Aortic Endothelial and Smooth Muscle Cell Co-Culture: An In Vitro Model of the Arterial Wall

Abstract

Interactions between vascular endothelial (EC) and smooth muscle cells (SMC) contribute both to the normal function of the vascular wall and to the pathogenesis of lesions such as atherosclerosis and fibrointimal hyperplasia. However, study of these interactions has been hampered by the difficulty in growing these two cell types in simultaneous culture. Methods using conditioned media, shared media, and bilayer culture have been described, but none is well suited to the study of vascular cell interactions. We report a method for EC-SMC co-culture that preserves bilayer morphology, allows independent study of the cells and their matrices after intervention, remains stable over long periods in culture, and permits study of changes in cell-cell interaction with growth of the cells to confluence. This simple bilayer co-culture system simulates the in vivo situation and may enhance our understanding of EC-SMC interactions.

Paracrine effect of human vascular endothelial cells on human vascular smooth muscle cell proliferation: Transmembrane co-culture method

“…This coculture method therefore may be more advanced than those employed in previous studies, in which conditioned media were used or co-culture was done on a single plate. Previous results describing the effect of endothelial cells on VSMC proliferation when endothelial conditioned media or other co-culture methods were used have been inconsistent [13][14][15][16][19][20][21][22]. The present study has demonstrated that cultured endothelial cells subseeded at a low cellular density exert a potent stimulatory effect on VSMC proliferation.…”

Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine TGF-β Signaling Pathway

“…13,[22][23][24] This regulation is growth state dependent, with subconfluent cultures of endothelial cells stimulating VSMC growth and postconfluent cultures inhibiting VSMC growth. 12,[25][26][27][28] Similarly, perlecan and endothelial-derived HSPGs have been shown to be essential in inhibiting the neointimal response to vascular injury. 14,29 -31 Our study adds a new dimension to these results, demonstrating that the regulation of perlecan by mechanical strain is an important mechanism in altering endothelial paracrine inhibition of VSMC proliferation in response to changes in mechanical environment.…”

Coculture of Endothelial Cells and Smooth Muscle Cells in Bilayer and Conditioned Media Models

“…Chamley-Campbell and Campbell have shown that EC-conditioned media can alter SMC phenotype toward a more contractile, less synthetic phenotype [7,8]. Additional investigators have demonstrated stimulation of SMC growth by ECconditioned media [16][17][18], while others have indicated that EC-conditioned media or ECs in coculture can stimulate or inhibit SMC proliferation at later time points [7,19,20,27,28]. With regard to the effect of ECs on SMCs in vivo, animal models have demonstrated that hyperplasia can occur under an intact endothelium [29] and neointimal hyperplasia within prosthetic grafts appears to occur only if there is an endothelial lining above the SMCs within the neointima [30].…”

“…During experinoted in systems where SMCs are exposed only to EC-ments, cells were kept in DMEM with 2.5% calf serum and 1% Lconditioned media, without physical EC/SMC contact glutamine. This medium produces approximately 50% of maximal [7,[16][17][18][19][20] [15,23,24]. The rate of [ 3 H]thymidine incorporation in each sample was reported as scintillation cpm/ng these ECs reached confluence (2 -3 days), SMCs were plated at 1 1 10 5 cells per well on the inner surface of the membrane opposite DNA.…”

“…model is useful for investigating the effects of physical Thus, in order for SMC projections in the bilayer model EC-SMC contact. Several EC/SMC coculture models to cross the membrane, they have to traverse extracelhave been developed [16,20,25,[32][33][34][35], and many lular matrix previously laid down by the ECs. In order investigators believe that the physical arrangement of to examine the effect of the ECs on SMC projections, ECs and SMCs in a bilayer coculture model is superior it is more appropriate to compare SMCs in the condito a conditioned media model, despite a dearth of quantioned media and SMC only groups.…”