Iris pigment epithelium attachment to aged submacular human Bruch's membrane.
OBJECTIVE
To determine whether iris pigment epithelium (IPE) cells can attach to aged submacular human Bruch's membrane and to assess whether IPE cells express the integrin subunits that may be necessary to bind to the known extracellular matrix ligands present in Bruch's membrane.


METHODS
IPE cells were seeded onto the RPE basement membrane (RPEbm) or inner collagenous layer (ICL) of aged submacular Bruch's membrane as microaggregates or were expanded in culture until enough cells could be obtained for seeding. Cell morphology and the percentage of cell coverage were determined 1 or 7 days after seeding. Messenger RNA was extracted from cultured and uncultured IPE cells and analyzed by RT-PCR. The expression of integrin subunits alpha1 to alpha6 and beta1 mRNA was examined.


RESULTS
Coverage by uncultured IPE was low on both surfaces at day-1 (RPEbm, 7.9% +/- 4.8%; ICL, 5.0% +/- 2.5%) with few intact cells present. Culturing IPE improved attachment with similar coverage on both surfaces and no significant difference between day-1 (RPEbm, 89.9% +/- 9.1%; ICL, 63.4% +/- 26.5%) and day-7 (RPEbm, 97.8% +/- 2.3%; ICL, 94.7% +/- 6.6%). By day-7, cell morphology and coverage on both surfaces was variable, ranging from few intact cells to a high degree of coverage by flattened cells. All integrin subunits studied were expressed in cultured cells, whereas alpha2, alpha3, and alpha4 showed less or no expression in uncultured cells.


CONCLUSIONS
Upregulation of integrin mRNA expression may be one explanation for the difference in coverage by cultured versus uncultured IPE cells. The presence of dead, dying, or flattened cells at day 7 indicates that IPE may not survive or differentiate on aged submacular Bruch's membrane.
Itaya
Hiroshi
H
Institute of Ophthalmology and Visual Science, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 90 Bergen Street, Newark, NJ 07101-1709, USA.
Gullapalli
Vamsi
V
Sugino
Ilene K
IK
Tamai
Makoto
M
Zarbin
Marco A
MA
D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology*; D016570:Bruch Membrane / Q000502:physiology*; D002455:Cell Division; D006801:Humans; D007150:Immunohistochemistry; D039001:Integrin alpha Chains / Q000235:genetics; D007498:Iris / Q000166:cytology / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D046509:Tissue Culture Techniques
D039001:Integrin alpha Chains; D020033:Protein Isoforms; D012333:RNA, Messenger