.65±0.25 pg/mg) in the vitreous corresponding to the PVR group were higher (p<0.001) than in the other three groups (IERM, other control, and RD).
Concentrations of IR-ET-1 in vitreous and in plasma correlated positively, for all patient groups (control: r=0.93, p<0.00001; RD: r=0.48, p<0.01; PVR: r=0.91, p<0.0001). As shown in Figure 3, the slope of the correlations was greater in the PVR group than in the RD and control groups.
The mean levels of IR-ET-1 (p<0.0001) and IR-ET-1 adjusted by protein (p<0.0001) in the vitreous were significantly higher in patients who had PVR than in those who did not (Table 3). Although protein concentration in the vitreous was also higher (p<0.0001) in PVR (2.51±0.05 mg/ml) than in RD (1.30±0.01 mg/ml) and/or control group (1.12±0.01 mg/ml), when we evaluated vitreous levels of IR-ET-1 adjusted by protein concentration, this ratio was even higher (p<0.0001) in PVR (7.65±0.25 pg/mg) than in RD (2.73±0.03 pg/mg) and/or in the control group (2.61±0.02 pg/mg).
Localization of ET-1 immunoreactivity in epiretinal membranes
Serial sections were cut through the ERMs and immunostained sequentially. Histologic examination showed that all ERMs contained heterogeneous cell populations that exhibited diverse morphologic characteristics (Figure 4). IERMs contained macrophages, glial cells, and fibroblastic cells (Figure 4A). ERMs from PVR contained similar cell populations, but also RPE cells (Figure 4E,F). All ERMs studied were positive for ET-1. Figure 4 shows the localization of ET-1 immunoreactivity in an IERM (Figure 4B) and in an ERM from PVR (Figure 4F,G). Diffuse ET-1 protein expression was observed in both stromal and cellular components of the membranes (Figure 4B,F,G). When serial sections from the sames specimens were immunostained for GFAP (Figure 4C,H) and cytokeratin (Figure 4J), positive staining for both ET-1 and GFAP by glial cells was found within IERMs (Figure 4B,C) and PVR ERMs (Figure 4G,H). Positive staining for both ET-1 and cytokeratin by RPE cells was found within PVR ERMs (Figure 4G,J). Because of the thinness of the ERM sections, structures in the photomicrographs appear similar but not identical. The positive immunostaining for ET-1 was abolished (negative control) when the sections were incubated either with the antibodies absorbed with the antigen or with the normal rabbit serum instead of primary antibody (Figure 4D,I). Thus, ET-1 appears to be localized in both, cellular (RPE, glial, macrophages, and fibroblastic cells) and stromal components of ERMs.
Epiretinal membranes: ETA and ETB mRNA expression
ETA and ETB receptors were expressed (Figure 5) both in IERMs (Figure 5A,B, lanes 1-4) and in PVR ERMs (Figure 5A,B, lanes 5-8). The amplified 84-bp ETA and 98-bp ETB PCR cDNA fragments were of predicted molecular size. The PCR products amplified for ETA and ETB were purified, their sequence determined, and verified to share 100% with their respective cDNA sequences (Table 2). β-actin, the housekeeping gene control, was expressed in all the specimens (Figure 5C; lanes 1-8).
Several endogenous growth factors and cytokines appear to participate in development of epiretinal angiogenesis and fibrosis, including vascular endothelial growth factor (VEGF), fibroblast growth factors, and insulin-like growth factors [24-27]. We speculated contribution of endothelin to epiretinal membranes (idiopathic and PVR).
Previously IR-ET-1 was detected in subretinal fluid of patients with retinal detachment complicated with PVR and concentrations of IR-ET-1 in the subretinal fluid of these patients correlated with plasma IR-ET-1 concentrations . In this paper we demonstrate a correlation between IR-ET-1 plasma and vitreous levels in the three groups of patients considered (control, including idiopathic ERMs, RD, and PVR) suggesting that intravitreal IR-ET-1 at least in part derives from the systemic circulation. The increase in vitreous IR-ET-1 compared with the increase in plasma IR-ET-1 was greater in patients with PVR, consistent with enhanced access of plasma proteins to the vitreous cavity in this condition due to disruption of blood-ocular barrier . It is unclear whether ET-1 enters the eye by passive transfer or by an active permissive mechanism. Alternatively, ET-1 may also be produced locally in the eye as a manifestation of retinal disease. Five observations support this suggestion. First, the ratio of vitreous to plasma ET-1 concentrations was higher in patients with PVR than in control patients, even though control patients had ocular conditions (dislocated crystallin or fragments, idiopathic ERMs) associated with enhanced permeability of ocular blood vessels. Second, vitreous ET-1 was higher in patients with ERMs consistent with PVR than in cases with idiopathic ERMs (without associated RD, in the control group), even though plasma ET-1 levels were comparable in both groups. Third, the presence of vitreous organization, because of some bleeding in RD cases with no PVR, did not result in a higher ratio of vitreous to plasma ET-1 concentrations, as would be expected if the bloodstream were the sole source of intraocular ET-1. Fourth, ET-1 was present (immunohistochemistry) in the ERMs from patients with PVR. Fifth and finally, human ET-1 gene expression appears to be enhanced by hypoxia [10,11], and retinal hypoxia is a consistent feature of RD and proliferative retinopathies . Interestingly, the application of ET-1 in the perineural region of the anterior optic nerve produced ischemia . Furthermore, several studies showed that ET-1 is involved in the regulation of retinal blood flow [14-16]. ET-1 concentrations within the retina or at the vitreoretinal interface may be greater if ET-1 is produced locally or diffuses into the vitreous chamber from the retinal or choroidal vascular beds and/or from RPE which recently has been demonstrated to be a source for ET-1 in the retina, being more important its increased release during breakdown of the blood-retinal barrier , as in PVR. We have found relatively low levels of vitreous ET-1 in cases of IERMs compared to PVR cases but qualitatively similar levels of IR-ET-1 in ERMs sections were found compared to samples from patients with PVR, this suggest that RPE cells present in PVR membranes are a significant producer of ET-1 compared to glial cells. Elevated vitreous ET-1 concentrations in our patients with recurrent PVR may be explained by the fact that those patients had prior scleral buckling surgery for rhegmatogenous retinal detachment (associated to PVR<=C3) and retinopexy may, in turn, disturb the blood-ocular barrier (BOB) . The breakdown of the BOB associated with RD and PVR increases chemotactic and mitogenic activity in the vitreous cavity . Plasma contains several agents known to stimulate migration  or proliferation of RPE and/or retinal glial cells , including fibronectin, epidermal growth factor and insulin growth factor. The role of glia in RD and PVR has been recently reported [39-41].
Initially, in rhegmatogenous RD, RPE dysfunction with loss of contact between RPE or between RPE cells and photoreceptors or loss of signalling from photoreceptors may result in the initiation of RPE cell proliferation and migration to repair the retinal defect . The end point of the cellular response must be to re-establish the BOB. Once the cells become established in the vitreous cavity or in ERMs, they become an important source of stimulatory factors. Platelet derived growth factor (PDGF) is produced by RPE cells in ERMs  and is a chemoattractant and mitogen for both RPE  and retinal glial cells , it may help to recruit new cells to ERMs and stimulate the proliferation of cells that are there. Vascular endothelial growth factor (VEGF) is also localized to many cells in PVR membranes . Therefore, both VEGF and PDGF may contribute to the progression of ERMs. In turn, VEGF enhances ET-1 mRNA expression and ET-1 secretion in endothelial cells  and ET-1 enhances VEGF mRNA expression and VEGF secretion in vascular smooth muscle cells . Stimulatory interaction between both peptides suggest its relationship in hypoxic and/or ischemic retinopathies.
For some reason, possibly because of a loss of contact with photoreceptors or a loss of some mediators provided by photoreceptors, rhegmatogenous RD causes enhanced expression of growth factors by RPE cells and enhanced responsiveness to growth factors. An autocrine loop involving PDGF factors , VEGF factor , and insulin-like growth factors  has been demonstrated in cultured RPE cells. They produce growth factors that in turn stimulate their own growth. Also, in vivo the same phenomenon is seen, because there is upregulation of PDGF expression in RPE cells adjacent to laser burns or beneath detached retina [22,49,51].
In our patients with rhegmatogenous RD complicated with PVR, the ratio of vitreous to plasma ET-1 levels was elevated compared to RD uncomplicated with PVR (p<0.0001) and to the control group (p<0.0001). Hypoxia and/or ischemia of the detached outer retina has been reported . In our case, increased ET-1 may be a consequence of both retinal hypoxia and the wound healing process scar tissue formation (PVR) as a complication of RD .
In a previous paper  we reported subretinal fluid (SRF) ET-1 levels almost five times higher in eyes with PVR<=C3 than in eyes with RD uncomplicated with PVR. SRF increased levels of ET-1 more than 27 pg/ml exhibited recurrent PVR. In the present paper, the vitreous ET-1 levels of cases with recurrent PVR were 29.19±1.09 pg/ml. These findings suggest that SRF ET-1 concentration and/or vitreous ET-1 concentration may be useful to designate patients at risk to develop severe PVR. Since it is impossible to remove all ERMs during surgery, autocrine and paracrine stimulation derived from the remaining ERMs may contribute to the high rate of recurrent PVR after successful surgery. As recurrent detachment is common in PVR cases  and usually it is caused by recurrent proliferation  could be those cases may benefit of pharmacological treatment.
In conclusion, we report in this paper that IR-ET-1 was present in vitreous humor, that IR-ET-1 levels were elevated in eyes with PVR, and that ET-1 was localized by immunohistochemistry within epiretinal proliferative tissue from both IERMs and PVR membranes. Secretion of ET-1 by glial cells was found also in both IERMs and PVR membranes, but secretion of ET-1 by RPE and RPE involvement was only found in PVR membranes. Expression of ETA and ETB receptors was demonstrated in both types of ERMs. These results expands the range of diseases associated with ET-1 to include proliferative complications of rhegmatogenous retinal detachment.
The authors thank M. S. D. Gomez-Donaire for her excellent technical assistence in the immunohitochemical studies. This work was supported by grant DGES 97/0028 from the Ministerio de Educación y Cultura. R. Rollin holds a fellowship from the Fondo de Investigaciones Sanitarias BEFI 00/9140.
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