The effects of 4-methylumbelliferone on hyaluronan synthesis, MMP2 activity, proliferation, and motility of human aortic smooth muscle cells

Abstract

Extracellular matrix remodeling after proatherosclerotic injury involves an increase in hyaluronan (HA) that is coupled with vascular smooth muscle cell (SMC) migration, proliferation, and with neointima formation. As such events are dependent on HA, in this study we assessed the effects on SMC behavior of 4-methylumbelliferone (4-MU). As previously described in other cell types, 4-MU reduced HA in cultures of primary human aortic SMCs (AoSMCs) as well as the cellular content of the HA precursor UDP-glucuronic acid. We found that SMCs increased UDP-glucuronyl transferase 1 enzymes, which can reduce the cellular content of UDP-glucuronic acid confirming that the availability of the UDP-sugar substrates can regulate HA synthesis. Interestingly, we reported that 4-MU reduced the transcripts coding for the three HA synthases as well as UDP glucose pyrophosphorylase and dehydrogenase. As HA synthase transcript reduction is common to other cell types, the 4-MU effect on gene expression may be considered a mechanism for HA synthesis inhibition. Moreover, we showed that 4-MU strongly inhibits AoSMCs migration, which was restored by the addition of exogenous HA indicating that the rescuing depends on the interaction of HA with its receptor CD44. Besides the decrease in HA synthesis and cell migration, 4-MU reduced AoSMCs proliferation, indicating that 4-MU may exert a vasoprotective effect.

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis

“…Migrated cells were quantified after 6 h as described previously (29). Monocyte adhesive properties of AoSMCs in different conditions were tested with U937 monocyte adhesion assays.…”

“…Monocyte adhesive properties of AoSMCs in different conditions were tested with U937 monocyte adhesion assays. This experiment was done essentially as described previously (29) with the exception that the binding phase of monocytes with AoSMCs was done at 37°C for 1 h. To quantify the pericellular HA coat, particle exclusion assays using paraformaldehyde-fixed human red blood cells were done on untreated and treated AoSMCs as described previously (15).…”

Oxidized Low Density Lipoprotein (LDL) Affects Hyaluronan Synthesis in Human Aortic Smooth Muscle Cells

“…Cell Culture-Human AoSMC were purchased from Lonza and grown in the complete SmGm2 culture medium (Lonza, Basel, Switzerland) supplemented with 5% FBS as described previously (25). 1.5 ϫ 10 5 cells were seeded in a 6-well plate at 70 -80% confluence.…”

“…25 with quantification by Image J software as follows. Wound area was measured at time 0 (t 0 ) and after 24 h (t 24 ), and the wound closure was determined by the equation: ((wound area t 0 Ϫ wound area t 24 )/wound area t 0 )ϫ100.…”

“…Wound area was measured at time 0 (t 0 ) and after 24 h (t 24 ), and the wound closure was determined by the equation: ((wound area t 0 Ϫ wound area t 24 )/wound area t 0 )ϫ100. AoSMC cultures were treated with 4-methylumbelliferone in DMSO at a final concentration of 1 mM (25).…”