The ghrelin receptor GHSR-1a is robustly expressed in the retina. (a) Retinal cross-sections stained with anti-GHSR-1a revealed the total distribution of the receptor in the rat retina. Scale bar, 30 μm. (b–d) Colocalization revealed GHSR-1a expression in both endothelial cells (lectin; b) and Müller cells (CRALBP; c) but not in astrocytes (GFAP; d). Scale bar, 30 μm. (e) Representative image of Western blot analysis indicating GHSR-1a receptor expression in various retinal cell types, confirming the predominant vascular expression of this receptor. (f) Densitometry quantification of relative GHSR-1a expression in various neural cell types (n = 3).

Ghrelin protects retinal endothelial cells against hyperoxia-induced vaso-obliteration through GHSR-1a. (a–c) Representative images of retinal whole mounts stained with G. simplicifolica lectin from P10 animals after intravitreal injection with vehicle (saline; a), [Dap3]-Ghrelin (81 nM; b), or GHRP-6 (24 nM; c) during hyperoxic exposure. Red tracings: borders of vaso-obliteration zone. Scale bar, 1 mm. (d) Quantification of vaso-obliteration area after intravitreal administration of GHSR-1a agonist confirm the vasoprotective effects of the agonist (mean ± SD; ***P < 0.001 compared with vehicle control; n = 6). (e–l) Representative high-magnification images of G. simplicifolica lectin–stained retinal whole mounts from P10 animals after intravitreal injection with vehicle (f), [Dap3]-Ghrelin (81 nM; g) JMV-2959 (56 nM; h), or both (81 nM [Dap3]-Ghrelin and 560 nM JMV-2959; (i), as well as GHRP-6 (24 nM; j), [D-Lys3]-GHRP-6 (24 nM; k), or both (24 nM GHRP-6 and 240 nM [D-Lys3]-GHRP-6; l), after hyperoxic exposure. Normoxic vehicle controls (e). Scale bar, 200 μm. Vessels were revealed by red fluorescence detected by fluorescence microscopy. (m) Quantification of vasculature density in hyperoxia-exposed (vaso-obliterated) retina confirm the protection of the ghrelin-GHSR1a pathway (mean ± SD; **P < 0.01, ***P < 0.0001 compared with hyperoxia vehicle control; n = 5–12). (n) Western blot of retinal protein extract demonstrated the induction of both IGF-1 and VEGF after intravitreal administration of the stable Ghrelin analog ([Dap3]-Ghrelin; 81 nM) or GHSR-1a agonist GHRP-6 (24 nM). The receptor antagonists (JMV-2959; 560 nM) and ([Lys3]-GHRP-6; 240nM) blocked this induction. Densitometry quantification of IGF-1 (o) and VEGF (p) expression levels after the administration of GHSR-1a agonists and antagonists (mean ± SEM; *P < 0.05, **P < 0.01 compared with vehicle-injected control; compilation of n = 5–12 animals).

Ghrelin levels rise in the neovascular phase of OIR. (a) Plasma and (b) retinal ghrelin expression level in P17 hyperoxia-exposed animals compared with normoxia control reveal a robust induction during the neovascular phase of OIR (mean ± SD; *P < 0.05, ***P < 0.0001; n = 3–8).

Ghrelin is generated directly in the retina and is modulated in OIR. (a) Retinal expression of ghrelin (mRNA) from P7 rats exposed to high oxygen for 48 hours (P5–P7) and age-matched animals raised in room air (mean ± SEM; **P < 0.01 compared with normoxia control; n = 4). (b) Expression of ghrelin mRNA in P18 OIR rats (50%/10% alternating oxygen; P0–P14) and age matched in room air raised animals (mean ± SEM; *P < 0.05 compared with normoxia control; n = 4). (c–e) Colocalization revealed Ghrelin expression in Müller cells (specifically in Muller cell end-feet) (CRALBP; c) but not in endothelial cells (lectin; d) or astrocytes (GFAP; e). Scale bar, 30 μm.

Ghrelin promotes retinal endothelial cell growth through GHSR-1a. (a) Quantification of PBMEC proliferation by thymidine incorporation after treatment with [Dap3]-Ghrelin (10⁻⁸M), JMV-2959 (10⁻⁸M), or both (10⁻⁸M [Dap3]-Ghrelin and 10^[minus7M JMV-2959; mean ± SD; ***P < 0.001 compared with vehicle control; n = 3). (b–f) Representative microphotograph (b–e) and quantification (f) of aortic explants sprouting treated with [Dap3]-Ghrelin (10⁻⁸ M), JMV-2959 (10⁻⁸ M), or both (10⁻⁸ M of [Dap3]-Ghrelin and 10⁻⁷ M JMV-2959; mean ± SD; ***P < 0.001 compared with vehicle control; n = 6). Scale bar, 1 mm. (g–n) Representative G. simplicifolica lectin–stained retinal whole mounts indicating the proangiogenic effects of ghrelin-GHSR-1a during retinal development (g–m) and quantification (n) of P6 retinal vascular density after intravitreal administration at P4 of [Dap3]-Ghrelin (81 nM; h) JMV-2959 (56 nM; i) or both (81 nM [Dap3]-Ghrelin and 560 nM JMV-2959; (j), as well as GHRP-6 (24 nM; k), [D-Lys3]-GHRP-6 (24 nM; l), or both (24 nM GHRP and 240 nM [D-Lys3]-GHRP-6; m) (mean ± SD; ***P < 0.001 compared with vehicle control; n = 6–20). Vessels were revealed by red fluorescence detected by fluorescence microscopy. Scale bar, 200 μm.

Pharmacologic inhibition of GHSR-1a prevents pathologic retinal neovascularization and improves retinopathy. (a–h) Representative images of G. simplicifolica lectin–stained retinal whole mounts from vehicle-injected normoxic controls (a, b) and vehicle-injected (saline; c, d), JMV-2959 (56 nM; e, f), and [D-Lys3] GHRP-6–injected (24 nM; g, h) rats during the neovascular phase (P18) of OIR. Top: 500 μm. Bottom: 1 mm. Vessels were revealed as red fluorescence detected by fluorescence microscopy. (i–k) Calculated total vessel buds (i), vessel tortuosity area (j), and retinopathy score (k) of OIR retinal vasculatures after intravitreal injection of vehicle (saline), JMV-2959 (56 nM), or [D-Lys3] GHRP-6 (24 nM; mean ± SD; *P < 0.05 compared with vehicle control; n = 6–16). (l) Summary table indicating the subscores of each parameter investigated.