Cell atlas of trabecular meshwork in glaucomatous non-human primates and DEGs related to tissue contract based on single-cell transcriptomics

Abstract

As the major channel of aqueous humor outflow, dysfunction of trabecular meshwork (TM) can lead to intraocular pressure elevating, which can trigger primary open-angle glaucoma (POAG). In this study, we use single-cell RNA sequencing (scRNA-seq) technique to build an atlas and further explore the spontaneous POAG and healthy macaques cellular heterogeneity associated with the dysfunction of TM contraction. We built the TM atlas, which identified 14 different cell types. In Beam A, Beam B, Beam C, and smooth muscle cell (SMC) cell types, we first found multiple genes associated with TM contraction (e.g., TPM1, ACTC1, TNNT1), determining their differential expression in the POAG and healthy groups. In addition, the microstructural alterations in TM of POAG non-human primates were observed, which was compact and collapsed. Thus, our study indicated that TPM1 may be a key target for regulating TM structure, contraction function, and resistance of aqueous humor outflow.

Keywords: Optometry; Transcriptomics.

Graphical abstract

Figure 1

Workflow in constructing trabecular meshwork cell atlas for non-human primates with POAG Step 1–2: Slit-lamp exam, gonioscope exam, fundus imaging as well as OCT were utilized to screen for spontaneous POAG models, which according with diagnostic criteria of POAG in humans. Step 3: TM tissues were detached integrally from inner surface of eyeball. Step 4: These figures illustrates the general process of single-cell sequencing and analysis.

Figure 2

Cell types and gene expression in the macaques trabecular meshwork cells (A) Cell atlas in TM between spontaneous POAG and healthy macaques. (B) Feature-plot of marker gene expression that showed the distribution of each cell cluster in the cell atlas. (C) Dot-plot of marker gene expression combinations that identified type of each cell cluster. (D) Difference of cells’ proportions in each cell cluster between POAG and healthy macaques. (E) Dot-plot of the POAG-associated disease genes' expression level between POAG and healthy macaques.

Figure 3

Functional enrichment analysis of DEGs in Beam A, Beam B, Beam C, and SMC clusters Multiple genes and pathways associated with tissue contraction function were identified in these four cell clusters (annotation with pentagram).

Figure 4

Exploring the pathway associated with tissue contraction (A) The diagram of smooth muscle contract pathway, in which the relationship between TPM, ACTC, and TNNT1 was shown. (B‒E) Feature-plot and violin-plot of genes that showed TPM1, ACTC1, and TNNT1 mainly located in the Beams and SMC clusters.

Figure 5

Immunohistochemical localization of TPM, TNNT1, and ACTC1 in the macaques’ TM tissues (A and B) Results of immunohistochemistry. The TPM1 was expressed in the sieve-like structure of TM, and the expression level was lower in POAG tissues than that in normal tissues (p < 0.01). (C and D) Results of immunofluorescence. The ACTC1 was expressed in the sieve-like structure of TM, and the expression level was lower in POAG tissues than in normal tissues (p < 0.01). (E and F) Results of immunofluorescence. The TNNT1 was expressed in the sieve-like structure of TM, and the expression level was lower in POAG tissues than in normal tissues (p < 0.01). White arrows point to the sieve-like structure of TM.

Figure 6

Exploring tissue contraction function in in macaque TM tissues and from human TM cells (A) The TM structure of POAG and normal macaques. In the figures we can see the sieve-like regions of TM were compact and collapsed, especially in the anterior section. In the figures of normal macaques we can see the integral sieve-like structures of TM were loose and of normal thickness. (B‒D) TPM1 expression in overexpression and knockdown C:ACTC1 relative expression in TM (qPCR). TPM1 overexpression elevated ACTC1 expression (p < 0.01) D: TNNT1 relative expression in TM (qPCR). TPM1 inhibition reduced TNNT1 expression (p < 0.01). (E and F) In the elevated TPM1 expression group, the expression level of ACTC1 protein was significantly increased, and the expression level of TNNT1 protein was also increased in a certain degree (p < 0.01). In the group with decreased TPM1 expression, the expression level of ACTC1 protein was decreased than normal group, and the expression level of TNNT1 protein was significantly decreased (p < 0.01).