ABSTRACT
Hyaluronan and versican-rich pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent pericellular matrices, and on the synthesis and interaction of several major pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that pericellular matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular matrix expansion and alterations in ASMC phenotype.
Subject(s)
Chondroitin Sulfate Proteoglycans/metabolism , Extracellular Matrix/drug effects , Glycosyltransferases , Hyaluronic Acid/metabolism , Membrane Proteins , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Platelet-Derived Growth Factor/pharmacology , Transferases , Xenopus Proteins , Aorta , Cell Adhesion/drug effects , Cell Size/drug effects , Cells, Cultured , Chondroitin Sulfate Proteoglycans/biosynthesis , Extracellular Matrix/metabolism , Gene Expression Regulation/drug effects , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Humans , Hyaluronan Synthases , Hyaluronic Acid/biosynthesis , Infant, Newborn , Lectins, C-Type , Muscle, Smooth, Vascular/metabolism , Protein Binding/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , VersicansABSTRACT
The separation of intact proteoglycans using high-performance liquid chromatography is not trivial because the high molarity denaturing buffers required to maintain proteoglycans in the disaggregated state create back-pressures higher than the limits of many HPLC systems. Until recently, low back-pressure requirements of HPLC size-exclusion columns precluded their use for the separation of intact proteoglycans. In this study we show that rapid size-exclusion chromatography is possible in 8 M urea buffers using a Dionex BioLC system equipped with a Bio-Rad BioSil Sec-400 column. This technique reduced the time required for size-exclusion chromatography of intact proteoglycans from approximately 18 h (Sepharose CL4B) to 25 min and in some cases improved resolution of the sample.
