Study of the effects of hyaluronan biosynthesis Inhibition by 4-methylumbelliferone on human Aortic smooth muscle cells.

After atherogenic injury aortic smooth muscle cells (AoSMCs) which are normally located within the tunica media of blood vessels begin to synthesize high levels of extracellular matrix (ECM) components such as hyaluronan (HA), that is involved in the modulation of AoSMCs behaviour. During aging the onset of cardiovascular diseases increases dramatically and, in a cell aging in vitro model we found that in aged AoSMC HA synthesis is upregulated, enhancing their migration. Since HA strongly affects neointima formation we investigated the effect of HA synthesis inhibition by 4-methylumbelliferone (4MU), a natural derivative of coumarin, known to inhibit HA synthesis in some cell lines. AoSMC treated with 4MU showed a dose dependent reduction of HA synthesis that could be related both to a reduced genetic expression of the key enzymes involved in its biosynthesis and to a reduction of the cellular pool of UDP-glucuronic acid, caused by the glucuronidation of this chemical by UGTs, that we found to be constitutively expressed and active in AoSMC. Gene expression analysis by Real-Time RT-PCR revealed that 4MU treatment dramatically reduces HAS2 expression: to confirm the key role of this enzyme in HA biosynthesis we abrogated its expression by means of siRNA, obtaining comparable results. As cellular migration is a key event in neointima invasion, we investigated 4MU effects on both cell migration and gelatinolytic activity. MMP2 is a MMP involved in matrix remodeling, playing a key role in AoSMC migration: 4MU does not affect MMP2 activity in AoSMC, moreover it determined a strong inhibition of cellular migration. As the addition of exogenous HA restored the migratory potential of this cells, we demonstrated, by means of a blocking antibody, the pivotal role of CD44 in this process. To investigate which cellular pathways were altered by 4MU treatment we performed a whole genome expression study using microarrays from Celera-Applied Biosystems and we found that 4MU treatment induced an alteration of p53 and cell cycle pathways. Moreover we found that 4MU treatment induced a reduction of cell proliferation and an increase of cell mortality by apoptosis, as shown by annexin-V staining, FACS analysis and PARP western blot. In order to verify the role of 4MU in the apoptotic process we performed rescue experiments by adding exogenous HMW-HA during treatment. Interestingly we found that 4MU induced apoptosis can be rescued by addition of exogenous HMW-HA to cell culture. Growing evidence of HA involvement in various human pathologies raises the question how to modulate its synthesis in tissues: these data introduce the possibility to reach this objective using a molecule already available in human therapy like 4MU.