Primary trabecular meshwork cells incubated in human aqueous humor differ from cells incubated in serum supplements.
OBJECTIVE
To determine whether aqueous humor, the in vivo source of nutrients for trabecular meshwork cells, alters cellular and molecular characteristics in primary trabecular monolayer cell cultures when compared with standard culture conditions.


METHODS
Human primary trabecular meshwork cell cultures were grown in DMEM supplemented with 50% human aqueous humor (DMEM-AH), heat-denatured DMEM-AH, 10% fetal bovine serum (DMEM-FBS, the standard culture supplement), or heat-denatured DMEM-FBS. Confluent trabecular cells were assayed for cell propagation and morphology for 21 days. Protein expression profiles of trabecular cell lysates were analyzed by two-dimensional polyacrylamide gel electrophoresis. Western blot analysis was used to determine the protein expression of myocilin and TIMP-1 in conditioned media collected from trabecular cells at 5, 10, 15, and 21 days. Myocilin expression was also analyzed by Western immunoblots after addition of dexamethasone (10(-7) M) or ascorbic acid (29 mg/dL).


RESULTS
Trabecular cells supplemented with DMEM-AH for 21 days showed decreased cell proliferation when compared with DMEM-FBS (11% vs. 141%). Cellular morphology was also altered: Trabecular cells incubated in DMEM-AH showed larger-, broader-, and flatter-appearing cells than did the more spindle-shaped cells grown in DMEM-FBS. Protein profiles of trabecular cell lysates isolated from cells incubated in DMEM-AH differed from those incubated in DMEM-FBS. In DMEM-AH-conditioned medium, myocilin expression was increased and TIMP-1 expression was decreased at day 21. Induction of myocilin by dexamethasone was observed in conditioned medium isolated from cells treated with DMEM-FBS (442%), but only a 10% increase in myocilin was observed beyond the normal induction in DMEM-AH. Daily administration of ascorbic acid to DMEM-AH failed to increase myocilin expression beyond that obtained with DMEM-AH.


CONCLUSIONS
Addition of human aqueous humor rather than FBS to trabecular monolayer cell cultures triggers significant changes in cellular and molecular characteristics. The protein component of aqueous humor is responsible for these changes. Aqueous humor supplementation may maintain cultured trabecular cells in a more physiologic state.
Fautsch
Michael P
MP
Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. fautsch.michael@mayo.edu
Howell
Kyle G
KG
Vrabel
Anne M
AM
Charlesworth
M Cristine
MC
Muddiman
David C
DC
Johnson
Douglas H
DH
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001082:Aqueous Humor / Q000502:physiology*; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D003470:Culture Media; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D044967:Serum; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000099179:Myocilin
D001205:Ascorbic Acid; D003470:Culture Media; D003598:Cytoskeletal Proteins; D003907:Dexamethasone; D005136:Eye Proteins; D006023:Glycoproteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D000099179:Myocilin