Abstract
Purpose :
One of the most striking features of the meibomian gland (MG) is the absence of blood vessels. Indeed, the location of nearest vasculature to MG acini is beyond the basement membrane. This distance, according to Krogh’s law, would limit oxygen diffusion and create a relatively hypoxic environment for the MG. Such a situation would seem to be counterintuitive, given that other lipid producing cells (e.g. adipocytes) in fat tissues require an abundant blood supply as the source of oxygen. However, we hypothesize that the MG is relatively hypoxic for physiological reasons and that this condition is actually beneficial for the MG. We tested this hypothesis.
Methods :
We studied human and mouse eyelid segments, and immortalized human MG epithelial cells (IHMGECs). To evaluate oxygen levels in the mouse MG and vicinity, we injected pimonidazole hydrochloride (PIMO; 100mg/kg) intraperitoneally 2 hours before sacrifice. This compound is a common marker used in vivo to stain hypoxic tissues. Mouse eyelids were removed, processed for histology, and counterstained with hematoxylin to delineate lid anatomy. Resected human eyelid samples, obtained from healthy patients following their lid surgeries, were stained with the hypoxia markers, glucose transporter 1 (Glut-1), carbonic anhydrase 9 (CA9) and hypoxia-inducible factor 1a (HIF1a). To determine the effect of low oxygen levels on IHMGECs, we cultured cells under proliferating and differentiating conditions in both normoxic (20% O2) and hypoxic (5% O2) environments for 5 or 14 days. IHMGECs were evaluated for cell number, neutral lipid content (LipidTOX), lysosome accumulation (LysoTracker), and expression of different proteins (proliferating cell nuclear antigen [PCNA], HIF1a) by Western blots. Experiments were approved by an IRB and IACUC.
Results :
Our results demonstrate that mouse MGs, and not adjacent tissue, feature intense staining for PIMO. Similarly, we discovered that human MGs, and not the surrounding tissue, show intense staining for Glut-1, CA9 and HIF1a. Hypoxic conditions did not influence the proliferation of IHMGECs, but appeared to reduce the extent of cellular differentiation. It is possible that this relative decrease reflected an acceleration of differentiation.
Conclusions :
MGs exist in a relatively hypoxic environment. In other tissues, low oxygen concentrations allow stem cells to maintain their stemness, which may also be true for MGs.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.