1. The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing 210000, China.
2. The Affiliated Eye Hospital, Nanjing Medical University, Nanjing 210000, China.
3. Department of Ophthalmology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092, China.
4. Eye Institute, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai 200030, China.
5. NHC Key Laboratory of Myopia (Fudan University), Key Laboratory of Myopia, Chinese Academy of Medical Sciences, and Shanghai Key Laboratory of Visual Impairment and Restoration (Fudan University), Shanghai 200030, China.
* These authors contributed equally to this work.
Background: Capillary dysfunction has been implicated in a series of life- threatening vascular diseases characterized by pericyte and endothelial cell (EC) degeneration. However, the molecular profiles that govern the heterogeneity of pericytes have not been fully elucidated.
Methods: Single-cell RNA sequencing was conducted on oxygen-induced proliferative retinopathy (OIR) model. Bioinformatics analysis was conducted to identify specific pericytes involved in capillary dysfunction. qRT-PCRs and western blots were conducted to detect Col1a1 expression pattern during capillary dysfunction. Matrigel co-culture assays, PI staining, and JC-1 staining was conducted to determine the role of Col1a1 in pericyte biology. IB4 and NG2 staining was conducted to determine the role of Col1a1 in capillary dysfunction.
Results: We constructed an atlas of > 76,000 single-cell transcriptomes from 4 mouse retinas, which could be annotated to 10 distinct retinal cell types. Using the sub-clustering analysis, we further characterized retinal pericytes into 3 different subpopulations. Notably, GO and KEGG pathway analysis demonstrated that pericyte sub-population 2 was identified to be vulnerable to retinal capillary dysfunction. Based on the single-cell sequencing results, Col1a1 was identified as a marker gene of pericyte sub-population 2 and a promising therapeutic target for capillary dysfunction. Col1a1 was abundantly expressed in pericytes and its expression was obviously upregulated in OIR retinas. Col1a1 silencing could retard the recruitment of pericytes toward endothelial cells and aggravated hypoxia-induced pericyte apoptosis in vitro. Col1a1 silencing could reduce the size of neovascular area and avascular area in OIR retinas and suppressed pericyte-myofibroblast transition and endothelial-mesenchymal transition. Moreover, Col1a1 expression was up-regulated in the aqueous humor of the patients with proliferative diabetic retinopathy (PDR) or retinopathy of prematurity (ROP) and up-regulated in the proliferative membranes of PDR patients.
Conclusions: These findings enhance the understanding of the complexity and heterogeneity of retinal cells and have important implications for future treatment of capillary dysfunction.
Keywords: Pericyte, Single-cell RNA sequencing, Capillary dysfunction, Pericyte-myofibroblast transition