Effect of simultaneous intra/extravascular administration of high dose diltiazem on reactive postangioplasty cell proliferation
The effect of simultaneous intra/extravascular administration of high dose diltiazem on relative reactive cell proliferation both in the neointima and the media of POC segments was studied 7 and 28 days after ex vivo ballooning.
Seven days after ex vivo ballooning in the neointima a 39.4 fold increase of proliferative activity (n.s.) was detected, the number of BrdU-positive cells was increased from 0.1 ± 0.34% in untreated controls to 4.3 ± 5.6% (Fig. 1 and 2). After simultaneous intra/extravascular administration of diltiazem for 1 and 2 days the number of BrdU-positive cells was 2.8 ± 6.1% and 1.1 ± 1.8% corresponding to an inhibition by 36 respectively 75%. Administration of diltiazem for 3 days resulted in an increase of BrdU-positive cells by 52%, 6.6 ± 10.4% of BrdU-positive cells were detected. After incubation of POC-segments with diltiazem for 4, 5, 6, and 7 days the percentage of BrdU-positive cells was 0.8 ± 1.1%, 1.9 ± 3.6%, 0.3 ± 0.7%, and 1.2 ± 1.8% corresponding to an inhibition by 81%, 57%, 93%, and 72%. 28 days after ballooning the number BrdU-positive cells was very small, 0.0 ± 0.0% of BrdU-positive cells were detected in untreated controls and in the POC balloon-group. Incubation with diltiazem for 1, 2, and 3 days resulted in 0.0 ± 0.0%, 1.2 ± 2.6%, and 0.1 ± 0.2% of BrdU-positive cells, respectively. 28 days after incubation with diltiazem for 4, 5, 6, and 7 days the number of BrdU-positive cells was 2.2 ± 5.9%, 3.2 ± 7.2%, 0.0 ± 0.0%, and 1.1 ± 2.4%.
In the media the number of BrdU-positive cells was always very low, no matter whether or not cells were incubated with diltiazem or treated with the balloon or both. 7 days after ballooning 0.0 ± 0.0% of BrdU-positive cells were detected in untreated controls, 0.2 ± 0.2 after 9 bar ballooning. Incubation with diltiazem for 1, 2, and 3 days resulted in 0.1 ± 0.0%, 0.0 ± 0.0%, and 0.7 ± 1.3% of BrdU-positive cells. After incubation with diltiazem for 4, 5, 6, and 7 days the number of BrdU-positive cells was 0.0 ± 0.0%, 0.1 ± 0.2%, 0.1 ± 0.1%, and 0.0 ± 0.0%. 28 days after ballooning 0.0 ± 0.0% were found in untreated controls and after 9-bar ballooning. Incubation of POC-segments with diltiazem for 1, 2, and 3 days resulted in 0.1 ± 0.1%, 0.1 ± 0.2%, and 0.0 ± 0.0% of BrdU-positive cells, after incubation for 4, 5, 6, and 7 days 0.1 ± 1.4%, 0.1 ± 0.3%, 0.1 ± 0.1%, and 0.2 ± 0.4%.
No statistical significance was reached seven and 28 days after ballooning between the number of BrdU-positive cells in the balloon-group in comparison to the balloon/diltiazem group.
Effect of simultaneous intra/extravascular administration of high dose diltiazem on neointimal thickening
The effect of simultaneous intra/extravascular administration of high dose diltiazem on relative neointimal thickening was analysed 7 and 28 days after 9 bar ballooning (Fig. 3 and 4).
7 days after ballooning neointimal thickening in the balloon treated group was significantly increased 1.8 fold (p < 0.05) from 5.5 ± 4.0% in the untreated group to 10.3 ± 4.8%. simultaneous intra/extravascular incubation with diltiazem for 1 and 2 days caused an inhibition of neointimal thickening by 59% (n.s.) and 21% (n.s.), corresponding to 4.2 ± 3.0% and 8.1 ± 8.8%. After simultaneous intra/extravascular administration of diltiazem for 3 days neointimal thickening was increased (n.s) by 24% (12.8 ± 11%). Incubation of POC-segments for 4, 5, 6, and 7 days resulted in a neointimal thickening of 9.1 ± 6.3%, 4.0 ± 5.0%, 4.0 ± 4.5%, and 3.7 ± 4.1%, corresponding to an inhibition (n.s.) by 12%, 61%, 61%, and 64%.
28 days after ballooning neointimal thickening of POC segments was significantly (p < 0.05) increased 2.6 fold from 10.5 ± 11.0% in untreated controls to 28.4 ± 17.8%. No effect was found after incubation with diltiazem for 1 day. Incubation with diltiazem for 2 and 3 days resulted in an inhibition of neointimal thickening by 25% (n.s.) and 86% (p < 0.05), corresponding to 21.2 ± 10.0% and 4.1 ± 5.9%. After Incubation of POC-segments for 4, 5, 6, and 7 days with diltiazem neointimal thickening was inhibited by 73%, 53%, 82% (p < 0.05) and 76% (p < 0.05), corresponding to 7.7 ± 8.8%, 13.3 ± 8.0%, 5.1 ± 10.2%, 6.8 ± 12.1%.
Identification of smooth muscle and endothelial cells
In the media of POC segments staining with antibodies against smooth muscle α-actin was always positive, no matter whether or not organ cultures were treated with ballooning or diltiazem or both. The number of smooth muscle α-actin positive cells in the media decreased with increased time in culture, only few POC-segements stained positive after 28 days in culture. In the neointima of POC segments staining against smooth muscle α-actin was negative after ballooning and negative after ballooning and EP diltiazem incubation.
Staining of POC-segments with antibodies against vWF was always positive in untreated controls, but decreased after ballooning, simultaneous intra/extravascular incubation with high dose diltiazem did not further decrease the number of cells with positive staining against vWF.