Abstract
Purpose :
Studies have shown that changes in αvβ3 integrin expression/activity in TM cells can induce a glaucomatous phenotype. This study was to determine if there were age-dependent differences in αvβ3 integrin expression in human anterior segments and TM cell cultures.
Methods :
Immunohistochemistry of paraffin embedded human anterior segments was performed by co-labeling with antibodies against αvβ3 integrin and fibronectin. Images were acquired with a Zeiss Z2 epifluorescence microscope. Image J was used to quantify the levels of αvβ3 integrin and fibronectin in the TM/SC. TM cells were cultured in low glucose DME with 10% FBS. One week post confluency, total RNA was isolated and relative levels of the β3 integrin subunit and TGFβ2 were determined using qPCR. An adenovirus vector expressing either wildtype or constitutively active αvβ3 integrin tagged with mCherry was used to determine if its expression in TM cells affected TGFβ2 expression.
Results :
Immunolabeling studies showed that αvβ3 integrin levels varied between age-matched donor eyes while fibronectin levels were more uniform. Both αvβ3 integrin and fibronectin could be found on the trabecular beams, in the juxtacanalicular (JCT) region, and both the inner (IW) and outer wall (OW) of SC. Quantification showed there was more αvβ3 integrin in the OW of SC than the beams or JCT/IW of SC. In contrast, there was more fibronectin in the JCT/IW of SC than the beams or OW of SC. Interestingly, the amount of αvβ3 integrin and fibronectin varied from quadrant to quadrant within the same donor eye indicating expression was segmental. Analysis of mRNA levels of isolated cells from normal donor eyes showed an age-dependent loss in αvβ3 integrin and TGFβ2 expression. In contrast, cells isolated from glaucomatous tissue showed an increase in αvβ3 integrin and TGFβ2 expression compared to age matched controls. TM cells overexpressing a constitutively active αvβ3 integrin also showed increased TGFβ2 mRNA levels.
Conclusions :
These studies show that expression of αvβ3 integrin appears to be segmental suggesting it may play a role in regulating segmental outflow and its expression/activity may be age dependent. These studies also support earlier findings and show that expression of αvβ3 integrin correlates with TGFβ2 expression in vitro and suggest that αvβ3 integrin signaling may be a gain of function that contributes to glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.