At 1 year of age,
Angpt2−/− mice had a decreased SC area and increased SC narrowing count when compared to WT and
Angpt4−/− mice (
Figs. 3A–D). Notably,
Angpt2−/−;Angpt4−/− mice had a smaller SC area and more SC narrowings than any of the other genotypes (
Figs. 3A–D). The limbal vascular plexus in
Angpt2−/− and
Angpt2−/−;Angpt4−/− mice was also less complex with loss of typical circular limbal arteries and perilimbal veins
40 (
Fig. 3C). Clearly distinguishable patterns of circulating parallel limbal arteries and veins described in mice with partial C57BL/6 background
40 were detected in 7/7 WT and 4/4
Angpt4−/− samples, but such arteries were very rarely seen when
Angpt2 was deleted (in 2/8
Angpt2−/− and 0/5
Angpt2−/−;Angpt4−/− samples), and no circulating veins were found when 8
Angpt2−/− and 5
Angpt2−/−;Angpt4−/− samples were analyzed (
Fig. 3C). Additionally, the collecting veins (determined as the branching venous plexus and collector channels enriched at the sites of episcleral and aqueous veins in comparison to the above-discussed perilimbal veins that circulate alongside the SC) and the corneal arcades of the capillary plexus appeared to be dependent on Angpt2 as both
Angpt2−/− and
Angpt2−/−;Angpt4−/− mice displayed similar reduction in collecting vein branch and corneal arcade number (
Figs. 3E,
3F). Both
Angpt2−/− and
Angpt2−/−;Angpt4−/− mice also completely lacked corneolimbal LVs while WT and
Angpt4−/− mice had no changes (
Fig. 3G), along with a previous study reporting reduced LV area in a postnatally induced
Angpt2 deletion model
26 and implying that Angpt2 is indispensable for the development of both limbal blood and lymphatic vasculature. The observed vascular phenotypes in the
Angpt2-deficient mice were consistent with the scRNAseq data, and additionally, immunostaining localized Angpt2 expression to the LVs (
Supplementary Fig. S6). Essentially similar, albeit slightly less pronounced, SC and corneolimbal vasculature phenotypes were also seen in 12-week-old
Angpt2−/− and
Angpt2−/−;Angpt4−/− mice (
Supplementary Fig. S7). All in all, our data imply that
Angpt2 is required for the proper SC and corneolimbal blood and lymphatic vasculature development, and additional
Angpt4 deletion aggravates the negative impact of
Angpt2 deletion on the SC phenotype.