Abstract
Purpose :
Excessive wound healing after glaucoma filtration surgery (GFS) is primarily caused by the transformation of subconjunctival Tenon fibroblasts into increased extracellular matrix-producing myofibroblasts. The extracellular matrix forms a barrier that causes inadequate absorption of excess aqueous humor by the conjunctiva and increases intraocular pressure. To test future fibrosis inhibitors in vivo after GFS in a mouse model, murine subconjunctival fibroblasts (mSCFs) were isolated and compared to human Tenon fibroblasts (hTFs).
Methods :
Methods:
(1) Tissue samples were taken from the subconjunctival space of C57BL/6 mouse eyes (n=3) or tenon from humans undergoing trabeculectomy surgery (n=3). Both cell types were compared morphologically, by immunocytochemistry (ICC) and Western blot analysis. An outgrowth of cells from both tissues was observed 5 to 7 days after initial culture and could be successfully expanded until passages 5-7.
(2) ICC staining of Vimentin, α-SMA, Collagen I (Col1), and Collagen VI (Col6) was performed with both cell types after 5 days of treatment without or with TGF-beta (10 ng/ml) to induce fibrosis or with the TGFBR2-inhibitor SB431542 (10 ng/ml) to block fibrosis induction.
(3) Western blots of α-SMA (with and without TGF-beta treatment for 5 days) and Col1 (without treatment) were performed.
Results :
The ICC analysis revealed that Col1 was present in every cell line in the perinuclear region's cytoplasm in mouse and human cultures. Extracellular Col1 could not be detected. Col6 could also be seen in the perinuclear area, and structures of Col6 connecting neighboring cells could be detected in all analyzed cell types. Vimentin showed a filamentous distribution in mSCFs and hTFs. As expected, the number of α-SMA-positive cells was increased in both cell types after 5 days of TGF-beta exposure. Western blots analysis showed higher α -SMA protein expression in TGF-beta treated mSCFs and hTFs compared to untreated or SB431542-treated cells.
Conclusions :
MSCFs and hTFs have a filamentous distribution of Vimentin. Both show Col1 and Col6 expressions in the perinuclear region. Both cell types share the origin and ability to transform into myofibroblasts as TGF-beta treatment for 5 days induced α-SMA protein compared to untreated or SB431542-treated cells. The similarities between mSCFs and hTFs are essential for conducting trabeculectomy or subconjunctival scarring studies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.