Hemangiogenesis and lymphangiogenesis in corneal pathology

Abstract

Purpose: We characterized the presence of hemangiogenesis (HA) and lymphangiogenesis (LA) in human corneal specimens exhibiting 13 underlying pathologies.

Methods: Human corneal specimens were obtained from consenting subjects (n = 2 or n = 3 for each pathology; total sample size, n = 35). The pathological specimens were stained with hematoxylin and eosin (H&E) to determine the presence or absence of corneal neovascularization (NV) and superficial or deep stromal distribution of NV. Immunohistochemical staining was then performed to differentiate HA (positive for CD31) from LA (positive for lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1]).

Results: The double-negative (CD31(-)/LYVE-1(-)) immunostaining, indicating the absence of NV, was exhibited by 21 specimens (60%). CD31(-)/LYVE-1(-), indicating the presence of HA and absence of LA, was exhibited by 12 specimens (34%). The double-positive (CD31(+)/LYVE-1(+)) phenotype, indicating both HA and LA, was exhibited by 2 specimens (6%). Notably, the CD31(-)/LYVE-1(-) phenotype, indicating the presence of LA and absence of HA, was not detected among the specimens. Deep stromal NV was exhibited in a 4:3 ratio to superficial stromal NV. The double-negative immunostaining was more prevalent in noninflammatory pathologies, particularly in comparison with combined neovascular phenotypes (ie, CD31(+) or LYVE-1(+)). Among the neovascular phenotypes, HA was 7 times more common than LA. Specimens exhibiting LA presented only with the double-positive phenotype.

Conclusions: HA is the predominant component of NV in corneal pathologies. LA accompanies HA; however, isolated LA (from lymphatics in the conjunctiva) does not occur in these corneal pathologies. Our results suggest the potential therapeutic utility of targeting antineovascular therapies specifically for corneal HA and/or LA pathology.

FIGURE 1

Characterization of corneas exhibiting NV in various underlying ocular pathologies. H&E–stained specimens were assessed for histopathological characteristics of corneal vasculature. Arrows indicate neovascular formations. Pterygium presented with superficial stromal NV in subject 33 (A) and subject 32 (B). C, Ulcerative keratitis (subject 35) was also associated with superficial stromal NV. Specimens with (D) fungal keratitis (subject 16), (E) HSV keratitis (subject 24), (F) graft rejection (subject 21), and (G) chronic keratitis (subject 8) presented with deep stromal NV. Specimens (H and I) with inflammatory pannus in these images show NV distributed to superficial stroma (subjects 26 and 27); scale bar, 47.62 µm.

FIGURE 2

Immunohistochemical visualization of the CD31⁺/LYVE-1⁻ phenotype. Specimens exhibiting vascular structures were immunostained for CD31 (green), LYVE-1 (blue), and propidium iodide (PI, red) to differentiate between HA and LA. Arrows indicate neovascular formations. Representative specimens showing (A–D) fungal keratitis (subject 16), (E–H) HSV keratitis (subject 24), (I–L) inflammatory pannus (subject 27), (M–P) inflammatory pannus (subject 25), and (Q–T) chronic keratitis (subject 8); scale bar, 47.62 µm.

FIGURE 3

Immunohistochemical visualization of the CD31⁺/LYVE-1⁺ phenotype. The specimens with pterygium exhibiting vascular structures were immunostained for CD31 (green), LYVE-1 (blue), and propidium iodide (PI, red). Arrows indicate neovascular formations. Immunostaining shows specimens positive for both CD31 and LYVE-1. Representative specimens showing pterygium (A–D) in subject 33 and (E–H) subject 32; scale bar, 47.62 µm.

FIGURE 4

Immunohistochemical visualization of the CD31⁻/LYVE-1⁻ phenotype. Specimens exhibiting no vascular structures were immunostained for CD31 (green), LYVE-1 (blue), and propidium iodide (PI, red). Representative specimens showing keratoconus (A–D) in subject 30, Fuchs dystrophy (E–H) in subject 14, and corneal scarring (I–L) in subject 11; scale bar, 47.62 µm.