Carbon Dioxide Inhibits Retinal Angiogenesis via NO–mediated Downregulation of the Prostaglandin Receptor EP3

Abstract

Abstract: : Purpose: Epidemiologic studies and animal models have revealed that hypercapnia impairs normal vascular development, yet little is known about its mechanisms. Given that any instance of ischemia is by physiological consequence accompanied by a local tissue hypercapnia, such mechanisms would provide insight into several ischemic retinopathies (e.g. retinopathy of prematurity, diabetic retinopathy, etc.), as well as into ischemia–related conditions in general. Recently, hypercapnia has been shown to elicit increased expression and activation of endothelial NO synthase (eNOS). Therefore, we further investigated how CO₂ and NO may contribute to retinal vascular impairment. Methods: Rat pups were exposed from birth until day 6 to 10% CO₂. Following exposure, the vessels were stained and the degree of vascularization quantified. Western blot analyses for NOSs, the angiogenic prostaglandin receptor, EP₃, and an EP₃ transcription factor, NFkappaB, were performed, as was immunohistochemistry on retinal sections for NOS localization. Intraocular injections of either the non–selective NOS inhibitor, L–NAME, or the selective EP₃ agonist, M&B28767, were used to examine their roles in hypercapnia–induced vasoattenuation. Lastly, retinas were cultured as ex vivo explants under control or hypercapnic conditions (5% and 10% CO₂, respectively) to determine the effects of hypercapnia independent of hemodynamics. Results: Hypercapnia led to increases in eNOS with L–NAME acting to protect the retina from the hypercapnia–induced arrest in vessel development. Hypercapnia resulted in a decrease of nuclear NFkappaB, and a downregulation of EP₃ which was inhibited by L–NAME. Stimulation of EP₃ diminished the deleterious effects of hypercapnia. Furthermore, ex vivo explants illustrated that hypercapnia can also have detrimental effects on EC survival completely independent of its effects on hemodynamics. Conclusions: This work provides novel insight into the genesis of ischemic retinopathies by describing a novel pathway of CO₂/NO–mediated inhibition of retinal angiogenesis via NFkappaB–related downregulation of the angiogenic prostaglandin receptor, EP₃. Moreover, these results unveil the adverse effects of hypercapnia on EC survival completely independent of hemodynamics.

Keywords: ischemia • eicosanoids • nitric oxide