Balancer chromosomes are convenient tools used to maintain lethal mutations in heterozygotes. We established a method for engineering new balancers in C. elegans by using the CRISPR/Cas9 system in a non-homologous end-joining mutant. Our studies will make it easier for researchers to maintain lethal mutations and should provide a path for the development of a system that generates rearrangements at specific sites of interest to model and analyse the mechanisms of action of genes.
Genetic balancers (including inversions, translocations and crossover-suppressors) are essential tools to maintain lethal or sterile mutations in heterozygotes. Recombination is suppressed within these chromosomal rearrangements. However, despite efforts to isolate genetic balancers since 19781,2,3,4,5, approximately 15% (map units) of the C. elegans genome has not been covered6 (Supplementary Fig. 1). Because the chromosomal rearrangements generated by gamma-ray and X-ray mutagenesis are random, it is difficult to modify specific chromosomal regions. Here, we used the CRISPR/Cas9 genome editing system to solve this problem. The CRISPR/Cas9 system has enabled genomic engineering of specific DNA sequences and has been successfully applied to the generation of gene knock-outs and knock-ins in humans, rats, mice, zebrafish, flies and nematodes7. Recently, the CRISPR/Cas9 system has been shown to induce inversions and translocations in human cell lines and mouse somatic cells8,9,10. Similarly, inversions up to 57.5 kb have been obtained in the zebrafish germline11. Although a large number of cells can be treated at once for effective CRISPR/Cas9 editing in cell lines, it is more difficult to do so in the germlines of model organisms because of limitations in the ability to introduce genome editing tools. Thus, researchers need an efficient way to engineer the chromosomal structure in multicellular organisms in vivo. In the present study, we established an editing method using the CRISPR/Cas9 system in C. elegans to generate genetic balancers at specific chromosomal sites. The inversions and crossover-suppressors produced were up to 6.7 Mb (~17 cM), lengths 2 orders of magnitude longer than produced in a previous work in the germline of a model organism. To facilitate genomic engineering, we targeted the genome rearrangements in a non-homologous end-joining (NHEJ) mutant background. Our method resulted in a higher proportion of successful rearrangements to generate new balancers. Moreover, we found that the inversion and crossover-suppressor balancers generated in heterozygotes did not result in interchromosomal effects.
Results and Discussion
Experimental design to screen for new balancers on chromosome IV
We designed two sgRNAs (single guide RNA) in the exons of two target genes that result in easily identifiable phenotypes when they are disrupted. We next constructed two targeting vectors that joined the chromosomal breakpoints together, each of which had 2 kb of sequence homologous to each predicted junction point, so that chromosomal rearrangements could be induced by homologous recombination (HR) between the targeted regions and homology vectors (Fig. 1b, Supplementary Fig. 2). A previous study has reported that disabling NHEJ via the RNAi inactivation of the cku-80 gene (a homologue of the human KU80), which acts as a DNA binding protein, significantly improves the HR efficiency in C. elegans12. Therefore, NHEJ disruption may allow for the efficient repair of DSBs by using targeting vectors via HR. One of the NHEJ genes, lig-4 (a human LIG4 homologue), is essential for the final ligation step of the DNA ends. A strain known to contain a disruption in lig-4, tm750, was used in the experimental procedures and is depicted in Fig. 1a.
Genetic engineering of new balancers by using the CRISPR/Cas9 system.
We first attempted to generate an inversion balancer on the left arm of chromosome IV (Fig. 1b), which includes part of the largest region of the C. elegans genome that is not covered by current genetic balancers (Supplementary Fig. 1). We co-injected sgRNAs targeting egl-4 and unc-17 with Cas9, the targeting vectors and a Pmyo-2::Venus transgene marker into the gonads of young adult P0 worms. We screened for fluorescent F1 worms that contained larval-arrested F2 progeny caused by unc-17 disruption and confirmed the rearrangements by PCR amplification of both junction points. To examine whether the candidates maintained the chromosomal inversion, F2 worms that laid larval-arrested F3 progeny were further investigated by using both PCR amplification and DNA sequencing. Through these experimental procedures, one chromosomal rearrangement was obtained in each of the 136 F1 worms in the lig-4(tm750) mutant background (Table 1). The rearrangement named tmIn1(IV) exhibited detectable egl-4::unc-17 fused genes at two junction points, as confirmed by PCR amplification of the breakpoints, but the WT did not (Fig. 1c). These breakpoints were verified by DNA sequencing (Fig. 1d). The rearrangement tmIn1(IV) exhibited a recessive larval arrest phenotype (Fig. 1e). Thus, these experimental techniques induced successful chromosomal rearrangements in the germline of a multicellular organism.
Summary of experimental efficiencies to generate the genetic balancers IV.
We further screened for the generation of large chromosomal balancers on the left arm of chromosome IV. We obtained two additional balancers, tmIn2(IV) and tmIn3(IV), which covered 13.5 cM and 16.4 cM, respectively (Fig. 1h, Table 1, Supplementary Figs 3 and 4). The probability of obtaining inversion strains appeared to decrease as the target size became larger (0.60% and 0.32% for tmIn2 and tmIn3, respectively: Table 1). The tmIn2(IV) and tmIn3(IV) worms exhibited a recessive larval arrest phenotype (Fig. 1f,g). Although target sites between csn-4 and egl-4 covering only 7.6 cM and sgRNA-specific mutations were observed, these chromosomal rearrangements could not be isolated (Fig. 1h, Table 1). One of the target genes (csn-4) was located near pairing centres (PCs), where the chromosome is stabilized by homologue pairing13. The generation frequency decreased at the end of the PC side of the chromosome (Table 1). Heterochromatin is important for maintaining the structural integrity of the genome14. However, tmIn1(IV), tmIn2(IV) and tmIn3(IV) rearrangements were generated on regions of the chromosome arm that are known to contain heterochromatin15 (Table 1). These results suggest that our approach can generate chromosomal rearrangements at specific sites even in heterochromatic regions, whereas these rearrangements were below the generation limit mainly because of the presence of PCs.
Confirmation of the suppression of recombination in tmIn3(IV)
We examined whether tmIn3(IV) could balance a recessive lethal mutation within the inversion interval, as described in Supplementary Fig. 5a. Heterozygous tmIn3(IV) hermaphrodites were mated with heterozygous males carrying a recessive lethal lin-1 mutation (tm5929). After the self-fertilization of F1 worms, the balanced strain lin-1/tmIn3(IV) segregated three phenotypes: WT (lin-1/tmIn3 heterozygotes), lethal (lin-1 homozygotes) and larval arrest (tmIn3 homozygotes) (Supplementary Fig. 5b–g). Thus, the new balancer is a useful tool for maintaining lethal mutations on the left arm of chromosome IV. The segregation of these phenotypes was maintained through more than 20 generations, suggesting that tmIn3 suppresses further recombination of the covered genomic region.
Whole-genome sequence analysis of tmIn3(IV)
To further assess whether the generated balancer strains exhibited rearrangements at unexpected loci, we extracted the genomic DNA from the lin-1(tm5929)/tmIn3(IV) worms and analysed it by whole-genome sequencing (Supplementary Fig. 6, Supplementary Table 1). We observed several structural variants consistent with target-specific rearrangements but no target-independent rearrangements. From these results, we conclude that our methodology can accurately induce target-specific rearrangements.
Isolation of an inversion balancer on chromosome II
Likewise, we sought to generate a chromosomal rearrangement on the left arm of chromosome II, which includes the second-largest region of the C. elegans genome that is not covered by known balancers (Supplementary Fig. 1). We obtained a new balancer named tmIn4(II), which covered 8.6 cM and extensively maintained recessive lethal mutations (Supplementary Figs 7 and 8, Supplementary Table 2). Thus far, we have not been able to generate a balancer near the PCs on chromosome II (Supplementary Fig. 7e, Supplementary Table 2). The results from chromosome IV also indicate that successful chromosomal rearrangements are mainly affected by the distance from the PC region (Fig. 1h).
Genetic engineering of crossover-suppressor and translocation balancers
In addition to the generation of inversions, we also engineered two crossover-suppressors, tmC1(X) and tmC2(X), composed of sequential inversions between lon-2 and mec-10, and between F53B1.2 and unc-18 (Fig. 2, Supplementary Table 3). This strategy was used to produce more stable balancers because multiple inversions more effectively prevent recombination6. The crossover suppressors tmC1(X) and tmC2(X) covered most of the left arm of the X chromosome from F53B1.2 to mec-10, covering 17 cM (Fig. 2h).
Genetic engineering of crossover-suppressors by using the CRISPR/Cas9 system.
Next, to examine whether our method was also effective between different chromosomes, we generated a chromosomal translocation named tmT3(III;IV) that arose between pal-1(III) and unc-17(IV) (Supplementary Fig. 9). There were no differences in the efficiency of generating inversions or translocations (Table 1, Supplementary Fig. 9d; Fisher’s test, P > 0.05). Thus, the results demonstrate that these experimental methodologies successfully provided a systematic approach to target chromosomal rearrangements at specific sites. Because the DNA repair pathways are highly conserved across species, our methodology may provide a universal approach for engineering chromosomal rearrangements.
Generation of transgene-integrated strains by using the CRISPR/Cas9 system
To facilitate balancer chromosome usage, we developed a technique using the CRISPR/Cas9 system that produced multi-copy fluorescent gene integration in tmC1(X) from extrachromosomal arrays16 (Supplementary Fig. 10a). We first generated an extrachromosomal line tmC1;tmEx4487 that expressed Pmyo-2::Venus together with Punc-18::unc-18 (which rescues unc-18 disruption) and a dpy-3 genome sequence as the sgRNA target. We co-injected the dpy-3 genome sequence-specific sgRNA with a Cas9 expression vector and a Pdpy-7::DsRed transgene marker into the gonads of tmC1;tmEx4487 worms. We isolated F1 worms with Venus and DsRed expression and screened F2 progeny for dumpy (Dpy) phenotypes and Venus expression. The breakpoints were examined by PCR amplification (Supplementary Fig. 10b). Venus fluorescent Dpy worms that carried the balancer chromosome and harboured the Pmyo-2::Venus transgene were isolated as tmC1[F53B1.2 lon-2 unc-18 mec-10 Pmyo-2::Venus Punc-18::unc-18] (Supplementary Fig. 10c,d).
Examination of the repair mechanisms that generate rearrangements
During the course of isolation of genetic balancers, we found that only a portion of the phenotype-positive lines yielded PCR-positive alleles; in the case of tmIn1, only 6 of the 40 phenotype-positive lines were PCR-positive (Table 1). This finding implies that DSBs are often repaired without inversion or with structural changes that are unable to be amplified by PCR, thus suggesting that breakpoints are often repaired by a mechanism other than HR. To determine whether the targeting vectors and lig-4 mutant background were truly necessary, we injected the genome-editing and marker plasmids without targeting vectors in the gonads of WT and lig-4 worms. In the absence of the targeting vectors, we still obtained inversions (in 0.33% of WT and 0.13% of lig-4(tm750) offspring), but the probability was decreased compared with that observed in the lig-4(tm750) background injected with targeting vectors (0.73%). Recent studies have shown that the CRISPR/Cas9-induced DSB repair of germ cells in C. elegans is often mediated by polymerase theta-mediated end-joining (TMEJ)17. These observations suggest that in the lig-4(tm750) mutant background, the targeting vectors may be required for HR, but TMEJ may also induce rearrangements. A previous report has also identified the generation of inversions that depended on the LIG4 gene in human cells without repair templates18, suggesting that NHEJ may also be involved in the process.
Upon closer inspection of the repaired regions in the rearrangements in 9 strains obtained from the lig-4 mutant background by using targeting vectors, we found that only one strain (tmIn26) contained complete copies of the targeting vector sequences at both breakpoints (Supplementary Table 4). Another strain tmT3 contained one complete copy at a breakpoint but contained an indel at another breakpoint. The other 7 strains had indels at both breakpoints. Thus, of 18 breakpoints, 3 appeared to be repaired by HR, whereas 15 were repaired by TMEJ. This phenomenon suggests that each breakpoint is repaired by either system stochastically.
In the case of 4 inversion strains without targeting vectors, we found that all the breakpoints contained some indels of the genome sequence (Supplementary Table 4 and Supplementary Fig. 11). This result suggests that TMEJ (for these 4 strains) or NHEJ (except for the case of tmIn45), might be used to repair the breakpoints.
It should also be noted that we were unable to obtain any inversions in the wild-type background by using targeting vectors (0/755 F1 animals). Although it is expected that all three repair mechanisms (HR using targeting vectors, NHEJ and TMEJ) can repair breakpoints, we could not find any evidence for successful rearrangements among the 102 phenotype-positive candidates (Table 1). The probability of successful rearrangements appeared even lower than that in the wild-type background without targeting vectors. Although the mechanisms for this phenomenon remain unclear, we speculate that the introduction of targeting vectors could mobilize NHEJ, thus quickly resulting in the repair of breakpoints without inversion19,20.
In summary, our strategy systematically generated chromosomal inversion, translocation and crossover-suppressor balancers at specific sites. These new balancers covered 8% of the C. elegans genome, remaining 7% of the 15% of the genome that was previously uncovered by balancers. It should be noted that our crossover-suppressor lines containing a fluorescent marker are ideal for the analysis of lethal mutations. Many of the common balancer lines used by the C. elegans research community have translocations and thus suffer from aneuploidy, which is inconvenient for phenotypic analyses6. In contrast, inversion and crossover-suppressor balancer lines have structural variations within their own chromosomes, are free from aneuploidy and are more straightforward to use for the examination of mutant phenotypes. Unfortunately, the crossover-suppressors used to date in the field have complex chromosomal structural changes. Our strategy using CRISPR/Cas9 resulted in minimal additional chromosomal changes. Our crossover-suppressors with double inversions covered a larger genomic region than did simple inversion balancers. Finally, we were able to introduce locus-specific fluorescent markers into these crossover-suppressor lines16.
Caenorhabditis elegans wild-type strain Bristol N2 was used in this study. Lines carrying lig-4(tm750), lin-1(tm5929), and mlt-7(tm1794) mutations were obtained previously21. Nematodes were grown by using standard genetic protocols22.
We used site-directed mutagenesis to insert the guide sequences into a Peft-3::Cas9 + sgRNA dual expression vector (pDD162, Addgene plasmid 47549, Cambridge, MA). We designed G(N)19–25NGG specific sgRNA sequences as previously described23 (Supplementary Table 5). The sgRNA sequences were designed to target the exons of genes with easily identifiable loss-of-function phenotypes, such as uncoordinated (Unc), dumpy (Dpy), long (Lon), or lethal (Let). The Cas9-sgRNA plasmids were made by using a Clontech In-Fusion PCR Cloning Kit (Clontech Laboratories, Palo Alto, CA) as previously described24.
Targeting vectors were constructed by inserting 2 kb of homologous sequences for each target site into pBluescript KS(+) by using a Clontech In-Fusion PCR Cloning Kit (Clontech Laboratories) as previously described24 (Supplementary Table 5). We designed targeting vectors to join two DNA sequences so that junction is the centre of predicted cleavage sites which are located within 3 bp of PAM (promoter adjacent motif) sequences25. For example, in the case for the tmIn1, the left targeting vector contained a chimeric fusion of 1 kb upstream sequence from the putative cleavage site in the egl-4 gene and the reverse-directed 1 kb upstream sequence of the unc-17 gene from the predicted cleavage site. The right targeting vector is composed of a chimeric fusion of the reverse-directed 1 kb downstream sequence from the putative cleavage site in the egl-4 gene and 1 kb downstream sequence of the unc-17 gene from the predicted cleavage site. These target vectors used together, can cause a inversion.
A Cas9 integration-site dpy-3 genome fragment containing approximately 500 bp of sequence homologous to the target site was inserted into pPD95.79, using EcoRI and BamHI sites as previously described16. Plasmids for the transgenic markers Pmyo-2::Venus and Pdpy-7::DsRed were generated as previously described26.
Plasmids were prepared for injection using Qiagen’s Midi Plasmid Purification Kit (QIAGEN, Hilden, Germany). The targeting vectors were linearized from purified plasmids by PCR amplification and were purified using Illustra GFX PCR DNA and a Gel Band Purification Kit (GE Healthcare, Little Chalfont, UK). To generate new balancers, the following concentrations of injection mix were used: 45 ng/μl Cas9-sgRNA #1 dual expression vector, 45 ng/μl Cas9-sgRNA #2 dual expression vector, 40 ng/μl targeting vector (left side), 40 ng/μl targeting vector (right side) and 30 ng/μl Pmyo-2::Venus transgene marker. To generate the integrated strain, the following concentrations of injection mix were used: 100 ng/μl Cas9-sgRNA dual expression vector and 40 ng/μl Pdpy-7::DsRed transgene marker. The injection mix was centrifuged for 3 min at 15,000 × g at 4 °C in Ultrafree-MC filter devices (Millipore, Massachusetts, MA). The injection mix was injected into the germ lines of adult hermaphrodite worms by using standard methods as previously described26. Importantly, the total Cas9-sgRNA plasmid concentration of the injection mix should be lower than 100 ng/μl. When the Cas9-sgRNA concentration exceeded 100 ng/μl, the F1 progeny were sterile.
Screening for the generation of new balancers using the CRISPR/Cas9 system
To screen for new genetic balancers, injected P0 worms were grown on NGM plates at 20 °C for three days. We picked fluorescent F1 worms to individual plates at 20 °C (for example, Table 1 F1 worms). First screening: after three days, we selected plates which contained phenotype-positive F2 worms (for example, Table 1 phenotype-positive worms). By this way, we chose the F1 worms whose genome was cut by Cas9 at the target sites. We then picked F1 worms to lysis buffer (500 μg/ml proteinase K, 100 mM NaCl, 50 mM Tris, 20 mM EDTA, and 1% SDS) and confirmed by nested-PCR amplification with primers (Supplementary Table 5), whose sequences are not included in the targeting vectors (for example, Table 1 F1 PCR). Second screening: To examine whether the rearrangements occurred in the germline of the animals and they were heritable, we then picked F2 animals and performed the same PCR as above (for example, Table 1 F2 PCR). We isolated positive bands and determined and aligned the sequences of both breakpoints. After we identified strains with heritable rearrangements, we singled their F2 progeny to individual plates at 20 °C and cultured them for three days and confirmed the presence of the phenotype-positive F3 in the plates.
Test for balancer chromosome
The tmIn3(IV) rearrangement was chosen to examine whether it could balance a recessive lethal mutation. Heterozygous tmIn3/+ hermaphrodites were mated with heterozygous lin-1/+ males. The F1 progeny from each cross plate were transferred to individual plates at 20 °C for three days. After self-fertilization, the lin-1/tmIn3 hermaphrodites produced offspring that segregated into three genotypes, lin-1/tmIn3, tmIn3/tmIn3 and lin-1/lin-1, and were distinguishable according to their phenotypes.
The tmIn4(II) rearrangement was also examined to determine whether it could balance a recessive lethal mutation. Heterozygous tmIn4/+ hermaphrodites were mated with heterozygous mlt-7/+ males. The F1 progeny from each cross plate were transferred to individual plates at 20 °C for three days. After self-fertilization, mlt-7/tmIn4 hermaphrodites produced offspring segregating into three genotypes, mlt-7/tmIn4, tmIn4/tmIn4 and mlt-7/mlt-7, which were distinguishable according to their phenotypes.
Genomic DNA was extracted from starved worms. Fragmentation of the genome into approximately 140 bp segments and preparation of genomic libraries were performed using automated Library Builder system (Thermo Fisher Scientific). Then, sequence templates were synthesised from the prepared libraries using the Ion Chef system, and the templates were sequenced by Ion Proton (Thermo Fisher Scientific, Massachusetts, MA) according to standard protocols (https://ioncommunity.thermofisher.com/docs/DOC-8775).
Detection of structural variants
Raw sequencing reads were primarily mapped to the reference sequence by using TMAP software (https://github.com/iontorrent/TMAP). The reference sequence was prepared by adding sequences of Pmyo-2::Venus, pDD162 (Addgene plasmid 47549) and pBlueScript II KS(+) to the C. elegans genome sequence (ftp://ftp.wormbase.org/pub/wormbase/species/c_elegans/sequence/genomic). After primary mapping, we calculated the mean value for all read lengths. The product of the average read length times the number of reads was divided by the length of the reference sequence. The result was defined as the coverage (Supplementary Table 1). Then, the genomic rearrangements were detected by following processes.
From the primary mapping results, we obtained clipped reads, which contained both mapped and unmapped sequences (Supplemental Fig. 6a, solid and broken lines). We selected unmapped sequences that were longer than 20 bp and extracted all the continuous 16-base sequences from the unmapped reads and their complementary sequences as queries for the following realignment. From the reference sequence, the regions that perfectly matched the queries were searched by using the Aho-Corasick algorithm27. Then, the unmapped sequences were compared and aligned to the neighbouring sequences of each matched regions using the Smith–Waterman algorithm28. For the algorithm, the values used for matching, mismatching and gap score were +2, −1 and −2, respectively. Through this alignment, we detected the most homologous regions for each unmapped sequence. If there was more than one candidate for the most homologous region of an unmapped sequence, we selected the one that was nearest to the mapped region of the original clipped read.
As a result, we obtained split reads (SR) whose 5′- and 3′-regions were mapped to different sites of the reference29. Next, the split reads were classified into the following 5 categories: deletion-, insertion-, inversion-, translocation- and translocational inversion-type SR. When the 3′-region of an SR was aligned downstream or upstream of the site where the 5′-region of the read was mapped, the SR was defined as a deletion- or insertion-type SR. Otherwise, when the 5′-region of an SR was aligned to the reverse strand of the 3′-region of the read in the same linkage group, the SR was defined as an inversion-type SR. If the 5′- and 3′-regions of an SR were aligned to different linkage groups, the read was defined as a translocation-type SR. If an SR was determined to be both translocation and inversion-type, the SR was defined as a translocational inversion-type SR (Supplementary Fig. 6a, middle panel). After classification, we eliminated SRs that were also detected in control data. If the number of deletion-type SRs that contained a common gap between the 5′- and 3′-regions was greater than 2, we defined the region as a deletion candidate. Additionally, we investigated the combination of two types of SRs to detect complicated variant candidates. When combined deletion- and insertion-type SRs were located on both sides of a region, the region was defined as an insertion candidate. Similarly, when two inversion-type SRs were located on both sides of a region, the region was defined as a local inversion or inverted insertion candidate. The translocated insertion and inverted translocational insertion candidates were also defined using two translocation- and translocational inversion-type SRs. If there were gaps near the border of a variant region, the variant was also defined as a deletion candidate (Supplementary Fig. 6a, lower panel). To improve the reliability, complicated variants were removed when fewer than ten reads contained common variant regions.
We also counted the number of reads covering each base of the reference sequence as the depth of sequence. Regions in which the depth values were greater than one were defined as mapped regions (Supplementary Table 1). In mapped regions, the depth values were divided by the coverage value, and the quotient was defined as the normalized depth (ND) (Supplementary Fig. 6b, left panel). Then, the ratio of the ND between the balanced strains and tmIn3 was calculated as the depth ratio (DR) value (Supplementary Fig. 6b, right panel). A low DR value meant that the copy number of the base was lower than that in the control, thus suggesting that the base was deleted in the balanced strain. Finally, we evaluated the variant candidates investigated by SR analysis using DR values. When the DR value of a deleted region was higher than 0.75, the variant was removed. Furthermore, when the DR value of insertion variants was greater than 1.75 or 2.5, the variants were defined as duplications or multiplications, respectively.
Generation of the tmC1;tmEx4487 transgenic line
To generate tmC1;tmEx4487 transgenic worms, 20 ng/μl Cas9 integration-site dpy-3 genome fragment, 160 ng/μl Pmyo-2::Venus and 20 ng/μl Punc-18::unc-18 (unc-18 rescue construct) were co-injected into tmC1 worms by using standard methods as previously described30.
Generation of integrated strains by using the CRISPR/Cas9 system
Integration of extrachromosomal arrays into a balancer line was performed as previously described16. To screen for integrated strains, we first removed the lig-4 (tm750) background, and the injected P0tmC1;tmEx4487 worms were grown on NGM plates at 20 °C for four days. After self-fertilization, we picked F1 worms with Venus and DsRed fluorescence and transferred them to individual plates, where they were incubated at 20 °C for four days. If their F2 progeny carried integrated Pmyo-2::Venus constructs in tmC1, Dpy progeny would express Venus in the pharynx. In contrast, in F2 progeny carrying only tmEx4487, Dpy progeny would not express Venus. To confirm integration, F2 Dpy animals were transferred to individual plates and grown at 20 °C for four days. After self-fertilization, if the F3 Dpy progeny carried the desired integration, all Dpy progeny would express Venus.
How to cite this article: Iwata, S. et al. Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9. Sci. Rep.6, 33840; doi: 10.1038/srep33840 (2016).
We thank our laboratory members for their helpful discussions and suggestions.
Author Contributions S.I. designed and performed the experiments, and drafted the manuscript. S.Y. participated in the design of the studies. Y.S. performed the computational analysis of whole-genome sequence. S.Y., Y.S. and S.H. helped to write the manuscript. S.M. conceived and coordinated the studies, and corrected the manuscript.
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Department of Ophthalmology, University “La Sapienza”, Polo Pontino, Latina, Italy
Copyright © 2010 Serena Salvatore and Enzo Maria Vingolo. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Endothelin is a potent vasoactive peptide occurring in three isotypes, ET-1, ET-2, and ET-3. Through its two main receptors, endothelin A and endothelin B, it is responsible for a variety of physiological functions, primarily blood flow control. Recent evidence from both human and animal models shows involvement of endothelin in diabetes, retinal circulation, and optic neuropathies. Increased circulating levels of endothelin-1 (ET-1) have been found in patients with diabetes, and a positive correlation between plasma ET-1 levels and microangiopathy in patients with type-2 diabetes has been demonstrated. In addition to its direct vasoconstrictor effects, enhanced levels of ET-1 may contribute to endothelial dysfunction through inhibitory effects on nitric oxide (NO) production. Experimental studies have shown that chronic ET-1 administration to the optic nerve immediately behind the globe causes neuronal damage, activation of astrocytes, the major glial cell in the anterior optic nerve, and upregulation of endothelin B receptors. This paper outlines the ubiquitous role of endothelin and its potential involvement in ophthalmology.
Endothelin 1 (ET-1) is a potent vasoconstrictor  peptide that is also expressed in neurons . The peptide comprises 21 amino-acids with two intrachain disulfide linkages and was first isolated from the culture supernatant of porcine aorta endothelium cells in 1988 . Endothelin derives from “big endothelin” a prepropeptide that is cleaved by endothelin- converting enzymes to produce mature endothelin .
Since its identification, endothelin has attracted intensive interdisciplinavy interest because of its unique profile as an endothelium-derived vasoactive factor with a powerful and characteristically long-lasting vasopressor activity. Thus, whereas cellular mechanism of endothelin action appear to be similar to classic vasoconstrictor substances such as angiotensin II and norepinephrine, the slow time course of the regulatory mechanisms of its biosynthesis and secretion resembles more that of inflammatory cytokines. These characteristics make this family of small peptides unique within the realm of intercellular mediators with cardiovascular relevance .
Endothelin is expressed in three isoforms called ET-1, ET-2, and ET-3, with slightly different amino-acid sequences and different distribution in various tissues. Accordingly, three different genes encoding the endothelins have been identified in the human, rat, and pig genome . Furthermore, three ET receptor subtypes called ETA, ETB and ETC have been described.
The presence of ET-1 has been demonstrated in the human brain, pituitary gland , spinal cord , lung , rat bladder , rat kidney, feline intestinal tissue  and human and rat eye . In rabbit, ET-1 has also been found in the tear glands . ET-1 is present in the aqueous humour at concentrations several times higher than in plasma, presumably because it is secreted by the ciliary epithelium and not derived from plasma . ET-3 is less ubiquitous and is found in the iris, ciliary body, and retina in some species. The presence of ET-2 in the eye has not been studied extensively. However, in the context of a possible role for this isoform in the eye pathophysiology Murata and coworkers found no evidence of ET-2 gene expression in the retina .
The vascular endothelium modulates local vascular tone by releasing relaxing factors such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factors as well as the potent vasoconstrictor peptide endothelin-1 . Although this local regulatory system can be found in nearly all vertebrates, a great heterogeneity exists between different species, particularly in the various chemical and hormonal agonists that can stimulate the release of these endothelium-derived factors and between large and small vessels and different vascular beds .
Haefliger and coworkers  demonstrated the presence of both the endothelium-derived relaxing factor, nitric oxide, and the vasoconstrictor peptide endothelin in the human ophthalmic artery. These authors found that in human ophthalmic arteries, the endothelial L-arginine/nitric oxide pathway is active under basal conditions and is further stimulated by bradykinin, acetylcholine, and histamine, whereas ET1 has a potent vasoconstrictor effect. The potency of these responses suggests an important physiological role for endothelium-derived vasoactive substances in the regulation of the human ophthalmic circulation. Furthermore, a dysfunction of these endothelial mechanisms, which occurs in diabetes and hypertension (at least in peripheral arteries) may play an important role in the pathophysiology of ophthalmic complications .
In addition, in a subpopulation of patients with glaucoma presenting ocular vasospasms, endothelial dysfunction may represent the underlying cause or at least contribute to alterations in ophthalmic blood flow .
Vascular ET-1 is synthesized primarily in the endothelium, although it can also be produced in vascular smooth muscle cells, macrophages, leukocytes, cardiomyocytes, and fibroblasts .
In the kidney, tubular epithelial cells, mesangial cells, and podocytes are capable of ET-1 release .
Several mechanism are involved in the clearance of ET-1 from plasma, including endocytosis in the lungs, enzymatic degradation, degradation of the endothelinB receptor ligand complex, and enzymatic processes in the kidney and liver [19, 20].
ET-1 is the most potent vasoconstrictive substance known, more potent than angiotensin II . It has been shown to be highly concentrated in human atherosclerotic plaques with coronary vasospasm  and is also present in human plasma . In addition, ET-1 mediates a wide variety of biological activities, including contraction of non vascular smooth muscle , thus leading, for example, to bronchoconstriction . ET-1 also stimulates mitotic activity of rat vascular smooth muscle cells  and is thought to act as neuropeptide .
Endothelins are also associated with neuronal apoptotosis in the central nervous system. Exposure of retinal ganglion cells (RGCs) or RGC-5 cells, a transformed cell line, to ET-1 causes apoptic cell death. In addition, it was demonstrated that ET-1 enhances the glutamate-induced death of cultured retinal neurons. Furthermore Syed and colleagues  showed that a nonselective ET antagonist is neuroprotective in the rat retina during ischemia/reperfusion. These findings indicate that ET-1 is involved in the cell death signalling pathway(s) in retinal neurons. Interactions between ET-1 and nitric oxide synthase (NOS) have been well documented. For example stimulation of ETB receptors leads to vasodilation through the formation of nitric oxide (NO), and an intravitreal injection of ET-1 enhances NO production in the optic nerve of rabbits. NOS is strongly coupled to the generation of superoxide through the uncoupling of NOS. NO can affect mitochondrial function and increase the formation of superoxide by mitochondria. Under these conditions, NO is inactivated by superoxide anion and changed into highly toxic peroxynitrite. Superoxide also causes neuronal apoptosis, and an inhibition of its formation has neuroprotective effects. Reactive oxygen species (ROS), for example, nitric oxide, superoxide, and peroxynitrite, contribute to neurodegenerative diseases including Parkinson disease and amyotrophic lateral sclerosis.
In a recent study, Oku and coworkers also demonstrated that ET-1 causes death of retinal neurons through activation of NOS and production of superoxide anion .
Although ET-1 appears to act mainly as a local paracrine/autocrine peptide, circulating levels of endothelin seem also to have biological significance, especially in pathological states of increased serum concentration.
So far there has been no comprehensive report on the multifunctional role of ET-1 in the human eye. Therefore, the present paper is specifically addressed to illustrate ET-1 functions in the healthy and diseased eye.
2. ET-1 and Human Corneal Epithelium
ET-1 was found in the human corneal epithelium . Interestingly, ET-1 has also been demonstrated in rabbit tear fluid . Furthermore, in the rabbit ET-1 is able to induce proliferation of cultured corneal epithelial cells  and to promote corneal wound healing. Bek and McMillen  found that ET-1 is angiogenic in the rat cornea and this effect appears to be direct and dependent on activation of the ETA receptor. Likewise, ET-1 might be involved in regulating growth of human corneal epithelial cells, which is important for the continuous renewal of cornea and recovery from corneal trauma and keratoplasty.
3. ET-1 and Retinal Circulation
It has been demonstrated that endothelins can modulate retinal pericyte contractility and, hence, retinal hemodynamics. Because of the potential importance of pericytes in maintaining normal retinal physiology and the involvement of pericyte dysfunction in diseases such as diabetes and diabetic retinopathy, studies were performed to characterize the local effects of endothelin on retinal hemodinamics in the rat eye. The retinal hemodynamic changes in response to intravitreal injections of endothelin were quantitated in diabetic and nondiabetic rats using a retinal video fluorescein angiography system. Bursell and coworkers  demonstrated that the retinal circulation responds directly, in a dose-dependent manner, to ET-1 injection. The vasoconstrictor activity appears to be associated primarily with the retinal arteries, whose diameter is reduced by 17% 15 minutes after 10−7 M ET-1 injection, compared to baseline preinfusion diameters. Correspondingly, the veins demonstrated a non significant (6%) reduction in diameter that was at least three times smaller than the arterial response. In the diabetic rat, the retinal response to ET-1 injection was approximately 10 times smaller () than that observed in the nondiabetic rats. This reduction in the physiological response in diabetic animals parallels that observed in retinal pericytes cultured under high-glucose conditions and suggests that retinal pericyte function is impaired at early stages in diabetes. It has also been shown that glucose can cause an enhanced secretion of endothelin from cultured endothelial cells and that retinal pericytes demonstrate prolonged desensitization to further stimulation by ET-1 after the first application of endothelin. The findings suggest that the blunted ET-1 response observed in diabetic rats results from ET-1 secretion enhanced by glucose, endothelin receptor desensitization, or both. Although this phenomenon is still under investigation the fact that retinal blood flow is decreased at an early stage in diabetes suggests that increased ET-1 secretion and the resultant vasoconstriction at the microcirculatory level, potentially mediated by the retinal pericytes, contributes to the decreased retinal blood flow observed in diabetic animals. This may represent one of the molecular mechanisms related to the subsequent development of microvascular disease and diabetic retinopathy.
4. ET-1 and Diabetic Retinopathy
Several studies support the hypothesis that endothelial dysfunction anticipates type 2 diabetes, indicating that vascular endothelial dysfunction may precede insulin resistance, although the features of insulin resistance syndrome include factors that have negative effects on endothelial function. Impaired endothelial-dependent and independent microvascular reactivity has also been demonstrated in healthy subjects with risk factors for type 2 diabetes. One important feature of endothelial dysfunction is an increased production and biological activity of the potent vasoconstrictor and proinflammatory peptide ET-1. Elevated levels of ET-1 are found in patients with type 2 diabetes,  (see Figure 1), and ET-1-induced reduction in insulin sensitivity may take part in the development of the metabolic syndrome. Therefore, in diabetes mellitus the disturbance in ET-1 production from vascular endothelium represents an early phenomenon rather than the result of advanced stage of the disease.
Figure 1: The plasma immunoreactive-endothelin concentrations were found to be greatly raised in the patients with diabetes (1,880 +/− 120 fmol/l, mean +/− SEM) compared with the healthy subjects (540 +/− 50 fmol/l, less than.005). Figure adapted from Takahashi et al. .
Furthermore, ET-1 may contribute to the development of endothelial dysfunction, and consequently insulin resistance, by increasing the production of reactive oxygen species, mainly superoxide anion, in the vasculature. This is mainly dependent upon activation of NADPH oxidase protein expression and activity. ET-1 levels in tissues are closely linked with reactive oxygen species (ROS) to serve as pro- inflammatory factors, and ROS are considered important factors in mechanisms underlying diabetic complications and cardiovascular derangement .
Su et al.  investigated at the molecular level the effects of total triterpene acids (TTAs) from Fructus Corni on early diabetic complications and whether the novel endothelin receptor antagonist CPU0213 could reverse these pathological changes via suppression of ET and inducible nitric oxide synthase (iNOS) in early diabetic retinopathy. Furthermore, they compared the effects of TTAs with those produced by blocking the receptors of advanced glycation end-products (RAGE) and iNOS by amino guanidine an advanced glycation end-products antagonist. They found that activated RAGE may accelerate the biosynthesis and release of inflammatory factors, including ET-1, ROS and iNOS. Endothelin-1 and ROS interaction results in a vicious cycle leading to mutual enhancement of vascular and cellular effects that converge to represent aetiological factors that damage macromolecules in tissues. This process may involve nuclear factor-(NF-) kB and tumor necrosis factor-.
The extracellular matrix of the retina, which accumulates in retinopathy, is composed of mainly fibronectin, which undergoes alternative splicing to produce embryonic isoforms of fetal fibronectin, and increased levels of fibronectin contribute to retinopathy .
Fibronectin and its isomers are involved in endothelial cell proliferation and the increase in the extracellular matrix can be prevented by endothelin receptor antagonism by bosentan .
Diabetes produces increased microvascular permeability along with increased vascular endothelial growth factor (VEGF) mRNA expression, resulting in the deposition of hard exudates in the retina, where VEGF and ET-1 are known to interact. Thus, upregulation of the ETA receptor can be considered a biomarker for the development of early retinopathy in diabetes. The endothelin receptor antagonist CPU0213 does not substantially affect fasting serum glucose, but it improves the vascular and retinal complications of diabetic rats, in agreement with its amelioration of diabetic cardiomopathy. Furthermore, TTAs reverse the abnormal upregulation of the ET system in the vasculature and the abnormal expression of the ETA receptor and iNOS mRNA in retinal tissues. Thus, TTAs are likely to exert antioxidative and antiproliferative effects on diabetic retinopathy and vasculopathy by suppression of the protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways and, indirectly, by suppressing the overexpression of ppET-1, ECE, ETA receptor and iNOS mRNA. Thus, the effects of TTAs resemble those of endothelin receptor antagonists and may be superior to amino guanidine in alleviating diabetic complications such as retinopathy and vasculpathy.
Compromised vasorelaxation reduces blood perfusion in the retina, leading to upregulation of the ET system and to higher levels of ET-1 in the vasculature. This will further impair relaxation of vasculature smooth muscle because ET-1 is a very potent vasoconstrictor. More importantly, ET-1 produces proliferation of the vascular smooth muscle, increasing the thickness in the vasculature, reducing lumen size, increasing its thickness of the basement membran and thus decreasing permeability in the retina. The proliferative actions of ET-1 are likely to increase extracellular matrix and stimulate fibrosis in the retina, eventually producing proliferative retinopathy. Golubovic-Arsovska  found that upregulation of ETA receptor is related, at least in part, to the development of preproliferative and proliferative retinopathy in clinical settings and correlates with the appearance of maculopathy and macular edema.
The overexpression of ET-1 serves as a key biomarker for endothelial dysfunction that is likely related to superoxide generation and activated phosphatidyl 3-kinase activity .
Makino et al.  suggested that an increase in the basal and -adrenoceptor agonist-induced release of ET-1 in diabetes can be attributed to an overexpression of the mRNA for the ppET-1. An excess of ET-1 binding to the ETA receptor stimulates the PKC and MAPK pathways, which leads to phenotypic abnormalities in the genes encoding for the ppET-1, iNOS, proliferation of matrix formation, and vascular smooth muscle .
The molecular mechanisms of NOS regulation in hyperglycemia are not fully known, but recent studies point to a decisive role of an activated PKC pathway. Hyperglycaemia markedly activates the BII isoform of PKC in endothelial cells by promoting de novo synthesis of diacylglycerol and increasing mitochondrial superoxide production .
Diabetic retinopathy is a potentially sight-threatening complication that develops in nearly all patients with diabetes. Ocular tissues, for example, vascular and extravascular sites in the retina are sources of ET-1, and ET-1 contributes to abnormal retinal hemodinamics in diabetic retinopathy. Results from several studies on streptozocin-induced diabetes in rats have suggested a role of ET-1 in the pathogenesis of diabetic retinopathy. An interaction between ET-1 and VEGF has also been reported, and preliminary positive results of treatment with endothelin receptor blockers have raised interest in these substances as potentially therapeutic agents. Most of the studies on the role of ET-1 in the pathogenesis of diabetic retinopathy and on the importance of endothelin blockers in the treatment of this serious complication have been done in animal models. Nevertheless, they provide sufficient evidence that strongly links ET-1 to the pathogenesis of diabetic retinopathy.
5. ET-1 and Retinitis Pigmentosa
Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous retinal degenerations characterized by chronic progressive loss of rod and cone photoreceptor function . The damage is actually explained with an invalid gene product or protein causing a metabolic wrong step in the phototransduction process. Different inheritance patterns of tapetoretinal degenerations were described: autosomal dominant, recessive and X-linked, all of them associated with point mutations and intragenic micro deletions as well as other molecular defects within over forty-five different RP and loci . Hemodynamic studies have demonstrated that RP is associated with a reduction in retinal and choroidal blood flow. Retinal hemodynamic impairment is also present in early stages of RP and among the various hypotheses advanced on the causative factors involved in this impairment the ET-1-mediated vasoconstriction is a likely candidate.
ET-1 has been shown to be expressed in the retina, including the photoreceptors, the inner plexiform layer and the ganglion cell layer . The choroid, the vascular smooth muscle in retinal blood vessels and choriocapillaris have also been shown to contain receptors for ET-1.
Secretion of ET-1 by the retinal pigment epithelium (RPE) could target receptors on the apical (photoreceptors) or basal (choroid) sides. Activation of ET receptors in the retinal or choroidal vasculature may be important in regulating blood flow at this region. Haemodynamic studies have demonstrated that RP is associated with a reduction of retinal and choroidal blood flow  even in early stages of RP.
These vascular abnormalities might be correlated with the increased plasma ET-1 concentration that was found in two studies on RP patients [43, 48] (Figure 2). Although these data require further investigation in a larger population of patients, these studies open the possibility that antiendothelin drugs could result therapeutically useful in RP administered either alone or in association with treatments directed to improving tissue oxygenation such as hyperbaric oxygen therapy .
Figure 2: (a) Scatter plot of ET-1 plasma values in patients with retinitis pigmentosa (RP), syndromic retinitis pigmentosa (syndromic RP), and healthy subjects (HS). (b) Mean (M ± SD) plasma ET-1 levels in patients with RP, syndromic RP, and HS. Figure adapted from Vingolo et al. .
6. Endothelin-1 and the Optic Nerve Head
Open angle glaucoma is the most common optic neuropathy causing retinal ganglion cell (RGC) soma and axon loss, optic nerve head (ONH) structural loss, and visual field damage. Elevated intraocular pressure (IOP) is the most potent risk factor known for causing glaucomatous damage. Decades of experimental and clinical research consolidated the notion that lowering IOP has a favourable impact in the majority of patients with glaucoma. This has been confirmed in randomized clinical trials that included untreated control arms . How IOP can lead to structural damage and produce clinical glaucoma is not fully understood. Much evidence points to the ONH, consisting of RGC axons, blood vessels, connective tissues and glia, as the primary site of damage in glaucoma . It is likely that the microenvironment in the ONH reacts to stressors such as IOP and other potential IOP-dependent or IOP-independent factors, such as ischemia, to ultimately cause RGC axonal damage (Figure 3). The potential of endogenous vasoconstrictors to cause ischemic insult in the ONH was proposed many years ago and endothelin represents one vasoconstrictor agent with a possible role in glaucoma and other neurodegenerative diseases (Figure 3). An impairment of the anterior optic nerve microcirculation has been suspected to contribute and/or to be a causal factor in a variety of optic neuropathies. Thus, endothelins that produce dose-dependent vasoconstriction in various vascular beds, including the anterior optic nerve microvasculature, become likely candidates able to produce neural damage. Indeed, these peptides have been shown to produce localized vasoconstriction when injected directly into perivascular cerebral tissues in vivo and to result in regional ischemic damage of the brain nervous tissue .
Figure 3: Potential mechanism of endothelin (ET) and ET receptor-mediated actions causing retinal ganglion cell (RGC) loss. Optic nerve astrocytes may become activated via ET actions, leading to extracellular matrix (ECM) changes in the optic nerve head and eventual RGC loss. Figure adapted from Chauhan BC .
Increased plasma ET-1 levels have been described in normal tension glaucoma patients, although this finding was not confirmed in every study dealing with normal tension glaucoma patients or in studies with high tension glaucoma patients. On the other hand, the fact that aqueous ET-1 concentration is increased in primary open-angle glaucoma and in animal models of glaucoma underscores the possible contribution of endothelin to the pathogenesis of primary open angle glaucoma. Furthermore, chronic administration of ET-1 has been shown to produce an optic neuropathy similar to glaucoma. In the rabbit animal model of glaucoma Orgül et al.  assessed optic nerve blood flow after local administration of endothelin-1 in vivo. In addition, the effect of optic nerve ischemia was monitored by means of a confocal scanning laser ophthalmoscope. Administration of endothelin-1 to the anterior optic nerve region induced a significant decrease in local blood flow of approximately 38% compared to the contralateral eye. Multivariate analysis disclosed a small, but statistically significant change in optic nerve morphology, as measured with a confocal scanning laser ophthalmoscope, after 8 weeks of local administration of endothelin-1, compared to the control eye. These changes were consistent with an optic nerve cupping and a decrease in optic nerve rim volume. Histologic analysis showed loss of myelin and gliosis of the prelaminar portion of the optic nerve in optic nerves subjected to endothelin-1 for 8 weeks (Figure 4). Blood flow and morphologic changes were independent of changes in intraocular pressure.
Figure 4: Light microscopic view of two pairs of optic nerves (toluidine blue stain). Optic nerves subjected to endothelin-1 during 8 weeks ((a) and (c)) showed a loss of myelin (dark areas) and a gliosis of the prelaminar portion of the optic nerve compared to the contralateral eyes ((b) and (d)). Arrows point to myelinated nerve fibers in the control eyes ((b) and (d)). Figure adapted from Orgul et al. .
In healthy young humans, the circulating levels of endothelin are low . In pathologic conditions such as an ischemic cerebrovascular insult, the plasma level of endothelin-1 has been reported to be elevated . Emre and coworkers  found increased plasma level of ET-1 in primary open-angle glaucoma patients with progressive damage when compared with primary open-angle glaucoma patients with stable visual fields, this difference was independent of sex, age, and mean blood pressure. Furthermore, they found that values obtained among patients with a stable visual field were above the reference values established in their laboratory (Figure 5). A possible explanation would be that ET-1 may contribute to the initiation of the damaging processes in glaucoma, but then remain increased as a consequence of the damage.
Figure 5: Plasma concentration of ET-1 in patients with glaucoma with and without visual field progression (triangle = mean; box = standard error od the mean; whiskers = standard deviation). Plasma levels of ET-1 at the end of the observation period were significantly higher in patients with progressive damage (3.47 (0.75) pg/ml; range 2.34–5.17 pg/ ml) compared with those with stable (2.60 (SD 0.54) pg/ml; range 1.91–3.45 pg/ml) visual fields (ANOVA: ; ). This difference was still significant after controlling for the interaction of sex and including age and MBP as covariates into the model (ANCOVA: ; ). Sex had a borderline significant effect (ANCOVA: ; ), but the interaction between sex and disease progression was not significant (ANCOVA: ; ). Figure adapted from Emre et al. .
Endothelin may also directly regulate the blood flow within the optic nerve . The ocular circulation is particularly sensitive to changes in local ET-1 concentration, even at doses that do not affect the systemic haemodynamics or flow velocity in the ophthalmic artery . In some individuals with primary open-angle glaucoma, higher-than normal plasma and asqueous humor concentrations of ET-1 have been observed [60–63]. This high concentration of ET-1 has been associated with reduced blood flow in the posterior ciliary arteries that supply the optic nerve vasculature. Peripheral vascular response to ET-1 is also altered in some individuals with glaucoma. Intravitreal injections of ET-1 into the rabbit eye produce marked effects on the anterior ciliary circulation, as well as constriction of the retinal vasculature. With ET-1 administration to the retrobulbar perineural space, localized constriction in the posterior ciliary arterial circulation has been produced in both the rabbit and primate eye .
7. Endethelin-1 and Retinal Detachment
ET-1 is found in the photoreceptor inner segments of the human retina where it may play a role in neuromodulation or neurotransmission [65, 66].
ET-1 immunoreactivity is present on the cell body of the astrocytes and both ET-1 mRNA and ET-1 immunoreactivity have been found in the retinal pigment epithelium (RPE) cells. Elevated subretinal fluid (SRF) and vitreous immunoreactive ET-1 (IR-ET-1)  levels were associated with RD (retinal detachment) and proliferative vitreoretinopathy (PVR) .
IR- ET-1 was localized in the cellular and stromal components of PVR membranes where, ETA and ETB receptor expression has also been demonstrated.
The significant correlation found between SRF-IR-ET-1 and plasma IR-ET1 suggests that intraocular IR-ET1 derives in part from systemic circulation, but the slope of the correlation, always greater for the PVR group, is consistent with enhanced access of plasma proteins in the eye due to a disruption of the blood-ocular barrier. The increased difference of SRF- plasma IR-ET1 in the PVR group may also suggest a local ET-1 production in PVR.
Endothelin could play a role in photoreceptor synaptic transmission, and this would require thight control of the endothelin extracellular concentration. Modulation of synaptic transmission might affect photoreceptor survival, perhaps by regulating glutamate release .
RPE and glial cells are the main contributors to membrane formation and contraction in PVR. The possibility that RPE plays a role in endothelin-mediated photoreceptor survival cannot be excluded because this retinal layer contains ET-1, prepro-ET-1, and ETA immunoreactivities .
ET-1 may also act as growth factor for astrocytes, inducing DNA synthesis and proliferation . Astrocytic proliferation together with an excessive secretion of ET-1 has been reported in cerebral focal ischemia in vivo . Sasaki et al.  demonstrated that ET-1 specifically stimulated the efflux of glutamate via ETB receptors from cultured rat astrocytes, suggesting that ET-1 may esacerbate neurodegeneration. Infusion of ETB selective antagonists attenuates the increase in astrocytes after injury of the brain cortex, indicating that induction of reactive astrocytes depends on the activation of ETB receptors . Reactive gliosis has been suggested as a clinically significant limiting factor in the recovery of vision after RD. Excess of ET-1 released by injured glial cells can be compensated by scavenging ETB receptors,  and it has been suggested that blockade of these receptors after central nervous system injury might modulate glial scar formation, providing a more permissive substrate for neural survival and regeneration . Roldán-Pallarés et al.  investigated the relationship between visual acuity (VA) and SRF-IR-ET1 levels in RD. They concluded that VA was inversely correlated with SRF- IR-ET1 levels. Nevertheless, the highest negative correlation between postoperative VA and VA difference and the SRF-IR-ET1 levels was found in the group with proliferative complication of retinal detachment (PVR). These findings support the idea of performing a primary vitrectomy at early stages of pathology in RD to eliminate the intraocular peptide and perhaps associate a pharmacologic therapy in RD, more importantly in PVR.
ET-1 can modulate anterograde fast axonal transport, which is essential for maintaining synaptic function and neuronal survival .
Recently experimental evidence that stimulation of endothelinergic receptors may modulate photoreceptor survival and glial activation has been provided .
Antiendothelin monoclonal antibodies as well as receptor antagonists BQ-123 (ETA selective, peptidic) and Ro 46-2005 (nonselective, nonpeptidic) have been repeatedly shown to ameliorate ischemic and cyclosporine-induced acute renal failure. Chronic administration of FR 139317, another ETA selective antagonist, was effective in preventing progressive proliferative renal disease and associated hypertension in a rat model of chronic glomerulonephropathy induced by surgical renal mass reduction. The blockade of endothelin action by neutralizing antibodies or BQ-123 has been shown to reduce the extent of experimental acute myocardial infarction in rat, rabbit, and dog. The peptidic ETA selective antagonist BQ-485 is effective in preventing delayed cerebrovascular spasm in a dog model of subarachnoid hemorrhage. In a similar model in the rabbit, the nonpeptidic antagonist Ro-47-0203 reverses delayed vasospasm. Furthermore, endothelin receptor antagonists lower basal blood pressure in spontaneously hypertensive and spontaneously hypertensive strokeprone strains of rats as well as in sodium-depleted squirrel monkeys, suggesting a possible role for endothelins in maintaining blood pressure control under certain conditions and in the development of genetically determined hypertension. The antagonist not only reduces blood pressure but also prevents secondary renal disease observed in DOCA/salt-treated, spontaneously hypertensive rats with malignant hypertension.
ECE (endothelin-converting enzyme) represents another target for pharmacological intervention on the endothelin system because ECE appears to be a metalloprotease that has a strict substrate specificity. A selective inhibitor for the enzyme can presumably inhibit the production of active endothelins in a highly fast manner. Unfortunately, this avenue of research has been severely hampered in the past because of the elusive molecular nature of ECE itself. However, now that ECE has been apparently purified to near-homogeneity, this major component of the endothelin system should soon be revealed at the molecular level.
Endothelin-1 itself could be the target molecule of new therapeutic drugs. In a recent study, Scorza et al.  analyzed the influence of aminaphtone, a 4-aminobenzoic acid derivative clinically used for the treatment of capillary disorders, on ET-1 protein production, pre pro endothelin (PPET-1) gene expression and ECE activity in human ECV304 cells after incubation with physiological concentrations of interleukin-1β (IL-1β). ECV304 cells originate from endothelial cells of human umbilical vein by spontaneous transformation and have been widely used for in vitro studies of the endothelium and ET-1 pathways, since they are able to produce ET-1 and express the mRNA and the α-isoform of ECE. They demonstrated that addition of different concentrations of aminaphtone to ECV304 cells reduces production of ET-1 and downregulates PPET-1 gene expression in a concentration-dependent manner. Moreover they observed that aminaphtone at a concentration of 6 μg/mL, which roughly represents the peak plasma concentration reached after oral administration of 75 mg of the drug, reverts production of ET-1 to baseline values . This may encourage clinical trials on the efficacy of this molecule in the downregulation of ET-1 levels in eye disorders. Development in the near future of nonpeptidic receptor antagonists with even higher potency and specific ECE inhibitors shoud further facilitate the understanding of the biological role of endothelins in health and disease. Those new biological insights should provide renewed promise for the progress toward this novel target for therapeutic intervention, which may include ocular diseases for which no effective drug treatment is currently available.