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Potential role for laminin 5 in hypoxia-mediated apoptosis of human corneal epithelial cells

¹ Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, USA
² Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI, USA

View larger version (14K): [in a new window]   Fig. 1. Colorimetric BrdU cell proliferation ELISA of human corneal epithelial cells cultured under normoxic (20% O2) or hypoxic (2% O2) conditions. Proliferation is represented as the mean absorbance (A450) in arbitrary units + s.e.m. (n=30 for each time point and oxygen level). For 3, 7 and 10 days in culture, proliferation is significantly higher (*, P<0.0001) in normoxic HCE-T compared with hypoxic HCE-T.

View larger version (147K): [in a new window]   Fig. 2. Light microscopic analysis of mitochondrial membrane function in normoxic and hypoxic human corneal epithelial cells. HCEC were cultured for 3 days in 20% (A) or 2% (B) oxygen, 5 days in 20% (C) or 2% (D) oxygen, or 7 days in 20% (E) or 2% (F) oxygen before processing for apoptosis detection using the MitoLightTM technique. In each overlay in this Figure the green fluorescence represents cytoplasmic pools of a lipophilic cationic dye. The red fluorescence represents dye that has accumulated in mitochondria. Cells containing red-labeled mitochondria are scored as viable cells while those that lack labeled mitochondria and are uniformly green in color are scored as apoptotic. Note that at 5 and 7 days in culture, the normoxic cells appear to be smaller in size compared with the hypoxic cells.

View larger version (31K): [in a new window]   Fig. 3. Western blot and densitometric analysis of PARP content in normoxic and hypoxic human corneal epithelial cells. HCE-T were cultured for 7 days under normoxic (N) or hypoxic (H) conditions. 15 µg of cell lysates were resolved using 7.5% SDS-PAGE and processed for western blot analysis using mAb to PARP. Both the 115 kDa full-length PARP and the 89 kDa PARP cleavage product characteristic of apoptosis are detected by immunoblot. The relative density values of each PARP species determined by densitometry are indicated (in arbitrary units/µg total protein) to the right of each protein. The total of the densitometry readings is also indicated.

View larger version (44K): [in a new window]   Fig. 4. Western blot and densitometric analysis of laminin 5 2 chain protein content in normoxic and hypoxic human corneal epithelial cells. HCE-T were cultured for 3 days (A) or 7 days (B) under normoxic (N) or hypoxic (H) conditions. 15 µg of cell lysates were resolved using 7.5% SDS-PAGE and processed for western blot analysis using mAb D4B5 to the 2 chain. The unprocessed 155 kDa protein and processed 105 kDa and 80 kDa proteins are detected by immunoblot. The relative density values of each band are indicated (in arbitrary units/µg total protein) to the right of each immunoreactive protein. The total of the three densitometry readings is also indicated.

View larger version (37K): [in a new window]   Fig. 5. Western blot analysis of laminin 5 protein levels in ECM produced and deposited by normoxic (N) or hypoxic (H) human corneal epithelial cells. The ECM was solubilized, separated by 7.5% SDS-PAGE and processed for Western blot analysis using mAb 10B5 to the laminin 5 3 chain (A), mAb Clone 17 to the ß3chain (B) or mAb D4B5 to the 2 chain (C). The molecular weight of each of the immunoreactive proteins is indicated.

View larger version (112K): [in a new window]   Fig. 6. Immunofluorescence microscopic analysis of laminin 5 localization in normoxic and hypoxic human corneal epithelial cells. HCE-T were cultured for 3 days in 20% oxygen (A) or 2% oxygen (B) before processing for immunofluorescence microscopy using mAb GB3 to the 2 chain of laminin 5. Under normoxic conditions, the cells secrete and deposit a robust extracellular trail of laminin 5 protein that demarcates the path of movement of cells along the culture substrate (A). Note that the fluorescence intensity of the corresponding structures in the hypoxic cells is decreased (B), suggesting that less laminin 5 is deposited into the matrix.

View larger version (71K): [in a new window]   Fig. 7. Light microscopic analysis of apoptosis in normoxic human corneal epithelial cells. HCEC were cultured for 3 days in 20% oxygen. Cells in A represent the non-treated control. Cells in B were treated with function-blocking antibody to laminin 5 for 2 of those 3 days. Cells in C were treated with control IgG for 2 of those 3 days. All cells were processed for apoptosis detection using the MitoLightTM technique. Cells treated with function-blocking antibody to laminin 5 display a higher degree of apoptosis compared with untreated or IgG control cells.

View larger version (51K): [in a new window]   Fig. 8. Colorimetric BrdU cell proliferation ELISA of human corneal epithelial cells cultured under normoxic (20% O2) conditions in the presence of function-blocking antibody to laminin 5 (P3H9-2) or control IgG. Proliferation is represented as the mean absorbance (A450) in arbitrary units + s.e.m. (n=30 for each culture treatment). Proliferation is significantly lower (*, P=0.0004) in normoxic cells treated with laminin 5 antibody compared with control IgG.

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