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Background: Restenosis after coronary angioplasty is considered to be caused mainly by increased migration and proliferation of smooth muscle cells (SMC). The concept of local, site-specific delivery of pharmacologic therapies has opened the door for new, high-dose drug regimes., Methods and Results: SMC were isolated by enzymatic disaggregation with collagenase/elastase from human coronary plaque tissue of 29 patients (pSMC) and post mortem from the coronary media of 33 corpses (mSMC). Endothelial cells were isolated from human umbilical veins by enzymatic disaggregation with collagenase/dispase. By positive reaction with antibodies against smooth muscle alpha-actin and von Willebrand factor cells were identified as SMC or endothelial cells. In proliferation studies 5-150 micrograms/ml diltiazem was added to the culture media of pSMC, mSMC and endothelial cells. After 5 days there was a significant dose-dependent inhibition of cell proliferation (for pSMC with > 50 micrograms/ml, for mSMC with > 25 micrograms/ml, and for endothelial cells with > 5 micrograms/ml). In migration studies the effect of 5-150 micrograms/ml diltiazem on the velocity of migration of pSMC was investigated over a period of 48 h. Administration of diltiazem at concentrations of 100 and 150 micrograms/ml caused a significant inhibition of the migration of pSMC. The cytoskeletal components smooth muscle alpha-actin, vimentin, and alpha-tubulin of pSMC and the expression of von Willebrand factor of endothelial cells were investigated after an incubation period of 5 days with 50 and 150 micrograms/ml diltiazem. In the transfilter coculture model the effect of 50 micrograms/ml diltiazem on mSMC was investigated after mechanical injury of cocultured endothelial cells. Administration of diltiazem at a concentration of 50 micrograms/ml inhibited the development of a neointimal proliferate in the transfilter coculture model significantly (P < 0.001)., Conclusions: A high dose of diltiazem inhibited the migratory and proliferative activities of coronary SMC significantly. In further experimental studies the effect of locally applied high doses of diltiazem on postangioplasty restenosis should be elucidated.

Background: Restenosis after angioplasty is generally considered to be caused mainly by an increased proliferation of smooth muscle cells, but recently this theory has been questioned., Methods: Fresh hearts of 25 pigs were obtained and the left anterior descending coronary arteries carefully prepared, cut into segments and treated with a 3 mm standard balloon catheter for 60 s with 3, 6, 9 and 12 bar. Immediately after angioplasty the specimens were cultured in a mixture of WM/F-12, supplemented with 15% fetal calf serum., Results: After staining with a modified Verhoeff-van Gieson technique, intimal wall thickening was analysed with a computerized morphometric system. After 14 days in culture, angioplasty with 6, 9 and 12 bar resulted in a significant (P < 0.05) increase of neointimal thickening. After 21 days, a significant increase of neointimal thickening was seen after ballooning with 3, 9 or 12 bar (P < 0.05). After 28 days angioplasty with 3 or 6 bar showed a dose-dependent increase of neointima, which was not further increased after ballooning with 9 or 12 bar. Significance was reached after ballooning with 6, 9 and 12 bar (P < 0.05). Smooth muscle cells were identified with a monoclonal antibody against smooth muscle alpha-actin. Endothelial cells were identified using an anti-human von Willebrand factor. To determine the number of cells undergoing DNA synthesis, bromodeoxyuridine, a thymidine analogue, was added to the culture media 18 h before fixation. It was detected with a monoclonal antibody, with a biotinylated horse-anti-mouse antibody as secondary antibody. After 4 and 7 days in culture bromodeoxyuridine-positive cells were observed, indicating an early proliferative response after angioplasty., Conclusions: The coronary organ culture model offers opportunities for investigations of complex cellular events that are considered important in the development of restenosis. The data reported emphasize the importance of an early proliferative response of smooth muscle cells after coronary angioplasty, resulting in a significant neointimal thickening.

Background: Calcification influences the outcome of various angioplasty techniques in the treatment of coronary artery disease. During angioscopic in vitro studies, we observed that dissections and perforations not caused by vessel bending frequently occurred at the boundary areas of plaque and adjacent vessel wall. This study investigated whether this is related to the distribution of calcific deposits., Methods: Postmortem excimer laser coronary angioplasty (308-nm XeCl) was performed in 51 stenotic coronary arteries. Twenty-three segments were further examined; these consisted of 11 perforations, six dissections, three segments with no ablative effect after the application of 20,000 laser impulses, and three successfully passed stenoses without complications. X-ray diffraction analysis and scanning electron microscopy were performed to detect calcium deposits and their spatial relationship to perforations and dissections., Results: X-ray diffractions analysis detected calcifications in 21 of 23 specimens. Postmortem angiography revealed calcifications only on 11 of 23 segments. Three of 11 perforations were located at the plaque border, as were three of six dissections. In all six complications at the plaque border, x-ray diffraction analysis revealed that the plaque border was identical with a border of calcium deposits. Eight of 11 perforations and three of six dissections could be explained by axis divergence between the laser catheter and the vessel orientation., Conclusions: Contributing factors for perforations and dissections during excimer laser coronary angioplasty are axis divergence and the distribution of plaque calcification. More sensitive methods are needed to detect local vessel wall calcium in vivo.