Comprehensive Transcriptomic Profiling Reveals Distinct Cell States and Molecular Interactions in Schlemm's Canal, a Critical Ocular Structure for Intraocular Pressure Regulation

Core Concepts

Schlemm's canal endothelial cells have a lymphatic-biased identity and can be classified into three distinct molecular states, with the inner wall cells exhibiting higher expression of genes involved in cell-cell junction formation and lymphatic-specific pathways. Predicted ligand-receptor interactions suggest key signaling between Schlemm's canal and the adjacent trabecular meshwork tissue.

Abstract

This study provides a comprehensive multi-modal transcriptomic analysis of Schlemm's canal endothelial cells (SECs), a critical ocular structure for regulating intraocular pressure and implicated in glaucoma.
The key findings are:

Bulk RNA sequencing shows that SECs have a transcriptomic profile more similar to lymphatic endothelial cells than blood vessel endothelial cells, with enrichment of pathways related to extracellular matrix interactions, TGF-β signaling, and immune cell interactions.

Single-cell and single-nucleus RNA sequencing identified three distinct SEC states - inner wall (IW), outer wall (OW), and collector channel (CC) cells. IW cells express higher levels of the lymphatic marker Npnt, while OW cells express Selp. IW cells are further divided into two subclasses (IW1 and IW2) based on differential expression of Npnt and Ccl21a.

Immunofluorescence validation confirmed the localization of NPNT in the anterior IW and CCL21A in the posterior IW, suggesting potential modulation by local environmental differences such as aqueous humor flow rates.

Pathway analysis highlighted the importance of cell-cell junctions, actin organization, and small GTPase signaling in SECs, processes likely critical for regulating outflow resistance and intraocular pressure.

Predicted ligand-receptor analysis revealed key signaling interactions between SECs and the adjacent trabecular meshwork cells, including ANGPT1/TEK and VEGF/VEGFR pathways, providing insights into the molecular crosstalk between these two intimately linked tissues.

This study significantly expands the molecular understanding of Schlemm's canal and its cellular heterogeneity, laying the groundwork for future functional studies and the development of new glaucoma therapeutics targeting this important ocular structure.

Stats

Schlemm's canal endothelial cells express higher levels of lymphatic markers compared to blood vessel endothelial cells.
Schlemm's canal endothelial cells are enriched in pathways related to extracellular matrix interactions, TGF-β signaling, and immune cell interactions.
Three distinct states of Schlemm's canal endothelial cells were identified - inner wall, outer wall, and collector channel cells.
Inner wall Schlemm's canal endothelial cells express higher levels of the lymphatic marker Npnt, while outer wall cells express Selp.
Inner wall Schlemm's canal endothelial cells can be further divided into two subclasses (IW1 and IW2) based on differential expression of Npnt and Ccl21a.

Quotes

"Schlemm's canal is a critical ocular drainage structure that maintains intraocular pressure with important consequences for glaucoma."
"Delineating the molecular control of outflow resistance and of inner wall fluid permeability is important for understanding IOP homeostasis and developing novel glaucoma treatments."
"Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences."

Key Insights Distilled From

Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

by Balasubraman... at www.biorxiv.org 09-03-2023

https://www.biorxiv.org/content/10.1101/2023.08.31.555823v2

Deeper Inquiries

How do the distinct SEC states identified in this study contribute to the regulation of aqueous humor outflow and intraocular pressure homeostasis

In this study, the identification of distinct Schlemm's canal endothelial cell (SEC) states, specifically the inner wall (IW) and outer wall (OW) SECs, provides crucial insights into the regulation of aqueous humor outflow and intraocular pressure (IOP) homeostasis. The IW SECs, characterized by the expression of Npnt and Ccl21a, are likely involved in regulating fluid permeability and drainage within Schlemm's canal. These cells are strategically positioned to respond to changes in aqueous humor flow rates and shear stress, crucial for maintaining the delicate balance of IOP. The OW SECs, marked by Selp expression, along with collector channel (CC) cells, play a role in modulating outflow resistance and potentially influencing the flow of aqueous humor towards the venous circulation. By delineating these distinct SEC states, the study sheds light on the intricate mechanisms by which Schlemm's canal contributes to the regulation of aqueous humor dynamics and IOP control.

What are the potential functional consequences of the predicted ligand-receptor interactions between Schlemm's canal and trabecular meshwork cells, and how could this knowledge be leveraged for developing new glaucoma therapies

The predicted ligand-receptor interactions between Schlemm's canal and trabecular meshwork cells uncovered in this study offer valuable insights into the molecular crosstalk that underlies the regulation of aqueous humor outflow and IOP. For instance, the interaction between ANGPT1 secreted by trabecular meshwork cells and its receptor TEK expressed in SECs is a key signaling pathway implicated in the development and maintenance of Schlemm's canal. Disruptions in this pathway have been associated with ocular hypertension and glaucoma, highlighting its significance in IOP regulation. Similarly, the interactions involving VEGF ligands and receptors suggest a role in modulating vascular permeability and angiogenesis within the aqueous humor outflow pathway. Understanding these signaling pairs can provide a foundation for developing novel glaucoma therapies targeting specific molecular pathways involved in maintaining ocular fluid homeostasis and IOP control.

Given the lymphatic-biased identity of Schlemm's canal, what insights can be gained from comparing its molecular profile to that of lymphatic vessels in other tissues, and how might this inform our understanding of its unique developmental origins and specialized functions

The lymphatic-biased identity of Schlemm's canal, as revealed in this study, offers valuable insights into its unique molecular profile and functional characteristics compared to lymphatic vessels in other tissues. By comparing the molecular signature of Schlemm's canal to that of lymphatic vessels, researchers can gain a deeper understanding of its specialized functions in ocular fluid dynamics and intraocular pressure regulation. The identification of lymphatic genes enriched in Schlemm's canal underscores its role in fluid drainage and immune cell trafficking, essential for maintaining ocular health. Furthermore, insights into the developmental origins of Schlemm's canal as a hybrid vessel with both blood and lymphatic endothelial characteristics provide a basis for exploring its distinct morphological and functional features. This comparative analysis can inform future studies on the pathophysiology of glaucoma and the development of targeted therapies aimed at modulating lymphatic-related pathways in Schlemm's canal to improve disease management.