Abstract
Purpose: Rho-kinase (ROCK) inhibitors is a novel class of anti-glaucoma agents, which act by increasing the aqueous humor outflow through conventional trabecular meshwork (TM) pathway. The downstream signaling consequences of ROCK inhibition in the TM are not fully understood. In this study, we evaluated the role of thrombospondin-1 (TSP-1) in triggering ROCK inhibitor-mediated increase in outflow facility.
Methods: Primary cultures of human TM (hTM) cells were used. Cell migration assay was conducted with or without Y39983 (1μM, a selective ROCK inhibitor), LSKL (a TSP-1 antagonist), and TSP-1 siRNA knockdown. Differential protein expression was quantified by LC-MS/MS using SWATHTM technologies. Expression of TSP-1 protein and mRNA was confirmed by Western blot analysis and qPCR, respectively. Conventional outflow facility was measured in ex vivo mouse eyes.
Results: Proteomic analyses identified 20 proteins whose expression were significantly altered after hTM cells were treated with Y39983 for 2 days. Of these, thrombospondin-1 (TSP-1) was downregulated 5-fold following Y39983 treatment. In addition, Y39983 elicited a dose-dependent inhibition of hTM cell migration. Similarly, LSKL and TSP-1 siRNA knockdown significantly reduced TSP-1 gene expression (~50% reduction and ~80% reduction, respectively) and hTM cell migration, respectively. In the presence of Y39983, no further inhibition of cell migration was observed after LSKL treatment and TSP-1 gene silencing. Likewise, LSKL increased the outflow facility in mouse eyes by 74%, similar to that of Y39983 (increase by 82%). There were no additive effects with simultaneous treatment with LSKL and Y39983.
Conclusions: Y39983 down-regulated the TSP-1 expression in hTM and silencing TSP-1 gene expression improved the outflow facility. Since there was no additive effect with combined treatment of Y39983 and TSP-1 blockade, the results suggests that TSP-1 is a downstream effector of ROCK inhibition that regulates outflow facility.
Methods: Primary cultures of human TM (hTM) cells were used. Cell migration assay was conducted with or without Y39983 (1μM, a selective ROCK inhibitor), LSKL (a TSP-1 antagonist), and TSP-1 siRNA knockdown. Differential protein expression was quantified by LC-MS/MS using SWATHTM technologies. Expression of TSP-1 protein and mRNA was confirmed by Western blot analysis and qPCR, respectively. Conventional outflow facility was measured in ex vivo mouse eyes.
Results: Proteomic analyses identified 20 proteins whose expression were significantly altered after hTM cells were treated with Y39983 for 2 days. Of these, thrombospondin-1 (TSP-1) was downregulated 5-fold following Y39983 treatment. In addition, Y39983 elicited a dose-dependent inhibition of hTM cell migration. Similarly, LSKL and TSP-1 siRNA knockdown significantly reduced TSP-1 gene expression (~50% reduction and ~80% reduction, respectively) and hTM cell migration, respectively. In the presence of Y39983, no further inhibition of cell migration was observed after LSKL treatment and TSP-1 gene silencing. Likewise, LSKL increased the outflow facility in mouse eyes by 74%, similar to that of Y39983 (increase by 82%). There were no additive effects with simultaneous treatment with LSKL and Y39983.
Conclusions: Y39983 down-regulated the TSP-1 expression in hTM and silencing TSP-1 gene expression improved the outflow facility. Since there was no additive effect with combined treatment of Y39983 and TSP-1 blockade, the results suggests that TSP-1 is a downstream effector of ROCK inhibition that regulates outflow facility.
| Original language | English |
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| Title of host publication | Investigative Ophthalmology and Visual Science |
| Pages | 1631 |
| Volume | 62 |
| ISBN (Electronic) | 1552-5783 |
| Publication status | Published - Jun 2021 |
| Event | Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting - Duration: 1 May 2021 → 7 May 2021 |
Conference
| Conference | Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting |
|---|---|
| Period | 1/05/21 → 7/05/21 |