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Leukocyte attack in a 3D human coronary in-vitro model
1Department of Internal Medicine II--Cardiology, University of Ulm, Germany. rainer.voisard@medizin.uni-ulm.de
Insights
Monocytes, not CD4+ lymphocytes, drive early atherosclerosis by adhering to and penetrating coronary smooth muscle cells. This model aids in studying treatments for cardiovascular diseases.
Area of Science:
- Cardiovascular Biology
- Immunology
- Cell Biology
Background:
- Peripheral blood leukocytes play a crucial role in atherosclerosis and restenosis.
- Understanding leukocyte interactions with coronary cells is vital for cardiovascular health.
Purpose of the Study:
- To investigate the distinct roles of monocytes and CD4+ lymphocytes in affecting human medial coronary smooth muscle cells (HCMSMC).
- To evaluate the utility of three-dimensional human coronary in-vitro units of leukocyte attack (3DLA-units) for studying these interactions.
Main Methods:
- 3DLA-units were constructed with polycarbonate membranes, human coronary endothelial cells (HCAEC), and HCMSMC.
- Leukocyte attack was simulated using monocytes (MC) and CD4+ lymphocytes (CD4+-LC), with and without TNF-alpha stimulation.
- HCMSMC proliferation was assessed via bromodeoxyuridine (BrdU) uptake.
Main Results:
- Monocytes adhered to HCAEC, traversed membranes, and reached HCMSMC.
- CD4+-lymphocytes primarily adhered to HCAEC without significant transmigration.
- Monocyte attack significantly increased HCMSMC proliferation (2.9-fold), further enhanced with TNF-alpha (3.5-fold).
- CD4+-lymphocyte attack showed minimal effect unless stimulated with TNF-alpha (2.1-fold increase).
Conclusions:
- Monocytes are more influential than CD4+ lymphocytes in early leukocyte-mediated HCMSMC adhesion, chemotaxis, and proliferation within 24 hours.
- 3DLA-units provide an effective in-vitro model for direct assessment of leukocyte-endothelial-smooth muscle cell interactions and potential therapeutic 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.