Journal: Frontiers in Cell and Developmental Biology

Article Title: A switch from α5β1 to αvβ3 integrin activity contributes to the development of a profibrotic mesenchymal phenotype in trabecular meshwork cells

doi: 10.3389/fcell.2025.1730542

Figure Lengend Snippet: Activity of αvβ3 integrin affects the expression of VIM , SNAI2 , and TWIST1 mRNA levels. (A) Flow cytometry showed that TM cells (Old α5-) derived from 74 to 77-year-old donor eyes expressed lower levels of α5 integrin compared to TM cells derived from young (young α5+) and other old donor eyes (old α5+). The designation α5+ refers to the fact that a large percentage of cells express the α5 integrin subunit while α5- refers to the fact that most of these cells do not express the α5 integrin subunit. (B) Despite differences in the levels of the α5 integrin subunit, all three populations of cells expressed similar levels of β3 integrin. (C) More old a5- TM cells (N74 and N77) expressed active αvβ3 integrin on the cell surface compared to young and old TM cells expressing α5β1 integrins; * p < 0.02. N = 10,000 cells per condition. N = 7 young α5+ biological replicates (ages 17–36), N = 5 old α5+ biological replicates expressing α5β1 integrin (ages 55–75). N = 2 old α5- biological replicates (ages 74–77). Cells were labeled with P1D6 (α5β1 integrin), LM609 (total αvβ3 integrin), or LIBS2 (active αvβ3 integrin) mAbs. (D,E) RT-qPCR showed that Old α5- TM cells expressed significantly higher levels ( p < 0.04) of mRNA for the EndMT markers VIM and SNAI2 compared to cells isolated from young α5+ donor eyes (N25 and N35). (F) TWIST1 mRNA levels were also higher in the old α5 integrin negative cells but the levels were not statistically significant ( p < 0.08). (G–I) Knockdown of β3 integrin using shRNA in the old N77 cells (Old α5-) that contained elevated levels of active αvβ3 integrin and low levels of α5β1 integrin mRNA had statistically reduced levels of VIM ( p < 0.0004) and TWIST1 ( p < 0.01) mRNA. Levels of SNAI2 mRNA levels were also reduced, but not statistically ( p < 0.07). All experiments were done in triplicates and repeated twice.

Article Snippet: Briefly, TM cells were lifted with Cell Dissociation Solution Non-enzymatic (Sigma-Aldrich Corp.), blocked for 30 min on ice with 1% BSA in PBS and labeled for 1 h on ice with 10 μg/mL α5 (P1D6, ThermoFisher Scientific, #12-4900-42, RRID:AB_10717080), total αvβ3 (LM609, Sigma-Millipore, mAb 1976, RRID:AB_2296419), and active αvβ3 (LIBS2, Millipore Sigma, MABT27, RRID:AB_10806476) integrin antibodies in 1% BSA/PBS.

Techniques: Activity Assay, Expressing, Flow Cytometry, Derivative Assay, Labeling, Quantitative RT-PCR, Isolation, Knockdown, shRNA