Purpose

PPARγ plays a critical role in the maturation of immortalized human meibomian gland epithelial cells (hMGEC). To further understand the molecular changes associated with meibocyte differentiation, we analyzed transcriptome profiles from hMGEC after PPARγ activation.

Methods

Three sets of cultivated hMGEC with or without exposure to PPARγ agonist, rosiglitazone were used for RNA-seq analysis. RNA was isolated and processed to generate 6 libraries. The libraries were then sequenced and mapped to the human reference genome, and the expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using DESeq2 and NOISeq. Gene ontology enrichment analysis (GOEA) was performed on gene sets that were upregulated or downregulated after rosiglitazone treatment. Five genes were selected for validation and differential expression was confirmed using quantitative PCR. The Differential expression of CK5 was evaluated using Western blotting.

Results

Expression data indicated that about 58,000 genes are expressed in hMGEC. DESeq2 and NOISeq indicated that 296 and 3436 genes were upregulated and 258 and 3592 genes were down regulated after rosiglitazone treatment, respectively. Of genes showing significant differences > 2 fold, GOEA indicated that cellular and metabolic processes were highly represented. Expression of ANGPTL4, PLIN2, SQSTM1, and DDIT3 were significantly upregulated and HHIP was downregulated by rosiglitazone. CK5 was downregulated by rosiglitazone.

Conclusions

The RNA-seq data suggested that PPARγ activation induced alterations in cell differentiation and metabolic process and affected multiple signaling pathways such as PPAR, autophagy, WNT, and Hedgehog.

Purpose

We have shown that nonlinear optical corneal crosslinking (NLO CXL) and stiffening can be achieved in ex vivo rabbit corneas using an 80-MHz, 760-nm femtosecond (FS) laser, however the required power was beyond the American National Standard Institute limit. The purpose of this study was to test the efficacy of amplified FS pulses to perform CXL to reduce power by increasing pulse energy.

Methods

A variable numerical aperture laser scanning delivery system was coupled to a 1030-nm laser with a noncollinear optical parametric amplifier to generate 760 nm, 50 to 150 kHz amplified FS pulses with 79.5-μm axial and 2.9-μm lateral two-photon focal volume. Ex vivo rabbit corneas received NLO CXL, and effectiveness was assessed by measuring collagen autofluorescence (CAF) and mechanical stiffening. NLO CXL was also performed in 14 live rabbits, and changes in corneal topography were measured using an Orbscan.

Results

Amplified pulses (0.3 μJ) generated significant CAF that increased logarithmically with decreasing scan speed; achieving equivalent CAF to UVA CXL at 15.5 mm/s. Indentation testing detected a 62% increase in stiffness compared to control, and corneal topography measurements revealed a significant decrease of 1.0 ± 0.8 diopter by 1 month (P < 0.05).

Conclusions

These results show that NLO CXL using amplified pulses can produce corneal collagen CXL comparable to UVA CXL.

Translational relevance

NLO CXL using amplified pulses can produce corneal CXL comparable to UVA CXL, suggesting a potential clinical application in which NLO CXL can be used to perform personalized crosslinking for treatment of refractive errors and keratoconus.

Purpose

To evaluate the role of PPARγ in regulating meibocyte differentiation and lipid synthesis in a human meibomian gland epithelial cell line (hMGEC).

Methods

HMGEC were exposed to the PPARγ agonist, Rosiglitazone, from 10-50 μM. Cultures were also exposed to specific PPARγ antagonist, T0070907, to block PPARγ receptor signaling. Cells were then stained with Ki-67 and LipidTox to determine the effects on cell cycling and lipid synthesis, respectively. Expression of meibocyte differentiation related proteins, ADFP, PPARγ, ELOVL4, and FABP4, were evaluated by quantitative PCR and western blotting. A human corneal epithelial cell line (hTCEpi) was used as a control.

Result

Rosiglitazone significantly decreased Ki-67 staining within 2 days in a dose-dependent manner (P = 0.003) and increased lipid accumulation in hMGEC in a dose dependent manner. T0070907 suppressed both lipid droplet synthesis and cell cycle exit. Rosiglitazone significantly upregulated expression of ADFP, PPARγ, ELOVL4, and FABP4 by 9.6, 2.7, 2.6, and 3.3 fold on average (all P < 0.05 except for FABP4, P = 0.057) in hMGEC. T0070907 significantly abrogated rosiglitazone-induced upregulation of these genes when treated prior to rosiglitazone treatment (all P < 0.05). The observed lipogenic differentiation response was not duplicated in hTCEpi after exposure to rosiglitazone.

Conclusion

Rosiglitazone induced cell cycle exit and upregulation of lipogenic gene expression leading to lipid accumulation in hMGEC. These effects were suppressed by PPARγ antagonist indicating that PPARγ signaling specifically directs lipogenesis in hMGEC. These findings suggest that PPARγ plays a critical role in meibocyte differentiation.

Purpose

The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC).

Methods

HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function.

Results

EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation.

Conclusions

EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.