BACKGROUND: Restenosis is a reparative process that is activated in response to injury induced by angioplasty. Despite numerous experimental models of restenosis the number of human arterial organ culture systems is very limited and long-term experiences do not exist. METHODS AND RESULTS: During routine nephrectomies parts of the renal arteries of 88 patients were extracted, 47 were suitable for organ culture preparations. Sections were made at 3 mm intervals perpendicular to the vessel wall axis. The arterial segments were treated with 3 mm standard balloon-catheters (Medtronic 14K2030E) for 60 s with 3, 6, 9, and 12 bar. After angioplasty, the organ segments were cultured in a mixture of Waymouth's MB 752/1 and Ham F-12, supplemented with 15% fetal calf serum. After 0, 4, 14, 21, 28, and 56 days the organ cultures were fixed in 4% para-formaldehyde and embedded in paraffin. After staining with a modified elastica-van Gieson technique the intimal wall thickening was analyzed with a computerized morphometric system. For the identification of smooth muscle cells (SMC) a monoclonal antibody against smooth muscle alpha-actin was used. Endothelial cells were identified using an anti-human von Willebrand factor. To determine the number of cells undergoing DNA synthesis, bromodeoxyuridine (BrdU), a thymidine analogue, was added to the culture media 18 h prior to fixation. BrdU was detected with a monoclonal antibody, as secondary antibody a biotinylated horse-anti-mouse antibody was used. After 14, 21, and 28 days in culture BrdU-positive cells were detected in the neointima of the organ cultures, indicating mitotic activity in this area. After 28 and 56 days in culture a clear increase of neointimal thickening was found in the morphometric analysis. By positive reaction with antibodies against smooth muscle alpha-actin these cells were partly identified as SMC. CONCLUSIONS: The organ culture model offers opportunities for in vitro investigations of postangioplasty restenosis. The data emphasize the importance of a relatively late proliferative response of SMC in the human arterial organ culture model.
BACKGROUND:Restenosis is a reparative process that is activated in response to injury induced by angioplasty. Despite numerous experimental models of restenosis the number of human arterial organ culture systems is very limited and long-term experiences do not exist. METHODS AND RESULTS: During routine nephrectomies parts of the renal arteries of 88 patients were extracted, 47 were suitable for organ culture preparations. Sections were made at 3 mm intervals perpendicular to the vessel wall axis. The arterial segments were treated with 3 mm standard balloon-catheters (Medtronic 14K2030E) for 60 s with 3, 6, 9, and 12 bar. After angioplasty, the organ segments were cultured in a mixture of Waymouth's MB 752/1 and Ham F-12, supplemented with 15% fetal calf serum. After 0, 4, 14, 21, 28, and 56 days the organ cultures were fixed in 4% para-formaldehyde and embedded in paraffin. After staining with a modified elastica-van Gieson technique the intimal wall thickening was analyzed with a computerized morphometric system. For the identification of smooth muscle cells (SMC) a monoclonal antibody against smooth muscle alpha-actin was used. Endothelial cells were identified using an anti-humanvon Willebrand factor. To determine the number of cells undergoing DNA synthesis, bromodeoxyuridine (BrdU), a thymidine analogue, was added to the culture media 18 h prior to fixation. BrdU was detected with a monoclonal antibody, as secondary antibody a biotinylated horse-anti-mouse antibody was used. After 14, 21, and 28 days in culture BrdU-positive cells were detected in the neointima of the organ cultures, indicating mitotic activity in this area. After 28 and 56 days in culture a clear increase of neointimal thickening was found in the morphometric analysis. By positive reaction with antibodies against smooth muscle alpha-actin these cells were partly identified as SMC. CONCLUSIONS: The organ culture model offers opportunities for in vitro investigations of postangioplasty restenosis. The data emphasize the importance of a relatively late proliferative response of SMC in the human arterial organ culture model.
Authors: Anna Lebedeva; Daria Vorobyeva; Murad Vagida; Oxana Ivanova; Eugeny Felker; Wendy Fitzgerald; Natalya Danilova; Vladimir Gontarenko; Alexander Shpektor; Elena Vasilieva; Leonid Margolis Journal: Atherosclerosis Date: 2017-10-07 Impact factor: 5.162