In-depth analysis of the molecular mechanisms underlying the differences in vascularization and inflammatory response in two extreme CC strains using total RNAseq

Abstract

Purpose : Lymphangiogenesis is involved in several diseases like corneal graft rejection, tumor metastasis, and dry eye disease. The healthy cornea is an avascular and alymphatic tissue with a distinct limbal lymphatic vascular arcade. It was already shown that the genetic background significantly influences the architecture of this limbal lymphatic arcade and corneal lymphangiogenesis in mice. The Collaborative Cross (CC) population is a large panel of recombinant inbred mouse strains derived from a genetically diverse set of eight founder strains.

Methods : Wholemounts of naïve corneas from 9 different CC mice were stained with the lymphatic vessel marker LYVE-1 and the vessel area was analyzed using cellF. In vivo, three 11-0 nylon sutures were placed into the cornea stroma of BOON (low-lymphangiogenic) and NUK (high-lymphangiogenic) mice for 14 days. Fourteen days after inflammatory insult by suture placement, the total surface area of ingrown vessels in the cornea was determined. From naive and inflamed central corneas RNA was extracted and total RNAseq was performed.

Results : The comparison of the different CC strains reveals significant differences in the lymphatic vascularized area with different CC lines. Two of the identified extreme lines, the low-lymphangiogenic BOON and high-lymphangiogenic NUK were analyzed in more detail. Both lines showed a dramatic increase in inflammation-induced lymphangiogenesis compared to their naïve controls. Total RNAseq was performed from both naïve and inflamed corneas of both strains. Analyzing the genes with significant interaction effect (adj. p-value <= 0.01) using GO enrichment, we found a particularly striking enrichment purely involved in neuronal development and processes, sensory perception, ion transport, and synaptic signaling.

Conclusions : The comparison of the extremely high-lymphangiogenic NUK with the extremely low lymphangiogenic BOON under both naïve and inflammatory conditions clearly shows that CC mice are a powerful tool for the identification of new endogenous modulators of lymphangiogenesis. The genes identified here could be used to develop new therapeutic targets for eye diseases associated with pathologic lymphangiogenesis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.