Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

Andrew P Voigt, Kelly Mulfaul, Nathaniel K Mullin, Miles J Flamme-Wiese, Joseph C Giacalone, Edwin M Stone, Budd A Tucker, Todd E Scheetz, Robert F Mullins, Andrew P Voigt, Kelly Mulfaul, Nathaniel K Mullin, Miles J Flamme-Wiese, Joseph C Giacalone, Edwin M Stone, Budd A Tucker, Todd E Scheetz, Robert F Mullins

Abstract

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Keywords: age-related macular degeneration; choriocapillaris; choroid; single cell.

Conflict of interest statement

The authors declare no competing interest.

Copyright © 2019 the Author(s). Published by PNAS.

Figures

Fig. 1.
Fig. 1.
Identification of RPE and choroidal cell populations. (A) UMAP dimensionality reduction was performed to visualize the 11 identified RPE and choroidal clusters. A total of 4,335 cells were recovered after filtering. (B) An overview schematic of the cell populations found within the choroid. SC, Schwann cell; SMC, smooth muscle cell. (C) For each cluster, the average RNA expression of each gene was calculated. The top 100 most enriched transcripts in each cluster were identified and compiled, and the average expression of these transcripts was used as input for hierarchical clustering analysis. Violin plots of genes previously reported to be enriched in RPE and choroidal cell populations are plotted alongside the dendrogram.
Fig. 2.
Fig. 2.
Differential expression of macular versus peripheral pericytes and RPE cells. Differential expression was performed between smooth muscle cells/pericytes (A) and RPE cells (B) of macular and peripheral origin. For each gene, the average log fold change and the percentage of cells that express the gene above background are compared between the 2 clusters (Right). For example, a delta percent of +0.25 indicates that 25% more cells of macular pericytes express the gene above background than peripheral pericytes. Expression patterns from the most enriched genes were compared to bulk RNA expression from pooled RPE/choroid cell lysates (19), which largely corroborates the regional expression differences observed in cell populations identified by single-cell RNA sequencing.
Fig. 3.
Fig. 3.
Retinal versus choroidal endothelial cell gene expression. (A) Two of the donors in this investigation (donors 1 and 2) were included in a single-cell RNA-sequencing study of human retina (donors 2 and 3 of that report). The RPE/choroid and retinal data were aggregated for these donors. Two clusters of endothelial cells (clusters 5 and 6) consisting of 413 total cells were identified. (B) The clusters were largely distinct based on tissue of origin, with retinal endothelial cells colored in blue and choroidal endothelial cells colored in red. (C and D) Differential expression between retinal and choroidal endothelial cells was performed. For each gene, the average log fold change and the percentage of cells that express the gene above background are compared between the 2 clusters (Right). For example, a delta percent of +0.25 indicates that 25% more cells of choroidal endothelial cells express the gene above background than retinal endothelial cells.
Fig. 4.
Fig. 4.
Identification of choroidal vascular cell populations. (A) A second single-cell RNA-sequencing study was performed on 4 additional human donors. Endothelial cells were enriched prior to sequencing. A total of 14,234 cells were recovered after filtering, of which 8,521 (clusters 5 to 8) were classified as endothelial cells. (B) Choroidal vasculature consists of large caliber arteries and veins and an intermediary superficial capillary system known as the choriocapillaris. (C) The vast majority of cells in clusters 5 to 8 express the endothelial-specific transcript VWF. (D–F) Differential expression was performed to identify genes enriched in arterial (D), choriocapillaris (E), and vein (F) clusters. For each gene, the average log fold change and the percentage of cells that express the gene above background are compared between the 2 clusters (Right). For example, a delta percent of +0.25 indicates that 25% more cells of arterial endothelial cells express the gene above background than vein and choriocapillaris endothelial cells. (G) Hierarchical clustering of the top 100 most highly expressed genes in each cluster reveals that the choriocapillaris cluster has a more similar expression pattern to veins opposed to arteries. Expression of known endothelial (VWF), vein (DARC, MMRN1), choriocapillaris (CA4, PLVAP), and arterial (HEY1, SEMA3G) genes are compared across each endothelial cluster. (H) Expression of RGCC, 1 of the top 10 most enriched genes in the choriocapillaris cluster, was assessed by qPCR in an immortalized human choroidal endothelial cell line after treatment with 10% normal human serum (NHS, black) and 10% heat-inactivated serum (Hi NHS, gray) in 3 experiments (N1, N2, and N3). RGCC expression is increased in response to NHS compared to Hi NHS. **P < 0.01 and ***P < 0.001. (I) Immunohistochemical localization of RGCC. A section through the macula of an 87-y-old donor with type I neovascular AMD was colabeled with RGCC, the endothelial-specific fucose binding lectin Ulex europaeus agglutinin-I (UEA-I), and DAPI. RGCC localizes to an endothelial cell (yellow arrow) within a choroidal neovascular membrane (CNVM). BrM, Bruch’s membrane. Red fluorescence in the RPE is due to lipofuscin (an autofluorescent pigment); RGCC expression was not detected in the RPE cluster. (Scale bar: 25 µm.)

References

    1. Redmond T. M., et al. , Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat. Genet. 20, 344–351 (1998).
    1. Young R. W., Bok D., Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. Cell Biol. 42, 392–403 (1969).
    1. Schmidt S. Y., Peisch R. D., Melanin concentration in normal human retinal pigment epithelium. Regional variation and age-related reduction. Invest. Ophthalmol. Vis. Sci. 27, 1063–1067 (1986).
    1. Cai J., Nelson K. C., Wu M., Sternberg P. Jr, Jones D. P., Oxidative damage and protection of the RPE. Prog. Retin. Eye Res. 19, 205–221 (2000).
    1. Ban Y., Rizzolo L. J., Regulation of glucose transporters during development of the retinal pigment epithelium. Brain Res. Dev. Brain Res. 121, 89–95 (2000).
    1. Nickla D. L., Wallman J., The multifunctional choroid. Prog. Retin. Eye Res. 29, 144–168 (2010).
    1. Nork T. M., et al. , Measurement of regional choroidal blood flow in rabbits and monkeys using fluorescent microspheres. Arch. Ophthalmol. 124, 860–868 (2006).
    1. Hasegawa T., et al. , The embryonic human choriocapillaris develops by hemo-vasculogenesis. Dev. Dyn. 236, 2089–2100 (2007).
    1. Abi-Hanna D., Wakefield D., Watkins S., HLA antigens in ocular tissues. I. In vivo expression in human eyes. Transplantation 45, 610–613 (1988).
    1. Mullins R. F., Skeie J. M., Malone E. A., Kuehn M. H., Macular and peripheral distribution of ICAM-1 in the human choriocapillaris and retina. Mol. Vis. 12, 224–235 (2006).
    1. Hageman G. S., Zhu X. L., Waheed A., Sly W. S., Localization of carbonic anhydrase IV in a specific capillary bed of the human eye. Proc. Natl. Acad. Sci. U.S.A. 88, 2716–2720 (1991).
    1. Pennington K. L., DeAngelis M. M., Epidemiology of age-related macular degeneration (AMD): Associations with cardiovascular disease phenotypes and lipid factors. Eye Vis. (Lond.) 3, 34 (2016).
    1. Nowak J. Z., Age-related macular degeneration (AMD): Pathogenesis and therapy. Pharmacol. Rep. 58, 353–363 (2006).
    1. Beatty S., Koh H., Phil M., Henson D., Boulton M., The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv. Ophthalmol. 45, 115–134 (2000).
    1. Chirco K. R., Sohn E. H., Stone E. M., Tucker B. A., Mullins R. F., Structural and molecular changes in the aging choroid: Implications for age-related macular degeneration. Eye (Lond.) 31, 10–25 (2017).
    1. Mullins R. F., Johnson M. N., Faidley E. A., Skeie J. M., Huang J., Choriocapillaris vascular dropout related to density of drusen in human eyes with early age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 52, 1606–1612 (2011).
    1. Biesemeier A., Taubitz T., Julien S., Yoeruek E., Schraermeyer U., Choriocapillaris breakdown precedes retinal degeneration in age-related macular degeneration. Neurobiol. Aging 35, 2562–2573 (2014).
    1. Mullins R. F., et al. , The membrane attack complex in aging human choriocapillaris: Relationship to macular degeneration and choroidal thinning. Am. J. Pathol. 184, 3142–3153 (2014).
    1. Whitmore S. S., et al. , Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-seq. Exp. Eye Res. 129, 93–106 (2014).
    1. Tian L., et al. , Transcriptome of the human retina, retinal pigmented epithelium and choroid. Genomics 105, 253–264 (2015).
    1. Chen H., Liu B., Lukas T. J., Neufeld A. H., The aged retinal pigment epithelium/choroid: A potential substratum for the pathogenesis of age-related macular degeneration. PLoS One 3, e2339 (2008).
    1. Newman A. M., et al. , Systems-level analysis of age-related macular degeneration reveals global biomarkers and phenotype-specific functional networks. Genome Med. 4, 16 (2012).
    1. Whitmore S. S., et al. , Altered gene expression in dry age-related macular degeneration suggests early loss of choroidal endothelial cells. Mol. Vis. 19, 2274–2297 (2013).
    1. Stanton C. M., et al. , Complement factor D in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 52, 8828–8834 (2011).
    1. Yuan K., et al. , Increased pyruvate dehydrogenase kinase 4 expression in lung pericytes is associated with reduced endothelial-pericyte interactions and small vessel loss in pulmonary arterial hypertension. Am. J. Pathol. 186, 2500–2514 (2016).
    1. Casey C. S., et al. , Apolipoprotein E inhibits cerebrovascular pericyte mobility through a RhoA protein-mediated pathway. J. Biol. Chem. 290, 14208–14217 (2015).
    1. Lu J., Chatterjee M., Schmid H., Beck S., Gawaz M., CXCL14 as an emerging immune and inflammatory modulator. J. Inflamm. (Lond.) 13, 1 (2016).
    1. Hayashi Y., et al. , CXCL14 and MCP1 are potent trophic factors associated with cell migration and angiogenesis leading to higher regenerative potential of dental pulp side population cells. Stem Cell Res. Ther. 6, 111 (2015).
    1. van Soest S. S., et al. , Comparison of human retinal pigment epithelium gene expression in macula and periphery highlights potential topographic differences in Bruch’s membrane. Mol. Vis. 13, 1608–1617 (2007).
    1. Shibuya M., et al. , Proteomic and transcriptomic analyses of retinal pigment epithelial cells exposed to REF-1/TFPI-2. Invest. Ophthalmol. Vis. Sci. 48, 516–521 (2007).
    1. Nguyen D. V., et al. , An ocular view of the IGF-IGFBP system. Growth Horm. IGF Res. 23, 45–52 (2013).
    1. Voigt A. P., et al. , Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing. Exp. Eye Res. 184, 234–242 (2019).
    1. Zheng P. P., et al. , Glut1/SLC2A1 is crucial for the development of the blood-brain barrier in vivo. Ann. Neurol. 68, 835–844 (2010).
    1. Hochgrebe T. T., Humphreys D., Wilson M. R., Easterbrook-Smith S. B., A reexamination of the role of clusterin as a complement regulator. Exp. Cell Res. 249, 13–21 (1999).
    1. Thiriot A., et al. , Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues. BMC Biol. 15, 45 (2017).
    1. Sabbagh M. F., et al. , Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells. eLife 7, e36187 (2018).
    1. Herrnberger L., Ebner K., Junglas B., Tamm E. R., The role of plasmalemma vesicle-associated protein (PLVAP) in endothelial cells of Schlemm’s canal and ocular capillaries. Exp. Eye Res. 105, 27–33 (2012).
    1. Fang J. S., et al. , Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification. Nat. Commun. 8, 2149 (2017).
    1. Buschmann I., et al. , Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis. Development 137, 2187–2196 (2010).
    1. Wilkerson B. A., Argraves K. M., The role of sphingosine-1-phosphate in endothelial barrier function. Biochim. Biophys. Acta 1841, 1403–1412 (2014).
    1. Goldblum S. E., Ding X., Funk S. E., Sage E. H., SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function. Proc. Natl. Acad. Sci. U.S.A. 91, 3448–3452 (1994).
    1. Aran A., et al. , Loss of function of PCDH12 underlies recessive microcephaly mimicking intrauterine infection. Neurology 86, 2016–2024 (2016).
    1. An X., et al. , Response gene to complement 32, a novel hypoxia-regulated angiogenic inhibitor. Circulation 120, 617–627 (2009).
    1. Hwang B., Lee J. H., Bang D., Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp. Mol. Med. 50, 96 (2018).
    1. Macosko E. Z., et al. , Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).
    1. Peng Y. R., et al. , Molecular classification and comparative taxonomics of foveal and peripheral cells in primate retina. Cell 176, 1222–1237.e22 (2019).
    1. Rheaume B. A., et al. , Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes. Nat. Commun. 9, 2759 (2018).
    1. Shekhar K., et al. , Comprehensive classification of retinal bipolar neurons by single-cell transcriptomics. Cell 166, 1308–1323.e30 (2016).
    1. Hu Y., et al. , Dissecting the transcriptome landscape of the human fetal neural retina and retinal pigment epithelium by single-cell RNA-seq analysis. PLoS Biol. 17, e3000365 (2019).
    1. Reiner A., Fitzgerald M. E. C., Del Mar N., Li C., Neural control of choroidal blood flow. Prog. Retin. Eye Res. 64, 96–130 (2018).
    1. Flügel C., Tamm E. R., Mayer B., Lütjen-Drecoll E., Species differences in choroidal vasodilative innervation: Evidence for specific intrinsic nitrergic and VIP-positive neurons in the human eye. Invest. Ophthalmol. Vis. Sci. 35, 592–599 (1994).
    1. Mullins R. F., Kuehn M. H., Faidley E. A., Syed N. A., Stone E. M., Differential macular and peripheral expression of bestrophin in human eyes and its implication for best disease. Invest. Ophthalmol. Vis. Sci. 48, 3372–3380 (2007).
    1. McLeod D. S., Lefer D. J., Merges C., Lutty G. A., Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid. Am. J. Pathol. 147, 642–653 (1995).
    1. Skeie J. M., Fingert J. H., Russell S. R., Stone E. M., Mullins R. F., Complement component C5a activates ICAM-1 expression on human choroidal endothelial cells. Invest. Ophthalmol. Vis. Sci. 51, 5336–5342 (2010).
    1. Zeng S., Hernández J., Mullins R. F., Effects of antioxidant components of AREDS vitamins and zinc ions on endothelial cell activation: Implications for macular degeneration. Invest. Ophthalmol. Vis. Sci. 53, 1041–1047 (2012).
    1. Goverdhan S. V., et al. , Association of HLA class I and class II polymorphisms with age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 46, 1726–1734 (2005).
    1. Chirco K. R., Tucker B. A., Stone E. M., Mullins R. F., Selective accumulation of the complement membrane attack complex in aging choriocapillaris. Exp. Eye Res. 146, 393–397 (2016).
    1. Sohn E. H., et al. , Loss of CD34 expression in aging human choriocapillaris endothelial cells. PLoS One 9, e86538 (2014).
    1. Sun H. Y., Wei S. P., Xu R. C., Xu P. X., Zhang W. C., Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis. Biochem. Biophys. Res. Commun. 395, 361–366 (2010).
    1. Badea T., et al. , RGC-32 increases p34CDC2 kinase activity and entry of aortic smooth muscle cells into S-phase. J. Biol. Chem. 277, 502–508 (2002).
    1. Chirco K. R., et al. , Evaluation of serum and ocular levels of membrane attack complex and C-reactive protein in CFH-genotyped human donors. Eye (Lond.) 32, 1740–1742 (2018).
    1. Zeng S., et al. , Molecular response of chorioretinal endothelial cells to complement injury: Implications for macular degeneration. J. Pathol. 238, 446–456 (2016).
    1. Zheng G. X. Y., et al. , Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).
    1. Butler A., Hoffman P., Smibert P., Papalexi E., Satija R., Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
    1. Giacalone J. C., et al. , Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter. Microvasc. Res. 123, 50–57 (2019).
    1. Voigt A. P., et al. , Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration. Gene Expression Omnibus. . Deposited 17 August 2019.

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