Basement membrane integrity maintains blood vessel networks in inflamed corneas

Abstract

Purpose :
The basement membrane is a specialized extracellular matrix sheet mainly composed of collagen IV and laminins. Laminins are heterotrimeric proteins forming a network which is directly linked to the cell surface via cell adhesion molecules. The secreted extracellular matrix protein netrin-4 is able to intercalate into the network via binding to the laminin γ1 chain. The resulting complex of netrin-4 and laminin γ1 disrupts the laminin network and the entire basement membrane. The functional role of the laminin network during angiogenesis and vessel maintenance in inflammatory eye diseases has not been addressed so far. We hypothesis that laminin networks are necessary for blood vessel maintenance in the inflamed cornea.

Methods : Three interrupted 11-0 nylon sutures were placed into the corneal stroma of Balb/C mice (6 weeks old) and left in place for 14 days to induce neovascularization. Two weeks after suture placement sutures were either removed (supportive regression) or left in place for another 7 days (active regression). At that time, to disrupt laminin networks, the treatment group (n=10) received eye drops (3µl/3x/day; 7 days) of either Netrin-4 (1mg/ml), a laminin-binding mutant of Netrin-4 (Net4^E195A,R199A, 1mg/ml) or PBS as negative control . For immunohistochemistry, corneal flat mounts were stained with CD31 as pan endothelial marker and laminin γ1. Image analysis was performed by fluorescence and confocal microscopy.

Results : Netrin-4 eye drops significantly supported the regression of preexisting corneal blood vessels compared to the control group (p= 0.0010; Net4: 13.03 ± 1.7%; control: 21.7±5.9%) where as Net4^E195A,R199A ,which is unable to disrupt preexisting laminin networks, could not support vessel regression (p= n.s.; Net4^E195A,R199A: 17.45 ± 3.1%; control: 21.7±5.9%). Furthermore, destabilization of the laminin network by Netrin-4 actively induced blood vessel regression (p= 0.0156; Net4: 14.29 ± 2.3%; control: 19.2±5.4%) whereas Net4^E195A,R199A could not induce blood vessel regression (p=n.s.; Net4: 16.07 ± 2.8%; control: 19.2±5.4%). Immunohistochemistry revealed that the target structure Laminin γ1 is co-localized with corneal CD31+ blood vessels.

Conclusions : We show that the basement membrane is pivotal to guarantee blood vessel maintenance in the inflamed cornea. Moreover, our data identify the laminin network as a promising therapeutic target for angiogenesis associated diseases.

This is a 2021 ARVO Annual Meeting abstract.