The combined effect of azithromycin and insulin-like growth factor-1 on cultured human meibomian gland epithelial cells

Abstract

Purpose: Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease, a prevalent disorder severely affecting patients' quality of life but has no cure. We have discovered that azithromycin, a topical antibiotic used off-label to treat MGD-associated posterior blepharitis, directly acts on the human meibomian gland epithelial cells (HMGECs) to promote their differentiation, and in doing so, reduces cell proliferation. We have also found that insulin-like growth factor-1 (IGF-1), a drug approved by the Food and Drug Administration primarily used to treat dwarfism, stimulates the proliferation and lipid accumulation in these cells. We hypothesize that the combination of azithromycin and IGF-1 will promote cellular differentiation and lipid accumulation, while preserving the normal proliferation of HMGECs.

Methods: We cultured immortalized HMGECs with vehicle, 10 nM IGF-1, 10 μg/mL azithromycin, or a combination of IGF-1 and azithromycin for 5 to 13 days. Cells were evaluated for intracellular neutral lipids and lysosome accumulation by different staining methods; lipid composition of cell lysates were analyzed using high-performance thin-layer chromatography; proteins of interest (sterol regulatory element binding protein-1 [SREBP-1], cyclins B1 and D1) were measured by immunoblotting, and cell numbers were counted using a hemocytometer.

Results: Our findings demonstrate that the combination of azithromycin and IGF-1 promotes the differentiation and lipid accumulation of HMGECs, while preserving their normal proliferation rate. This combined treatment also increased the levels of neutral lipids, phospholipids, and SREBP-1, and restored cyclin B1 content to control amounts.

Conclusions: Our results support our hypothesis, and this combination regime may represent a unique and effective treatment of MGD.

Keywords: IGF-1; azithromycin; meibomian gland.

Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Figures

Figure 1

Effect of IGF-1, AZM, and IGF-1+AZM combination on intracellular accumulation of lipids and lysosomes. Cells were treated with vehicle, 10 nM IGF-1, 10 μg/mL AZM, or IGF-1+AZM combination for 13 days. (a) The green color represents LipidTOX green neutral lipid staining, and red color LysoTracker staining for lysosomes. (b) The fluorescence intensity of LipidTOX staining was quantified using ImageJ. Two-way ANOVA showed significant effect of AZM (****P < 0.0001). (c) The fluorescence intensity of LysoTracker staining was quantified using ImageJ. Two-way ANOVA showed significant effect of AZM (P < 0.0001). The experiments were repeated four times with similar results; data shown here are from a single experiment.

Figure 2

Effect of IGF-1, AZM, and IGF-1+AZM combination on the accumulation of CE, TG, FC, PE, and PC. (a) Cells were treated with vehicle, 10 nM IGF-1, 10 μg/mL AZM, or IGF-1+AZM combination for 7 days before performing chromatographic analyses of total lipid extracts. (b) Band intensity was quantified using ImageJ. Control band intensity was set to 1, and data (mean ± SE) were reported as fold-change compared with control values. The IGF-1 showed a significant effect on CE (*P < 0.05) and TG (****P < 0.0001). The AZM showed a significant effect on CE, TG, PE, PC (P < 0.0001 for all four), and FC (**P < 0.01). Other bands are unidentified lipids. Band intensity analysis included data from three independent experiments. STD, lipid standard.

Figure 3

Effect of IGF-1, AZM, and IGF-1+AZM combination on the expression of SREBP-1, cyclins B1 and D1. Cells were incubated with vehicle, 10 nM IGF-1, 10 μg/mL AZM, or IGF-1+AZM combination for 5 days. Cell lysates were evaluated on immunoblots for precursor and mature forms of SREBP-1, cyclins B1 and D1 (a), and protein band intensities quantified using ImageJ (b). The IGF-1 significantly affected the expression of pre-SREBP-1 and mature SREBP-1 (*P < 0.05) and cyclin B1 (****P < 0.0001). The AZM showed significant effect on mature SREBP-1 (**P < 0.01) and cyclin B1 (P < 0.0001). These experiments were repeated at least three times with similar results.

Figure 4

Effect of IGF-1, AZM, and IGF-1+AZM combination on the proliferation of IHMGECs. Cells were seeded (50,000 cells/well in12-well plates, n = 3 wells/group) and treated with vehicle, 10 nM IGF-1, 10 μg/mL AZM, or IGF-1+AZM combination for 13 days before cell counting. Results were reported as mean ± SE. The IGF-1 and AZM both exerted a significant but opposite effect on cell proliferation (****P < 0.0001 for both). Data from one experiment were shown as a representative of three studies performed under the same conditions.