Chronic inflammation is now accepted to have a critical role in the onset of several diseases as well as in vascular pathology, where macrophage transformation into foam cells contributes in atherosclerotic plaque formation. Endothelial cells (EC) have a critical function in recruitment of immune cells, and proinflammatory cytokines drive the specific expression of several adhesion proteins. During inflammatory responses several cells produce hyaluronan matrices that promote monocyte/macrophage adhesion through interactions with the hyaluronan receptor CD44 present on inflammatory cell surfaces. In this study, we used human umbilical chord vein endothelial cells (HUVECs) as a model to study the mechanism that regulates hyaluronan synthesis after treatment with proinflammatory cytokines. We found that interleukin 1␤ and tumor necrosis factors ␣ and ␤, but not transforming growth factors ␣ and ␤, strongly induced HA synthesis by NF-B pathway. This signaling pathway mediated hyaluronan synthase 2 (HAS2) mRNA expression without altering other glycosaminoglycan metabolism. Moreover, we verified that U937 monocyte adhesion on stimulated HUVECs depends strongly on hyaluronan, and transfection with short interference RNA of HAS2 abrogates hyaluronan synthesis revealing the critical role of HAS2 in this process. Hyaluronan (HA)3 is a linear glycosaminoglycan consisting of a disaccharide (glcUA-␤1,3-glcNAc-␤1,4) repeated several thousand times without any other chemical modifications (i.e. sulfation and epimerization) that are typical of the other glycosaminoglycans (1). HA is a multifunctional molecule in the extracellular matrix. In addition to its viscoelastic properties that modulate tissue hydration, HA can interact with cell surface receptors, including CD44, receptor for HA-mediated motility (RHAMM), Lyve-1 (lymphatic vessel endothelial receptor 1), HARE (HA receptor for endocytosis), intercellular adhesion molecule-1 (ICAM-1), and Toll-like receptor 4 (TLR4), and HA can initiate several signal transduction pathways (1). Chain lengths can depend on the activity of different isoforms of HA synthases (HAS1, -2, and -3) (2), or from the activity of degrading enzymes (i.e. hyaluronidases) (1). Short HA fragments produced after injuries or inflammation can interact with TLR4 and stimulate synthesis of macrophage chemokines and cytokines (3).In vascular pathologies, HA accumulation can regulate the behavior of smooth muscle cells and contribute to vessel wall thickening by inducing cell migration and proliferation (4). Moreover, in the media and neointima, HA exerts a proatherosclerotic effect by promoting adhesion of immune cells and by recruiting monocytes/macrophages (5) that, through cholesterol rich lipoproteins endocytosis, contribute to progression of atherosclerotic plaque. The molecular mechanism involved in the interaction of immune cells with HA depends on CD44. Interestingly, the organization of HA in the extracellular matrix has a critical role in this process, and cells subjected to various stresses (endopl...

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan (GAG) involved in cell motility, proliferation, tissue remodeling, development, differentiation, inflammation, tumor progression, and invasion and controls vessel thickening in cardiovascular diseases. Therefore, the control of HA synthesis could permit the finetuning of cell behavior, but the mechanisms that regulate HA synthesis are largely unknown. Recent studies suggest that the availability of the nucleotide-sugar precursors has a critical role. Because the formation of UDP-sugars is a highly energetically demanding process, we have analyzed whether the energy status of the cell could control GAG production. AMP-activated protein kinase (AMPK) is the main ATP/AMP sensor of mammalian cells, and we mimicked an energy stress by treating human aortic smooth muscle cells (AoSMCs) with the AMPK activators 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside and metformin. Under these conditions, HA synthesis, but not that of the other GAGs, was greatly reduced. We confirmed the inhibitory effect of AMPK using a specific inhibitor and knock-out cell lines. We found that AMPK phosphorylated Thr-110 of human HAS2, which inhibits its enzymatic activity. In contrast, the other two HAS isoenzymes (HAS1 and HAS3) were not modified by the kinase. The reduction of HA decreased the ability of AoSMCs to proliferate, migrate, and recruit immune cells, thereby reducing the pro-atherosclerotic AoSMC phenotype. Interestingly, such effects were not recovered by treatment with exogenous HA, suggesting that AMPK can block the pro-atherosclerotic signals driven by HA by interaction with its receptors.

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.

With the goal of assessing the environmental risk of pharmaceuticals, we have previously observed that a mixture of 13 different drugs at environmentally relevant concentrations had adverse consequences on human and zebra fish cells in vitro. Here we aimed to identify both main and interaction effects within the same environmentally relevant mixture of pharmaceuticals. We studied in vitro cytotoxicity in Escherichia coli, human embryonic HEK293, and estrogen-responsive OVCAR3 tumor cells using fractional-factorial experimental design. Our approach identified a subset of compounds of primary environmental concern, namely atenolol, bezafibrate, ciprofloxacin, and lincomycin, that had statistically significant effects on prokaryotic and eukaryotic cells at environmentally relevant exposure levels (ng/l). Drugs could interact and behave as chemosensitizers, with joint effects representing a statistically significant element of mixture toxicity. Effects and interactions were concentration dependent, confirming the difficulty of dose extrapolation in mixture toxicity data. This study suggests that a thorough investigation of mixture effects can direct environmental concerns toward a handful of pharmaceuticals, which may represent an actual risk at environmental concentrations. We indicate that risk identification may strongly depend on the use of environmentally relevant exposure scenarios. Antagonistic-synergistic interactions and dose dependency of effects may hamper the modeling and prediction of mixture toxicity with pharmaceuticals. Hazard identification for micropollutants depends heavily on appropriate study designs, and we indicate the use of in vitro cytotoxicity threshold and statistical design of experiments (DOEs) as a valid approach.

The glycosaminoglycan hyaluronan (HA) modulates cell proliferation and migration, and it is involved in several human vascular pathologies including atherosclerosis and vascular restenosis. During intima layer thickening, HA increases dramatically in the neointima extracellular matrix. Aging is one of the major risk factors for the insurgence of vascular diseases, in which smooth muscle cells (SMCs) play a role by determining neointima formation through their migration and proliferation. Therefore, we established an in vitro aging model consisting of sequential passages of human aortic smooth muscle cells (AoSMCs). Comparing young and aged cells, we found that, during the aging process in vitro, HA synthesis significantly increases, as do HA synthetic enzymes (i.e. HAS2 and HAS3), the precursor synthetic enzyme (UDP-glucose dehydrogenase), and the HA receptor CD44. In aged cells, we also observed increased CD44 signaling that consisted of higher levels of phosphorylated MAP kinase ERK1/2. Further, aged AoSMCs migrated faster than young cells, and such migration could be modulated by HA, which alters the ERK1/2 phosphorylation. HA oligosaccharides of 6.8 kDa and an anti-CD44 blocking antibody prevented ERK1/2 phosphorylation and inhibited AoSMCs migration. These results indicate that, during aging, HA can modulate cell migration involving CD44-mediated signaling through ERK1/2. These data suggest that age-related HA accumulation could promote SMC migration and intima thickening during vascular neointima formation. Hyaluronan (HA)2 is a linear, unsulfated glycosaminoglycan (GAG) that is composed of repeating units of D-glucuronic acid and N-acetylglucosamine linked together through alternating ␤1,4 and ␤1,3 glycosidic bonds. The amount and the molecular weight of HA are important factors that regulate the physiopathological effects that this molecule displays on cells (1). In mammals, three specific HA synthases (HAS1, -2, and -3) and three hyaluronidases (HYAL1, 2, and PH20) regulate HA synthesis and degradation with specific biochemical properties and distributions in adult as well as in embryonic tissues (2, 3). Therefore, these enzymes have a critical role in HA metabolism and are responsible for HA balance in the extracellular matrix (ECM).Hydrated HA makes the ECM an ideal environment in which cells can move and proliferate. Moreover, HA is an important space filling molecule as is evident in the vitreous humor, the dermis and the synovial fluid of joints. Besides its chemical and mechanical properties, HA interacts with several receptors at the cellular level that specifically trigger various signal transduction responses (4). The HA receptor CD44 is expressed on the surface of most cells, including immune system cells, and it mediates cell adhesion, proliferation and migration (5). Receptor for HA-mediated motility (RHAMM) mediates cellular motility (6). Lyve-1 is the specific HA receptor of the lymphatic system although very recent evidences indicate a more complex function of this protein unrelated to HA...