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Published on: August 20, 2016
Inhibition of mTOR differently modulates planar and subepithelial fibrogenesis in human conjunctival fibroblasts
Megumi Watanabe1, Yuri Tsugeno2, Tatsuya Sato3,4
1Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo Ika Daigaku, Hirosaki, Japan. watanabe@sapmed.ac.jp.
Purpose:
In the current investigation, the effects of the mTOR inhibitors, Rapa and Torin1 on the TGF-β2-induced conjunctival fibrogenesis were studied.
Study Design:
Experimental research.
Methods:
2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D).
Result:
Rapa or Torin1 both significantly increased planar proliferation in the non-TGF-β2-treated 2D HconF cells, but in the TGF-β2-treated cells, this proliferation was inhibited by Rapa and enhanced by Torin1. Although Rapa or Torin1 did not affect cellular metabolism in the non-TGF-β2-treated HconF cells, mTOR inhibitors significantly decreased and increased the mitochondrial respiration and the glycolytic capacity, respectively, under conditions of TGF-β2-induced fibrogenesis. Subepithelial proliferation, as evidenced by the hardness of the 3D spheroids, was markedly down-regulated by both Rapa and Torin1 independent of TGF-β2. The mRNA expressions of several ECM molecules and their regulators fluctuated in the cases of 2D vs 3D and TGF-β2 untreated vs treated cultures.
Conclusion:
The present findings indicate that mTOR inhibitors have the ability to increase and to reduce planar and subepithelial proliferation in HconF cells, depending on the inhibitor being used.
Insights
MTOR inhibitors Rapa and Torin1 differentially affect conjunctival fibrogenesis. These compounds impact planar and subepithelial proliferation, offering potential therapeutic avenues for fibrotic conditions.
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- Conjunctival fibrogenesis is a pathological process contributing to ocular surface scarring.
- Transforming growth factor-beta 2 (TGF-β2) is a key mediator in fibrotic diseases.
- Mammalian target of rapamycin (mTOR) signaling pathways play critical roles in cell growth and proliferation.
Purpose of the Study:
- To investigate the effects of mTOR inhibitors, Rapa and Torin1, on TGF-β2-induced conjunctival fibrogenesis.
- To analyze the impact of these inhibitors on cellular proliferation, metabolism, and extracellular matrix (ECM) production in conjunctival fibroblasts.
Main Methods:
- Experimental research utilizing 2D and 3D cultures of human conjunctival fibroblasts (HconF).
- Evaluations included Trans-Epithelial Electrical Resistance (TEER) for planar proliferation, Seahorse metabolic analyses, 3D spheroid size and hardness for subepithelial proliferation, and mRNA expression of ECM proteins and regulators.
Main Results:
- Rapa and Torin1 differentially modulated planar proliferation in HconF cells, with Rapa inhibiting and Torin1 enhancing proliferation in TGF-β2-treated cells.
- mTOR inhibitors altered cellular metabolism under TGF-β2 conditions, decreasing mitochondrial respiration and increasing glycolytic capacity.
- Both Rapa and Torin1 significantly reduced subepithelial proliferation, as indicated by decreased 3D spheroid hardness, irrespective of TGF-β2 treatment.
Conclusions:
- mTOR inhibitors exhibit distinct effects on planar and subepithelial proliferation in conjunctival fibroblasts.
- The findings suggest that specific mTOR inhibitors may offer targeted therapeutic strategies for managing conjunctival fibrotic conditions.
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