James B. Hurley

Vascular endothelial growth factor-A (VEGF) is a critical determinant of retinopathy due to vascular complications. Its expression is chronically regulated by transcription factors notably hypoxia-inducible factor-1α (HIF-1α) in response to decreased availability of oxygen [O ₂ ]. We considered whether there is a distinct rapid response component of VEGF secretion in response to low O ₂ , faster than could be achieved by HIF-1α. To investigate this possibility, we measured real time responses of VEGF, lactate, and HIF-1α by retinas and other tissues to rapid and precise decreases in dissolved O _2. and showed that retinas, retinal pigment epithelium, and endothelial cells all can secrete VEGF within 5 min after a decrease in [O ₂ ]. HIF-1α levels in retinas did not increase until 20 to 30 min after a decrease in dissolved [O ₂ ]. Moreover, increased HIF-1α induced by the drug Roxadustat, did not stimulate VEGF secretion at atmospheric (21%) O ₂ and a steady rate of VEGF secretion was sustained for over 120 min following elimination of HIF-1α and protein synthesis by exposure to the protein synthesis inhibitor cycloheximide. We then investigated the mechanism of VEGF release and found that VEGF was stored in the plasma membranes of retinas at 21% O ₂ . These stores are depleted within 30 min once [O ₂ ] is decreased. Taken together, these findings indicate that VEGF is constitutively made (under the influence of HIF-1α and other transcription factors) and stored within the retina, and rapid release occurs after a substantial decrease in available O ₂ independent of HIF-1α.

Photoreceptors are among the most metabolically active cells in the retina and are therefore highly sensitive to fluctuations in oxygen availability. Age-related tissue changes in the eye affect oxygen delivery to the outer retina, which may result in hypoxic stress within photoreceptors and can contribute to disease development and retinal degeneration. To investigate how chronic hypoxic signalling affects photoreceptor metabolism, we examined a rod-specific Vhl knockout mouse (Rod ^{Δ Vhl} ), in which constitutive HIF activation mimics the molecular response to hypoxia. Combining a cell-type–enriched multi-omics approach with metabolic flux analysis, we identified an early metabolic response in the retina of Rod ^{Δ Vhl} mice prior to degeneration. This response was characterized by a shift towards an oxidative redox environment indicated by a decrease in nucleotide precursors and an increased antioxidant response. While steady-state glycolytic flux remained unchanged, the dynamic ¹³ C-glucose tracing revealed accelerated carbon flow through the three-carbon glycolytic intermediates, indicating a carbon rerouting. Outer segment lipidomics revealed selective remodelling of phosphatidylcholine and phosphatidylethanolamine species toward more oxidation-resistant and elongated acyl chains, supported by early gene upregulation of essential enzymes involved in fatty acid elongation, desaturation and oxidation. Together, these findings indicate a coordinated shift in metabolic and lipid pathways in photoreceptors under chronic hypoxic stress, consistent with an adaptive response that may help preserve outer segment integrity and improve stress resilience.

Induced pluripotent stem cell-derived retinal pigment epithelium (iPSC RPE) has become a widely used model for studying the mechanisms of age-related macular degeneration (AMD). However, the nutrient composition of currently used RPE culture media is highly variable, posing a major challenge to reproducibility in RPE metabolism and phenotype. We systematically investigate how six distinct nutrient microenvironments shape RPE phenotype, function and metabolism in both iPSC RPE and fetal RPE (fRPE). These included MEMα, DMEM-HG/F12 basal media, physiological human plasma-like medium (HPLM) supplemented with FBS or B27, and X-VIVO 10. Although canonical RPE markers were expressed across all conditions, B27 supplementation and X-VIVO 10 increased RPE cell size, hexagonality, and transepithelial resistance. Culture in HPLM+FBS induced accumulation of lipid droplets and sub-RPE deposits, whereas X-VIVO 10 resulted in the formation of large intracellular vacuoles. B27 supplementation enhanced basal respiration, while X-VIVO 10 increased glycolytic capacity. Amino acid consumption was broadly conserved across media types, including complete depletion of proline in all conditions by 48 hours; however, lipid and nucleotide metabolism varied substantially between conditions. Notably, B27 supplementation in specific media types reversed the net direction of several metabolites, with creatine, serine and taurine shifting from consumption to production, while riboflavin and guanine shifted from production to consumption. These findings establish the nutrient environment as a key determinant of RPE phenotype, function and metabolism. Our work provides a valuable resource for media selection and interpretation of cellular and metabolic phenotypes relevant to RPE disease modeling.

Photoreceptors in the retina of a vertebrate’s eye are supported by a tissue adjacent to the retina, the retinal pigment epithelium (RPE). The RPE delivers glucose to the outer retina, consumes photoreceptor outer segments discs, and regenerates 11-cis-retinal. Here we address the question of whether photoreceptors also provide metabolic support to the RPE. We use complementary approaches and animal models to show that glucose is the primary fuel for the retina, that photoreceptors are the primary cell type in the retina to consume glucose, and that lactate derived from photoreceptor glucose consumption is transported to and catabolized by the RPE. These data rigorously support and extend the concept of a metabolic ecosystem between photoreceptors and RPE.

Neuroretinal degenerations including retinitis pigmentosa (RP) comprise a heterogeneous collection of pathogenic mutations that ultimately result in blindness. Despite recent advances in precision medicine, therapies for rarer mutations are hindered by burdensome developmental costs. To this end, Von Hippel-Lindau (VHL) is an attractive therapeutic target to treat RP. By ablating VHL in rod photoreceptors and elevating hypoxia-inducible factor (HIF) levels, we demonstrate a path to therapeutically enhancing glycolysis independent of the underlying genetic variant that slows degeneration of both rod and cone photoreceptors in a preclinical model of retinitis pigmentosa. This rod-specific intervention also resulted in reciprocal, decreased glycolytic activity within the retinal pigment epithelium (RPE) cells despite no direct genetic modifications to the RPE. Suppressing glycolysis in the RPE provided notable, noncell-autonomous therapeutic benefits to the photoreceptors, indicative of metabolically sensitive crosstalk between different cellular compartments of the retina. Surprisingly, targeting HIF2A in RPE cells did not impact RPE glycolysis, potentially implicating HIF1A as a major regulator in mouse RPE and providing a rationale for future therapeutic efforts aimed at modulating RPE metabolism.

THT is an additive for culture medium used to maintain retinal pigment epithelial (RPE) cells. The individual components of THT are taurine, hydrocortisone, and triiodothyronine. The effects of THT on RPE health had not been defined. To determine how THT affects RPE cell physiology, we grew induced pluripotent stem cell (iPSC)-derived RPE cells with a normal concentration of THT (“high” THT) or in a medium with a lower concentration of THT (low THT). We determined the impact of THT on the extracellular flux of glucose, lactate, palmitate, and VEGF. We assessed RPE function by measuring trans-epithelial electrical resistance (TEER). RPE cells grown in low THT are more glycolytic, oxidize fatty acids more slowly, have a higher TEER, and release more VEGF basally than RPE cells grown in high THT. Our findings show that THT impacts metabolism and function. Further studies will be needed to fully define the impact of each THT component on RPE metabolism, function, and health.

Purpose: In age-related macular degeneration (AMD) and Sorsby’s fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating β-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate β-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. Methods: We probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used ¹³ C6-glucose and ¹³ C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. ¹³ C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). Results: ACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsby’s fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. Conclusions: Firsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

Photoreceptors in the retina of a vertebrate’s eye are supported by a tissue adjacent to the retina, the retinal pigment epithelium (RPE). The RPE delivers glucose to the outer retina, consumes photoreceptor outer segments discs, and regenerates 11-cis-retinal. Here we address the question of whether photoreceptors also provide metabolic support to the RPE. We use complementary approaches and animal models to show that glucose is the primary fuel for the retina, that photoreceptors are the primary cell type in the retina to consume glucose, and that lactate derived from photoreceptor glucose consumption is transported to and catabolized by the RPE. These data rigorously support and extend the concept of a metabolic ecosystem between photoreceptors and RPE.