Expression of ATP-Binding Cassette Transporter A1 (ABCA1) in Eyelid Tissues and Meibomian Gland Epithelial Cells

Abstract

Purpose: To validate the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and distribution in human eyelid tissues and meibomian gland epithelial cells.

Methods: Meibomian gland tissues from human eyelids were isolated by collagenase A digestion and cultured in defined keratinocyte serum-free medium (DKSFM). Infrared imaging was used to analyze the general morphology of meibomian glands. Hematoxylin and eosin (H&E) staining and Oil Red O staining were used to observe the morphological structure and lipid secretion in the human meibomian gland tissues. Quantitative real-time polymerase chain reaction, western blotting, and immunostaining were used to detect the mRNA and protein expression and cytolocalization of ABCA1 in the meibomian gland tissues and cultured cells.

Results: The degree of loss of human meibomian gland tissue was related to age. Meibomian gland lipid metabolism was also associated with age. Additionally, human meibomian gland tissues express ABCA1 mRNA and protein; glandular epithelial cells express more ABCA1 mRNA and protein than acinar cells, and their expression in acinar cells decreases with differentiation. Furthermore, the expression of ABCA1 was downregulated in abnormal meibomian gland tissues. ABCA1 was mainly localized on the cell membrane in primary human meibomian gland epithelial cells (pHMGECs), whereas it was localized in the cytoplasm of immortalized human meibomian gland epithelial cells (iHMGECs). The mRNA and protein levels of ABCA1 in pHMGECs were higher than those in iHMGECs.

Conclusions: Meibomian gland tissues of the human eyelid degenerate with age. ABCA1 expression in acinar cells decreases after differentiation and plays an important role in meibomian gland metabolism.

Figure 1.

Infrared photography of human meibomian glands after separation and culture of primary meibomian gland epithelial cells.

Figure 2.

(A) Slit-lamp photography of the meibomian glands. (B) Infrared photography of the meibomian glands. The tarsal plates were separated from the cadaveric eyelids and divided into three age groups: teenager, middle-aged, and elderly. They were photographed directly after trimming some muscles and fascia. Note that the thickness of the tarsal plate and the setting of photographic lighting result in different transparencies. The two older donors (middle-aged and elderly) showed mild to moderate meibomian gland atrophy, accompanied by gland shortening (red arrow) or loss (black arrow).

Figure 3.

Human meibomian gland structure and lipid metabolism performance. (A) H&E staining of meibomian gland ducts and acini (n = 3). Abbreviations: a, acini; du, central duct; de, connecting ductule. Scale bar: 100 µm. (B) Oil Red O staining of meibomian glands (n = 3). Scale bar: 100 µm.

Figure 4.

Localization and expression of ABCA1 in the meibomian gland. (A) Immunofluorescence staining showed reduced expression of ABCA1 in abnormal meibomian glands. Compared to other eyelid tissues, ABCA1 staining was strong in meibomian gland duct epithelium, acinar basal cells, and progenitor cells. In acinar cells, nuclear colocalization (DAPI) showed that the intensity of ABCA1 expression decreased as the degree of cell differentiation increased (direction of red arrow). (B) qPCR results showed that ABCA1 mRNA levels were higher in the normal meibomian gland function group compared to the abnormal meibomian gland function group (P < 0.05, n = 3).

Figure 5.

Representative images of human P0 primary eyelid gland epithelial cells under an inverted microscope. (A) Human eyelid gland cultured ex vivo at day 3 and day 5. Fibroblast growth was seen around the glands (white arrows). (B) On days 10 to 12, cells fused to form many cell clusters (red arrows and yellow circles), with a confluence rate of 80%–90%. (C) After achieving a confluence of more than 90%, the cells became flat and enlarged, showing signs of cellular aging (black arrows).

Figure 6.

Expression and cytolocation of ABCA1 in meibomian gland epithelial cells in culture. (A) PPAR-γ (green), ABCA1 (green), Nile Red (red), and cell nucleus DAPI (blue) staining in P2 pHMGCs. (B) Immunofluorescence staining showed that ABCA1 was mainly expressed on the cell membrane in pHMGCs rather than in the cytoplasm in iHMGCs. (C) qRT-PCR showed that the expression of ABCA1 mRNA in pHMGECs was higher than that in iHMGECs. (D) Western blot analysis of ABCA1 in pHMGECs and iHMGECs.