F. Galambo1, H. Bass1,2, R. Beard2, B. J. Cha2, P. R. Nelson1,2 2University Of South Florida College Of Medicine,Department Of Molecular Pharmacology And Physiology,Tampa, FL, USA 1University Of South Florida College Of Medicine,Division Of Vascular And Endovascular Surgery/Department Of Surgery/Morsani College Of Medicine,Tampa, FL, USA
Introduction: Vascular injury disrupts normal vessel wall architecture causing de-endothelialization, and SMC dedifferentiation. Unregulated SMC migration leads to neointimal hyperplasia and eventually restenosis. We sought to develop an in vitro co-culture model to study SMC physiology under conditions of injury. We hypothesized that disrupting quiescent co-cultures would lead to stimulation of SMC chemotaxis.
Methods: SMC/EC co-cultures were established by growing human aortic EC to confluence onto the bottom surface of 0.45µm porous polycarbonate membranes in 6-well Transwell® inserts. Human aortic SMC were then grown to confluence on the top/inner side of the membranes and the co-cultures were then incubated for 48-72 hours to reach equilibrium. The porous membrane allows both chemical and physical communication between the cells, but maintains the layered architecture found in the vessel wall in vivo. To confirm this, the model was characterized using multiphoton fluorescent microscopy. Migrating SMCs were grown to confluence, serum starved for 48 to 72 hours, and seeded onto a second 24-well Transwell® insert with 8.0 µm pores. These inserts were then suspended into SMC and EC monocultures, as well as our co-culture model, both uninjured and injured. Injury of co-culture was created using a cell scraper. Migration was measured using a 4 hour modified Boyden chemotaxis assay. Comparisons were performed using a Student’s T-test.
Results: Using a Z-stacked technique, 3-Dimensional renderings and transverse sections of co-culture membranes demonstrated not only the establishment of healthy co-culture, but also the presence of cell-cell contact through the pores. Under serum-free stimulant-free conditions, SMC demonstrated a low baseline level of migration. SMC exposed to either EC or SMC monoculture alone demonstrated significantly increased migration (P< .0001). SMC exposed to uninjured SMC/EC co-culture demonstrated migration that returned to unstimulated control levels (* P< .0001). SMC exposed to injured SMC/EC co-culture exhibited significantly increased migration levels compared to uninjured conditions (** P< .0001). Migrations results are summarized in Figure 1.
Conclusion: Confocal microscopy demonstrated the viability and utility of our co-culture model in studying vascular injury physiology in vitro. SMC and EC grown in co-culture induce a quiescence compared to either cell type alone, and as such had no influence on SMC chemotaxis. In contrast, disrupting this quiescence, by injuring the co-culture lead to a significant stimulation of SMC migration. This model holds promise to more accurately study the mechanism of restenosis.
