Vascular derived endothelin receptor A controls endothelin-induced retinal ganglion cell death

Endothelin (EDN, also known as ET) signaling has been suggested to be an important mediator of retinal ganglion cell (RGC) death in glaucoma. Antagonism of EDN receptors (EDNRA and EDNRB, also known as ET-A and ET-B) prevented RGC death in mouse models of chronic ocular hypertension, and intravitreal injection of EDN ligand was sufficient to drive RGC death. However, it remains unclear which cell types EDN ligands directly affect to elicit RGC death. Multiple cell types in the retina and optic nerve express EDNRA and EDNRB and thus could respond to EDN ligands in the context of glaucoma. Here, we systematically deleted Edn receptors from specific cell types to identify the critical EDN receptor mediating RGC death in vivo. Deletion of both Ednra and Ednrb from retinal neurons (including RGCs) and macroglia did not prevent RGC loss after exposure to EDN1 ligands, suggesting EDN1 ligands cause RGC death via an indirect mechanism involving a secondary cell type. Deletion of Ednra from the full body, and then specifically from vascular mural cells, prevented EDN1-induced vasoconstriction and RGC death. Together, these data suggest EDN ligands cause RGC death via a mechanism initiated by vascular mural cells. It is possible RGC death is a consequence of vascular mural cell-induced vasoconstriction and its pathological sequelae. These results highlight the potential importance of neurovascular dysfunction in glaucoma.


INTRODUCTION
Glaucoma is a neurodegenerative condition affecting the output neurons of the retina-the retinal ganglion cells (RGCs). One of the most important risk factors for developing glaucomatous neurodegeneration is elevated intraocular pressure (IOP) [1]. To date, elevated IOP is the only clinically treatable component of glaucoma, and unfortunately, normalizing IOP does not prevent glaucoma progression or development in many patients [2]. Therefore, understanding the molecular signaling pathways that lead from ocular hypertensive injury to RGC death is critical for understanding the pathobiology of glaucoma. Recent evidence has suggested the importance of RGC-extrinsic signaling events (e.g., neuroinflammation, neurovascular dysfunction) in triggering glaucomatous RGC injury [3][4][5][6]. Molecular clustering analysis of ocular hypertensive DBA/2J retinas and optic nerves revealed several candidate mechanisms that are potentially critical in driving RGC injury in glaucoma, including activation of the endothelin system [5,7].
As with many signaling systems that have a physiological role, endothelin signaling has also been broadly implicated in the pathophysiology of numerous diseases, including retinal diseases and glaucoma [25,26]. EDN ligands and receptors are known to be expressed by glaucoma-relevant cell types. EDN ligands have been shown to be expressed by retinal and optic nerve macroglia [6,27] and myeloid-derived cells [5,6], while both EDN receptors are expressed by retinal neurons (including RGCs) [12,14,25,28,29] and macroglia [26,[30][31][32][33]. Endothelin signaling has been hypothesized to play a role in human glaucoma. Levels of EDN ligand were found to be higher in the aqueous humor and plasma of glaucoma patients [34,35]. Changes in blood flow have been documented in human [36][37][38][39] and animal models [3,5] of glaucoma, and it is hypothesized that these changes could be important factors in the development and progression of glaucoma. Animal models of ocular hypertension have also indicated a potential role for endothelin signaling in glaucoma. Edn ligands and receptors were significantly upregulated in retinas and optic nerve heads of ocular hypertensive DBA/2J mice before the onset of glaucomatous neurodegeneration [3,5,6]. Similar patterns of endothelin system upregulation were found in models of acutely induced ocular hypertension [28] and after glaucoma-relevant optic nerve crush [40]. EDN ligands are sufficient to cause RGC death-intravitreal injection or transgenic overexpression of EDN ligands caused significant RGC loss and axonal degeneration [3,5,11,12,[41][42][43][44]. Caspase 3 activation in RGCs and later RGC loss after EDN1 exposure was dependent upon JUN activation, similar to RGC death after glaucoma-relevant injuries including optic nerve crush [45] and ocular hypertension [46]. Importantly, pan-antagonism of EDN receptors with Bosentan or Macitentan conferred significant protection from glaucomatous neurodegeneration in DBA/2J ocular hypertensive mice [5,6]. Thus, targeting endothelin signaling may have potential as a neuroprotective treatment for glaucoma.
Despite their apparent role in glaucoma pathology, it is unclear how EDN ligands act in the retina or optic nerve to ultimately drive RGC death. It is possible EDN ligands cause RGC death directly via RGC-expressed EDN receptors, as has been demonstrated in vitro [12]. But it is also possible EDN ligands bind to receptors expressed by astrocytes or vasculature, thereby triggering a neurotoxic response. Understanding the cell types important in EDN-induced RGC death will provide insight into early, critical pathways of glaucomatous neurodegeneration and can identify potential therapeutic targets for neuroprotective glaucoma treatments. The present work utilized cell-specific deletions of Ednra and/or Ednrb to investigate the mechanisms by which EDN ligands drive RGC death in vivo.
To determine whether neuronal and/or macroglial EDN receptors are required for EDN-induced RGC death, EDN1 was intravitreally injected into the eyes of WT (Six3-cre − Ednra +/+ Ednrb +/+ , Six3cre + Ednra fl/fl Ednrb +/+ , and Six3-cre + Ednra +/+ Ednrb fl/fl mice. PBS was injected into the contralateral eye as a vehicle-matched control. As expected,`Six3-cre-mediated deletion of Edn receptors did not interfere with EDN1-induced vasoconstriction (vascular smooth muscle-expressed EDNRA is known to induce vasoconstriction upon ligand binding [8][9][10][11]). Intravitreal EDN1 injection caused similar levels of vasoconstriction in WT, Six3-cre + Ednra fl/fl , Six3cre + Ednrb fl/fl , and Six3-cre + Ednra fl/fl Ednrb fl/fl retinas (Fig. 1A). Previous reports have shown EDN1 caused caspase 3 activation (cleavage; cCASP3) in RGCs 5 days post-intravitreal injection, which corresponded to RGC dropout by 28 days [44]. Genetic manipulations that prevented later RGC dropout also prevented early caspase 3 activation [44]-a pattern which is also observed after other glaucoma-relevant injuries [46,50]. Therefore, the presence of cCASP3+ RGCs was used to assess RGC injury after EDN1 injection. As reported previously, intravitreal injection of PBS did not drive appreciable caspase 3 activation in RGCs, and intravitreal injection of EDN1 ligand drove significant caspase 3 activation in RGCs (Fig.  1B). Surprisingly, deletion of either Ednra or Ednrb from retinal neurons (including RGCs) and macroglia did not prevent EDN1induced caspase 3 activation (cleavage) in RGCs (Fig. 1B). To address the possibility that both EDN receptors expressed by retinal neurons and/or macroglia are required to cause RGC death, EDN1 was injected into the eyes of Six3-cre + Ednra fl/fl Ednrb fl/fl mice. Deletion of both Edn receptors from macroglia and retinal neurons did not prevent caspase 3 activation in RGCs in response to EDN1 (Fig. 1B). These data suggest EDN1 ligands did not directly affect neurons (including RGCs) or macroglia to drive RGC death. Rather, EDN1 ligands acted through either EDNRA or EDNRB expressed by a different cell type. These results necessitated the identification of the EDN receptor, regardless of the cell type expressing it, which ultimately drives EDN1-induced RGC death.
Endothelin ligand acted through non-neuronal, nonmacroglial EDNRA to elicit RGC death Given EDN1 did not elicit RGC death via RGC-or macrogliaexpressed EDN receptors, EDN1 must directly affect a different cell type through either EDNRA or EDNRB. Beyond retinal neurons and macroglia, Ednra is known to be expressed by vascular mural cells [6,[12][13][14][15][16][17][18][19], and Ednrb is known to be expressed by endothelial cells [20][21][22]. It is also possible Ednra and/or Ednrb are expressed at low levels by another cell type (e.g., myeloid cells) and are able to pathologically respond to EDN ligand exposure. To determine whether EDN acts through EDNRB or EDNRA to cause RGC death, EDN1-induced RGC death was assessed in mice with global deletions of Ednra or Ednrb (Cag-creER T2+ Ednra fl/fl and Cag-creER T2+ Ednrb fl/fl mice were treated with tamoxifen to produce full-body knockouts).

DISCUSSION
Glaucoma is a multifactorial, heterogeneous neurodegenerative condition. Often In glaucoma, an increase in IOP leads to RGC injury and subsequent death. Several hypotheses have been postulated as to how ocular hypertension leads to RGC injury in glaucoma. Recent work has provided strong evidence for the role of endothelin signaling in causing RGC injury and subsequent death in models of chronic ocular hypertension [5,6]. EDN ligands were upregulated in human [34,54] and animal models [3,5,25,40] of glaucoma, and intravitreal injection of EDN ligand was sufficient to drive caspase 3 activation in RGCs, which corresponded with later RGC death [44]. Similar to models of glaucoma-relevant axonal injury [45] and ocular hypertension [46], deletion of Jun from RGCs prevented caspase 3 activation in RGCs and prevented later RGC loss after intravitreal EDN1 injection [44]. Pan-antagonism of EDN receptors significantly slowed RGC loss in the DBA/2J model of ocular hypertension [5,6], suggesting a causal role for the endothelin system in glaucoma pathogenesis. However, it was unknown which cell type EDN ligands directly affect in order to ultimately drive RGC death, and through which receptor (EDNRA or EDNRB) this occurs.
Previous in vitro studies have suggested EDN ligands can cause primary RGC death, suggesting EDN ligands are directly neurotoxic to RGCs (acting through RGC-expressed EDN receptors) [12]. Here, we demonstrate EDN1 ligands did not cause RGC death directly and did not cause RGC death by affecting other retinal neurons or macroglia in vivo (Fig. 1). Also, in contrast with previous studies suggesting EDN ligands act through EDNRB to drive RGC death in vitro, after EDN injection in vivo, and in a model of chronic ocular hypertension [12,25], EDNRB was not required for EDN1-induced RGC death. Rather, EDNRA was the receptor that was necessary for EDN1-induced RGC death (Fig. 3). Given the canonical role of EDNRA is to mediate vasoconstriction [8][9][10][11], we investigated the importance of mural cell-expressed EDNRA in EDN-induced RGC death. We demonstrated EDN1induced RGC death was driven by vascular mural cell (smooth muscle and pericyte)-expressed EDNRA (Fig. 5). These data do not preclude the possibility that vascular mural cells respond to EDN1 by eliciting neurotoxic paracrine or endocrine signaling. However, it is likely EDN1-induced RGC death is a result of EDNRA-mediated vasoconstriction and its sequalae.
Because Edn ligands were upregulated in DBA/2J glaucoma [3,5,7], pan-antagonism of EDN signaling lessened RGC loss after ocular hypertensive insults [5,6], and EDN1-induced RGC death was driven by vascular mural cell-expressed EDNRA (Fig. 5), it is possible that chronic vascular pathology or vasoconstriction is an important mediator of RGC death in glaucoma. Vascular involvement is consistent with several observations in human and animal models of glaucoma. Reduced ocular and retinal blood flow have been documented in human [36][37][38][39] and animal models [3,5] of glaucoma, and it is hypothesized that these changes could be important factors in the development and progression of glaucoma. Hypoxic glia and RGCs were present after acute [55,56] and chronic [57] ocular hypertension in rodents, suggesting the potential importance of hypoxia in driving glaucoma-relevant pathology.
If vascular EDNRA-induced vasoconstriction causes RGC death in response to EDN ligand, it will be important to investigate the pathological cellular events that lead from vasoconstriction to RGC death. While EDN1 injection was shown to cause regional RGC and glial hypoxia [44] (similar to glaucomatous ocular hypertension [55,57]), oxygen deprivation itself is unlikely to cause this RGC death. Oxygen deprivation severe enough to cause RGC death is also known to cause loss of amacrine neurons [58][59][60][61][62]. Previous work has demonstrated that, similar to ocular hypertension [63], EDN1 injection was not sufficient to drive the death of amacrine cells [44]. Therefore, if vasoconstriction is important in EDNinduced RGC death, it most likely drives secondary neurotoxic pathological events.
Chronic low-level hypoxia mediated by endothelin signaling may lead to compromise of the blood-brain barrier after EDN1 exposure and in glaucoma. In vitro, hypoxic conditions were sufficient to degrade endothelial cell tight junctions and cause barrier permeability [64][65][66]. Chronic mild hypoxia [67] and transgenic overexpression or injection of EDN ligand [43,68,69] led to loss of blood-brain barrier integrity and vascular leakage in vivo. Breakdown of the blood-brain barrier and subsequent infiltration of peripheral immune cells has been suggested to drive neurodegeneration in glaucoma-prevention of immune cell infiltration with radiation therapy protected from glaucoma in DBA/2J mice [3]. Similarly, deletion of Cd11b (also known as Itgam -a cell adhesion protein critical for tissue infiltration of monocytes) was shown to lessen monocyte infiltration into the optic nerve head and protect from glaucomatous neurodegeneration in DBA/2J mice [70]. Consistent with these results, deletion of Glycam (a proteoglycan ligand for L-selectin known to prevent transendothelial migration of leukocytes) promoted monocyte infiltration into the optic nerve head and weakened the protection afforded to DBA/2J retinas by radiation treatment [4]. Given the importance of vascular compromise in glaucoma, it is possible that EDNRA-induced vasoconstriction and its sequelae damages the blood-brain barrier and plays a role in neurodegeneration in response to EDN1 ligand and in glaucoma.
It is also possible that EDN-induced regional mild hypoxia can affect immune cells in the retina or optic nerve. Astrocytes took on a reactive phenotype in response to hypoxia in vitro [71,72] and in vivo [73,74], which could potentially lead to a neurotoxic gliotic response. Furthermore, astrocytes aid in maintaining blood-brain barrier integrity. Chronic hypoxic conditions led to a loss of astrocyte-endothelial cell contacts [65]. Astrocytes are also known to upregulate and secrete VEGF upon hypoxic insult [75]. Astrocyte-specific VEGF was critical for pathological neovascularization after retinal hypoxic injury in vivo [76]. VEGF was required for hypoxia-induced blood-brain barrier breakdown [66], and astrocyte-specific VEGF was shown to cause blood-brain barrier breakdown in vitro [77]. Together, these data suggest hypoxia can cause changes in retinal astrocytes, which can in turn drive neurotoxic signaling and/or contribute to breakdown of the blood-brain barrier. The mechanisms by which EDN-EDNRA signaling drive RGC death must be elucidated, and the importance of these events in driving glaucomatous neurodegeneration upon chronic ocular hypertension merits future investigation.

MATERIALS AND METHODS Mice
All mice used were 1.5-6 months of age. Mice were fed chow and water ad libitum and housed on a 12-hour light-to-dark cycle. All experiments were conducted in adherence to the Association for Research in Vision and Ophthalmology's statement on the use of animals in ophthalmic and vision research and were approved by the University of Rochester's University Committee on Animal Resources. C57BL/6N-At m1Brd Ednra tm1a(EUCOMM)Hmgu / JMmucd knockout-first mice with promoter-driven alleles were obtained through UC Davis KOMP Repository. These mice were crossed with flippase recombinase transgenic mice (Flp tg , URMC genomics research core) to generate offspring with Ednra fl alleles. Ednrb tm1.1Nat /J alleles were obtained from the Jackson Laboratory (Ednrb fl ; Stock #011080 [47]). Mice with Ednra fl and Ednrb fl alleles were bred to Tg(Six3-cre)69Frty/GcoJ transgenic mice (Six3-cre + ; Jackson Laboratory, Stock# 019755) [48] to generate mice with a conditional deletion of Ednra and/or Ednrb from retinal neurons and macroglia. Mice with Ednra fl or Ednrb fl alleles were also bred to Tg(CAG-cre/ Esr1*)5Amc/J transgenic mice (Cag-creER T2+ ; JAX Stock #004682) [78] to produce offspring with full-body deletions of Ednra or Ednrb upon tamoxifen treatment. Mice with Ednra fl alleles were bred to Tg(Myh11-cre/ ERT2)1Soff/J transgenic mice (Myh11-creER T2+ ; Jackson Laboratory, Stock# 019079) [79] to generate mice with a conditional deletion of Ednra from vascular mural cells upon tamoxifen treatment. Mice transgenic for Cag-creER T2 or Myh11-creER T2 recombinase were bred to Gt(ROSA)26Sor tm75.1 (CAG-tdTomato*)Hze /J conditional reporter mice (TdTomato + ; JAX stock 025106) to generate offspring as TdTomato reporters of cre expression.

Statistical analysis and experimental rigor
Power calculations were performed before experiments were conducted to determine the appropriate sample size. Data were analyzed using GraphPad Prism9 software. Data from experiments designed to test differences between two groups were subjected to an F test to compare variance and a Shapiro-Wilk test to test normality to ensure appropriate statistical tests were utilized. For non-normally distributed data designed to test differences between two groups, a Mann-Whitney test was utilized. Data from experiments designed to test differences among more than two groups across one condition were subjected to a Brown-Forsythe test to compare variance and a Shapiro-Wilk test to test normality to ensure an appropriate statistical test was utilized. Data from experiments designed to detect differences among multiple groups and across one condition were analyzed using a Kruskal-Wallis test with Dunn's post hoc test. Data from experiments designed to detect differences among multiple groups and across two conditions were analyzed using a two-way analysis of variance followed by Holm-Sidak's post hoc test. For these statistical tests, every possible comparison was made when relevant, and multiplicity adjusted P values are reported. In all cases, data met the assumptions of the statistical test used. P values < 0.05 were considered statistically significant. Throughout the manuscript, results are reported as mean ± standard error of the mean (SEM).
Roughly equal numbers of male and female mice were used for each experimental group, except for Myh11-creER T2 Ednra fl mice (the Myh11-creER T2 transgene is inserted into the Y chromosome, thus, all animals used for this line of experiments were male). Phenotypically wild-type (WT) controls included tamoxifen treated and untreated cre + and cre − animals. Littermate controls were used wherever possible. Animals were randomly assigned to experimental groups. Before experiments were performed, it was established that animals with pre-existing abnormal eye phenotypes (e.g., displaced pupil, cataracts) would be excluded from the study. All procedures were conducted by an observer masked to genotype and condition.
Tamoxifen treatment and animal procedures At 6 weeks of age or older, animals were intraperitoneally injected with 125 mg/kg tamoxifen (Sigma, T5648) dissolved in corn oil at a concentration of 20 mg/mL once per day for five consecutive days. Experiments were conducted no earlier than 7 days after the last tamoxifen dose to allow for recombination of floxed alleles and degeneration of endogenous protein.
Intravitreal injections and fluorescein angiography were performed as previously described [44]. EDN1 (Sigma, E7764) was dissolved in sterile PBS at a concentration of 500 μM. As previously performed, 2 μL of 500 μM EDN1 dissolved in sterile PBS was intravitreally injected into one eye. Sterile PBS was injected into the contralateral eye as a volume-matched vehicle control.

DATA AVAILABILITY
The datasets used in the current study are available from the corresponding author on reasonable request.