Porcine Endothelial Cells Cocultured with Smooth Muscle Cells Became ProcoagulantIn Vitro

Abstract

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC=smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Endothelial vascular smooth muscle cell coculture assay for high throughput screening assays to identify antiangiogenic and other therapeutic molecules

“…By quantifying fluorescence intensity and including suitable controls, the following can be assessed: surface expression of adhesion molecules following a stimulus such as a cytokine or other inflammatory agent 38, 84; NO release; redox reactions 85, and reporter gene expression.…”

Hydraulic Conductivity of Endothelial Cell-Initiated Arterial Cocultures

“…Those findings illustrated that serum supplemented and time-dependent arterial coculturing methods stimulate heterotypic activities. Also, in directly cocultured porcine arterial ECs and SMCs (without an intervening porous membrane), proximity, serum content, and culture time were all demonstrated to be precursors in the chain of events regulating indicators of thrombogenicity 33 .…”

In vitro 3D angiogenesis assay in egg white matrix: comparison to Matrigel, compatibility to various species, and suitability for drug testing

“…In the case of endothelial and mural cell coculture, vascular smooth muscle cells (VSMCs) secrete basement membrane proteins that enable adhesion of endothelial cells (ECs). [1][2][3][4] In addition, proangiogenic factors released by VSMCs promote development and stabilization of capillaries. VSMCs and ECs grow in a three-dimensional (3D) multilayered structure similar to vascular wall structure.…”