1. Interaction of Sickle Erythrocytes With Endothelial Cells in the Presence of Endothelial Cell Conditioned Medium Induces Oxidant Stress Leading to Transendothelial Migration of Monocytes
by Chand Sultana, Yamin Shen, Vinod Rattan, Cage Johnson, and Vijay K. Kalra
Blood
Volume 92(10):
November 15, 1998
©1998 by American Society of Hematology

2. Effect of incubation of RBC and inhibitors on NF-kB activity in HUVEC nuclear extracts by gel-shift assay.
Effect of incubation of RBC and inhibitors on NF-kB activity in HUVEC nuclear extracts by gel-shift assay. HUVEC were incubated with RBC (2% Hct) in the presence of E-CM (100 μg/mL) for 60 minutes, unless otherwise indicated in the presence and absence of inhibitors (genistein, 25 μg/mL; KYRGDS, 100 μmol/L; HFPA, 10 μmol/L; and probucol, 50 μmol/L). Nuclear extracts were prepared and incubated with a double-stranded 32P-labeled NF-kB oligonucleotide probe. Where indicated, excess cold competitor oligonucleotide was added to the nuclear extracts and incubated for 10 minutes before addition of radiolabeled DNA probe. The data are representative of four independent experiments.
Chand Sultana et al. Blood 1998;92:
©1998 by American Society of Hematology

3. Relative NF-kB activation in HUVEC in response to interaction of RBC
Relative NF-kB activation in HUVEC in response to interaction of RBC. HUVEC were incubated with either AA RBC (n = 3; two different donors ) for 1 hour and 4 hours or with SS RBC (n = 4; three different donors and one replicate) for 1 hour in the absence an...
Relative NF-kB activation in HUVEC in response to interaction of RBC. HUVEC were incubated with either AA RBC (n = 3; two different donors ) for 1 hour and 4 hours or with SS RBC (n = 4; three different donors and one replicate) for 1 hour in the absence and presence of E-CM. Nuclear extracts were probed for NF-kB activity. The NF-kB activity in the gel (not shown) was quantified by densitometric scan of the autoradiograph by Alpha Image 2000 Documentation and Analysis system. The data shown is relative change in NF-kB activity compared to untreated HUVEC. NF-kB activity increased to 365% ± 34% (range, 320% to 425%) on incubation of HUVEC with SS RBC and E-CM. Replicate of the same donor sample showed less than 10% variation.
Chand Sultana et al. Blood 1998;92:
©1998 by American Society of Hematology

4. Cell adhesion molecule expression in HUVEC in response to interaction with RBC. HUVEC grown to confluence in 24-well plates were incubated with RBC (2% Hct) and E-CM (100 μg/mL) for 4-hour time periods, unless otherwise indicated.
Cell adhesion molecule expression in HUVEC in response to interaction with RBC. HUVEC grown to confluence in 24-well plates were incubated with RBC (2% Hct) and E-CM (100 μg/mL) for 4-hour time periods, unless otherwise indicated. ICAM-1, E-selectin, and VCAM-1 expression was determined by ELISA. (A) Time period of expression of CAMs in response to SS RBC plus E-CM; results are expressed as mean ± SD of OD at 405 nm of triplicate determinations, (n = 5; including three different donors). (B) Effect of AA RBC (n = 3), AS RBC (n = 3), and SS RBC (n = 6; including four different donors) on CAMs expression at 4 hours. (C) HUVEC were preincubated for 30 minutes with peptides (KYRGDS, 100 μmol/L; AGDV, 100 μmol/L) and polymyxin sulfate (5 μg/mL) before incubation with SS RBC plus E-CM or E-CM (100 μg/mL) alone for 4 hours. Incubation with LPS (100 ng/mL) was performed for 4 hours. (D) HUVEC were preincubated for 30 minutes with inhibitors (Probucol, 50 μmol/L; vitamin E, 25 μmol/L; SOD, (200 U/mL); catalase, (200 U/mL); GSH-EE, 0.5 mmol/L; and BSO (100 μmol/L) before incubation with SS RBC for 4 hours, followed by measurement of VCAM-1 expression.
Chand Sultana et al. Blood 1998;92:
©1998 by American Society of Hematology

5. Migration of monocyte-like HL-60 cells and PBM across HUVEC monolayer in response to incubation with RBC. HUVEC were grown to confluence on fibronectin-coated porous membranes (Transwell, Cat. #40492, Biocoat cell culture inserts, Becton-Dickinson).
Migration of monocyte-like HL-60 cells and PBM across HUVEC monolayer in response to incubation with RBC. HUVEC were grown to confluence on fibronectin-coated porous membranes (Transwell, Cat. #40492, Biocoat cell culture inserts, Becton-Dickinson). To the upper compartment, RPMI-1640 containing FCS was added, followed by RBC (2% Hct) and E-CM (100 μg/mL) and fluorescently labeled vitamin D3-differentiated HL-60 cells or PBM (0.5 × 106 cells per well). At the indicated time points, aliquots were removed from the lower compartment of the Transwell chamber for counting of monocyte-like HL-60 cells. Data are expressed as mean ± SD of SS RBC, n = 4 (three different donors), and three independent determinations for AA RBC. (A) Time course of HL-60 cells transendothelial migration. (B) HUVEC were incubated with inhibitors (MoAb to human PECAM-1 (10 μg/mL); MoAb to ICAM-2 (10 μg/mL); GF X (20 nmol/L); and Calyculin A, (2 nmol/L) for 45 minutes before the addition of SS RBC (2% Hct) and E-CM. (C) HUVEC were preincubated with either antioxidant (probucol, 50 μmol/L) for 45 minutes or GSH effectors (GSH-EE, 0.5 mmol/L and BSO, 100 μmol/L) for 24 hours, before the addition of SS RBC (2% Hct) and HL-60 cells. (D) HUVEC were incubated with MoAb to human PECAM-1 (10 μg/mL); KYRGDS, (100 μmol/L); and AGDV, (100 μmol/L) for 45 minutes before the addition of SS RBC (2% Hct) and E-CM (100 μg/mL). This was followed by the addition of fluorescently labeled PBM. The transmigrated monocyte cells were counted at 2 hours. Data are expressed as mean ± SD for three different donors RBC.
Chand Sultana et al. Blood 1998;92:
©1998 by American Society of Hematology

6. Time course of phosphorylation of PECAM-1 in HUVEC in response to incubation with SS RBC. HUVEC were labeled with32P and incubated with SS RBC (2% Hct) for the indicated time period and then processed for immunoprecipitation with PECAM-1 antibody.
Time course of phosphorylation of PECAM-1 in HUVEC in response to incubation with SS RBC. HUVEC were labeled with32P and incubated with SS RBC (2% Hct) for the indicated time period and then processed for immunoprecipitation with PECAM-1 antibody. The immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity quantitated in the gel lane corresponding to PECAM-1 (130 kD) by Ambis radiogel scanner as described in Materials and Methods. The data is presented as percent increase in the incorporation of 32P in PECAM-1, assigning the value of 100% for 32P incorporated into PECAM-1 in untreated HUVEC. Data are mean ± SD of n = 3, with each experiment run in duplicate for indicated time points, except for 1-hour period (n = 7). 32P incorporated into PECAM-1 was 638% ± 220% at 1-hour time point, with a range of 410% to 850%. The replicate data for the same donor sample showed less than 15% difference in the 32P incorporation into PECAM-1.
Chand Sultana et al. Blood 1998;92:
©1998 by American Society of Hematology

7. Effect of inhibitors on the 32P incorporation in PECAM-1 in HUVEC on incubation with RBC. 32P-labeled HUVEC were incubated with inhibitors either for 45 minute (GF X, 20 nmol/L; SQ 22536, 2 μmol/L; calyculin A, 2 nmol/L; probucol, 50 μmol/L; or for 24...
Effect of inhibitors on the 32P incorporation in PECAM-1 in HUVEC on incubation with RBC. 32P-labeled HUVEC were incubated with inhibitors either for 45 minute (GF X, 20 nmol/L; SQ 22536, 2 μmol/L; calyculin A, 2 nmol/L; probucol, 50 μmol/L; or for 24 hours (GSH-EE, 0.5 mmol/L and BSO, 100 μmol/L), followed by 1-hour incubation with SS RBC (2% Hct) and E-CM. Data are expressed as mean ± SD for three different SS RBC donors, in duplicate determinations, relative to untreated HUVEC (none).
Chand Sultana et al. Blood 1998;92:
©1998 by American Society of Hematology