Lipopolysaccharide (LPS) exposure alters the phenotype of cultured arterial smooth muscle cells

Abstract

Experiments were designed to determine if short-term exposure to LPS affected transdifferentiation of arterial smooth muscle cells to express bone-related proteins. Smooth muscle cells were grown to 90% confluency (passage 4 to 5), in M199 medium (control solution). Either LPS (100pg/ml, low; 100ng/ml, mid; 10μg/ml, high) or equal volume of vehicle was added to the media for 3 days at which time the media was exchanged for control solution. Cells were collected at 3, 6, and 12 days after exposure to LPS or vehicle. Expression of smooth muscle phenotypic proteins (desmin, vimentin, αSM-actin) and bone-associated phenotypic proteins (matrix gla protein, MGP; osteoprotegrin, OPG, and bone sialoprotein, BSP) were determined by Western blot. Intracellular calcium was measured by colorimetric method and alkaline phosphatase activity was measured by pNPP phosphatase assay. Exposure to mid-concentration of LPS for 3 days transiently increased then decreased only desmin expression. LPS at mid- and high concentrations significantly increased expression of MGP, BSP and OPG, an increase which was sustained for 9 days after removal of LPS. Intracellular calcium concentration showed a time dependent decrease in all groups. Exposure to LPS augmented the decrease 3 days after removal of LPS. Alkaline phosphatase activity decreased at 6 days in the mid and high LPS groups and in all groups at day 12. These results indicate that short-term exposure (3 days) of smooth muscle cells to LPS specifically modulates a SMC-phenotypic protein and dose-dependently facilitates expression of bone-associated proteins. These findings suggest that infection may contribute to arterial calcification by facilitating transdifferentiation of smooth muscle cells.