Phase I and II biotransformations in living CaCo 2 cells cultivated under serum-free conditions. Selective apical excretion of reaction products.

CaCo 2 cells, cultivated in a synthetic, serum-free nutritive medium on poly (ethylene terephthalate) membranes, form a confluent monolayer of differentiated cells, with the apical and basolateral poles exposed to the upper and lower compartments, respectively, of bicameral culture inserts (Halleux and Schneider, In Vitro Cell Dev Biol, 27A: 293-302, 1991). This cell culture system allows the passage of intact mannitol by the paracellular route and the transcellular diffusion of testosterone which appears mainly as a biotransformed unconjugated metabolite. When ethoxyresorufin is added to either the apical or basolateral poles of living CaCo 2 cells, resorufin is formed, and more than 80% is excreted at the apical pole. Under our experimental conditions, no detectable amounts of glucurono- or sulfconjugates are found. Methylcholanthrene and phenobarbital increase the biotransformation of ethoxyresorufin 50 and 3 times, respectively, and induce that of benzoxyresorufin, but not of pentoxyresorufin which remains absent under all conditions. These substances do not affect the proportion of resorufin recovered at the apical role. Verapamil inhibits by 25% the release of resorufin but does not affect its distribution. Chlorodinitrobenzene is conjugated with glutathione and at least two-thirds of the product is excreted at the apical pole; methylcholanthrene and phenobarbital do not increase this activity. These results demonstrate that differentiated CaCo 2 cells, under serum-free conditions, perform phase I and II reactions and that the biotransformation products are selectively excreted at the apical pole

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

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

Smooth muscle cells influence monocyte response to LDL as well as their adhesion and transmigration in a coculture model of the arterial wall.

We investigated the possible interference of smooth muscle cells with monocyte response to LDL as well as with their adhesion and transmigration in a coculture of porcine endothelial and smooth muscle cells. Lysophosphatidylcholine (LPC), a component of oxidized LDL (oxLDL), stimulated the adhesion of THP-1 cells to endothelial cells both in mono- and in coculture with smooth muscle cells. When THP-1 cells were incubated with endothelial cells in the presence of copper oxLDL, their adhesion was increased, but only in coculture. The addition of sodium nitroprusside (SNP) together with oxLDL markedly increased the adhesion of THP-1 cells in coculture. Close proximity between endothelial and smooth muscle cells was necessary to observe that effect. Furthermore, this increase in adhesion of THP-1 cells can, at least in part, be attributed to the augmented production of monocyte chemoattractant protein-1 (MCP-1) observed in coculture under the influence of oxLDL and SNP. The passage of THP-1 cells through the coculture was stimulated by MCP-1 and LPC. These results show that physical contacts or close proximity between endothelial and smooth muscle cells play a key role in the adhesion of monocytes and their infiltration into the intima in response to oxLDL