OBJECTIVE: The importance of peripheral blood leukocytes for the development of early atherosclerosis and restenosis has confronted cardiologists with classical hematologic issues. Three-dimensional human coronary in-vitro units of leukocyte attack (3DLA-units) open the field for exact studies of leukocyte attack and its subsequent effects on human medial coronary smooth muscle cells (HCMSMC). METHODS: Central part of 3DLA-units are polycarbonate membranes with a pore size of 5 microm that correspond to the internal elastic membrane. Human coronary endothelial cells (HCAEC) were cultured on one side of the membranes, HCMSMC on the other side. Before leukocyte attack expression of adhesion molecules was up-regulated by tumour necrosis factor-alpha (TNF-alpha). Leukocyte attack was mimicked by selective adding of human monocytes (MC), respectively human CD4+-lymphocytes (CD4+-LC) to the HCAEC side of the 3DLA-units. Three-dimensional leukocyte attack units were fixed and stained after a period of 30 min, 1, 2, 3, 4, 6, and 24 h. Cell divisions of HCMSMC were analysed by measuring the uptake of bromodeoxyuridine (BrdU). RESULTS: Monocytes were able to adhere to the endothelial surface, pass through the filter-pores, and penetrate the HCMSMC side of the 3DLA-units. Human CD4+-lymphocytes (CD4+-LC) only attached to the HCAEC side, and no chemotaxis to the HCMSMC side was detected. Proliferation of HCMSMC was increased 2.9-fold (P< 0.001) after selective MC-attack and 3.5-fold after selective MC-attack and TNF-alpha stimulus. No significant increase was found after selective CD4+-LC attack, a significant increase (2.1-fold; P < 0.001) was seen after selective CD4+-LC attack and TNF-alpha, stimulus. CONCLUSIONS: Within the given limitations of the model the study emphasizes a predominance of MC in comparison to CD4+-LC in the process of adhesion, chemotaxis, and triggered reactive proliferation of co-cultured HCMSMC within the first 24 h after leukocyte attack. 3DLA-units offer an elegant method to study directly the effects of intravascular and intramural treatment strategies.
OBJECTIVE: The importance of peripheral blood leukocytes for the development of early atherosclerosis and restenosis has confronted cardiologists with classical hematologic issues. Three-dimensional human coronary in-vitro units of leukocyte attack (3DLA-units) open the field for exact studies of leukocyte attack and its subsequent effects on human medial coronary smooth muscle cells (HCMSMC). METHODS: Central part of 3DLA-units are polycarbonate membranes with a pore size of 5 microm that correspond to the internal elastic membrane. Human coronary endothelial cells (HCAEC) were cultured on one side of the membranes, HCMSMC on the other side. Before leukocyte attack expression of adhesion molecules was up-regulated by tumour necrosis factor-alpha (TNF-alpha). Leukocyte attack was mimicked by selective adding of human monocytes (MC), respectively human CD4+-lymphocytes (CD4+-LC) to the HCAEC side of the 3DLA-units. Three-dimensional leukocyte attack units were fixed and stained after a period of 30 min, 1, 2, 3, 4, 6, and 24 h. Cell divisions of HCMSMC were analysed by measuring the uptake of bromodeoxyuridine (BrdU). RESULTS: Monocytes were able to adhere to the endothelial surface, pass through the filter-pores, and penetrate the HCMSMC side of the 3DLA-units. Human CD4+-lymphocytes (CD4+-LC) only attached to the HCAEC side, and no chemotaxis to the HCMSMC side was detected. Proliferation of HCMSMC was increased 2.9-fold (P< 0.001) after selective MC-attack and 3.5-fold after selective MC-attack and TNF-alpha stimulus. No significant increase was found after selective CD4+-LC attack, a significant increase (2.1-fold; P < 0.001) was seen after selective CD4+-LC attack and TNF-alpha, stimulus. CONCLUSIONS: Within the given limitations of the model the study emphasizes a predominance of MC in comparison to CD4+-LC in the process of adhesion, chemotaxis, and triggered reactive proliferation of co-cultured HCMSMC within the first 24 h after leukocyte attack. 3DLA-units offer an elegant method to study directly the effects of intravascular and intramural treatment strategies.
Authors: Carlos Casas-Arozamena; Alberto Otero-Cacho; Bastian Carnero; Cristina Almenglo; Maria Aymerich; Lorena Alonso-Alconada; Alba Ferreiros; Alicia Abalo; Carmen Bao-Varela; Maria Teresa Flores-Arias; Ezequiel Alvarez; Alberto P Munuzuri; Miguel Abal Journal: Sci Rep Date: 2021-12-01 Impact factor: 4.379