Compartmentalized coculture of porcine arterial endothelial and smooth muscle cells on a microporous membrane

Abstract

Endothelial and smooth muscle cells were harvested from porcine pulmonary arteries and grown to two passages from primary culture in serum-containing medium. Thereafter, the cells were plated on the opposite sides of microporous poly-(ethylene terephthalate) membrane and cultivated in a chemically defined, serum-free medium. The membrane with pores of 1 microgram diameter allowed the passage of molecules and the extension of cell processes, while maintaining separate homogeneous cell populations. Pores of 3 microgram diameter permitted the crossing of smooth muscle cells through the membrane. The coating of the polymer with constituents of the extracellular matrix optimized cell adhesion. Morphological analysis of the model showed typical cobblestone pattern and ultrastructure of endothelial cells, which lost rapidly the expression of von Willebrand factor but kept that of angiotensin-converting enzyme. Smooth muscle cells were spindle shaped and specific alpha-actin was revealed by immunochemistry and quantitated by enzyme-linked immunosorbent assay (ELISA). Their ultrastructure featured an intermediate contractile-synthetic phenotype. Permeability studies to different molecules showed a marked reduction of the albumin clearance. Finally, in coculture in the presence of endothelial cells, the smooth muscle cells proliferation was increased, whereas it was not the case in autologous cocultures. In conclusion, such a coculture model may help to a better understanding of the interactions between endothelial and smooth muscle cells that may be important in the pathogenesis of vascular diseases.

Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation

“…Although paracrine signaling and direct cell-cell contact between arterial ECs and SMCs have been shown in numerous studies to regulate the phenotype of SMCs (4,10,15,18,19,26,27,31,32,34), the signals exchanged during the direct cell-cell contact have not been fully defined. Using the EC/ SMC coculture system established by Isakson communication between the cocultured cells was blocked by pharmacological inhibitors or genetic ablation of the gap junctional protein Cx-43, the differentiation of PASMCs was prevented.…”

Myoendothelial gap junctional signaling induces differentiation of pulmonary arterial smooth muscle cells

“…THE COORDINATED INTERACTIONS between systemic arterial endothelial cells (ECs) and smooth muscle cells (SMCs) are highlighted by many in vivo and in vitro studies, where changes in EC function or activity are associated with phenotypic changes in SMCs, or vice versa (10,15,21,24,31,32). The reciprocal changes in ECs and SMCs have been attributed primarily to paracrine signaling (5,11,23), but direct cell-cell interaction through myoendothelial gap junctional signaling is also important (8,26,35,36,48).…”

A Genetically Modified Adenoviral Vector Exhibits Enhanced Gene Transfer of Human Smooth Muscle Cells

“…Primary rabbit SMC were isolated in the same fashion as described above for the rat. Primary porcine SMC were isolated from the abdominal aorta of large white pigs approximately 3–6 months of age as described by Kinard et al [24]. Briefly, porcine aortas were incubated with increasing concentrations of collagenase as described [24]until SMC were obtained.…”

“…Primary porcine SMC were isolated from the abdominal aorta of large white pigs approximately 3–6 months of age as described by Kinard et al [24]. Briefly, porcine aortas were incubated with increasing concentrations of collagenase as described [24]until SMC were obtained. The isolated porcine SMC were cultured in DMEM and 15% heat-inactivated fetal calf serum (HI-FBS) and confirmed by α-actin immunohistochemistry to be vascular SMC using the FITC-conjugated monoclonal anti-α-smooth muscle actin monoclonal (Sigma Chemical Co., St. Louis, Mo., USA).…”