The effects of metabolism on the control of hyaluronan synthesis both in healthy and pathologic conditions are critical and still not completely understood. The hyaluronan capacity to bind several receptors triggering specific pathways may represent a valid target for new approach in several therapeutic strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Background: UDP-GlcNAc is a precursor of glycoconjugates, including hyaluronan, and induces protein glycosylation to form O-linked GlcNAc (O-GlcNAcylation). Results: UDP-GlcNAc induces hyaluronan synthesis through O-GlcNAcylation of hyaluronan synthase 2, which stabilizes the enzyme and prevents its proteasomal degradation. Conclusion: O-GlcNAcylation of hyaluronan synthase 2 can control synthesis of extracellular matrices with hyaluronan. Significance: UDP-GlcNAc could control cell microenvironments that are altered in many pathologies, including vascular diseases and cancer.

Background: Intracellular proteins glycosylation with O-GlcNAc is able to influence cell microenvironment. Results: O-GlcNAcylation increases hyaluronan synthase 2 (HAS2) transcription via its natural antisense transcript HAS2-AS1. Conclusion: A novel mechanism to regulate hyaluronan synthesis via long non-coding RNA is described. Significance: This finding highlights a new target to regulate HA synthesis, critical in many pathophysiological processes.

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.

Cell behavior is determined by both genetic and environmental factors. The cell microenvironment is not only a scene in which various actors play a role, but is itself an active participant, able to influence many cellular responses by binding signaling molecules or by modulating intracellular signaling cascades. Further, extracellular matrix remodeling is a critical step to allow physiological as well as pathological processes. As environmental factors are able to modulate gene expression by epigenetic modifications, this review focuses on new aspects of the regulation of extracellular matrix remodeling enzymes. Moreover, as one of the main components of cell microenvironment is the glycosaminoglycan hyaluronan, novel findings regarding the control of hyaluronan synthesis are discussed in terms of epigenetics and from the post‐translational point of view.

Background: OxLDL and the high level of hyaluronan are major triggering factors of atherosclerosis. Results: The oxLDL load of the aortic human smooth muscle cells (SMC) via the scavenger receptor LOX-1 causes ER stress, overexpression of HAS2, and hyaluronan deposition. Conclusion: the oxidized sterols driven into the SMC by oxLDL have a role in hyaluronan metabolism. Significance: oxLDL influences extracellular matrix hyaluronan.

Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2. Hyaluronan (HA)3 is one of the most abundant glycosaminoglycans (GAGs) in extracellular matrices (ECMs) and is composed of linear, unsulfated repetitions of D-glucuronic acid and N-acetylglucosamine. In mammals, two specific HA synthases (HAS1 and -2) produce high molecular weight HA (HMW-HA), in the range of millions of Da, whereas the other isoenzyme (HAS3) synthesizes HA of lower molecular mass, in the range of several thousands of Da (1). The size of HA depends also on specific degrading enzymes (i.e. hyaluronidases) that can produce bioactive HA oligosaccharides. Therefore, in vivo, HA chains can greatly vary in lengths and can differently regulate cell behavior through interactions with several receptors, including CD44, RHAMM (receptor for HA-mediated motility), lymphatic vessel endothelial receptor 1 (Lyve-1), HA receptor for endocytosis (HARE), and Toll-like Receptor 4 (TLR4) (2).In cardiovascular pathologies, HA accumulates during neointima formation and alters smooth muscle cell (SMC) behavior (3). In some pathological conditions, contractile SMCs dedifferentiate to form a synthetic phenotype characterized by a high production of ECM components, including HA and versican, by synthesis of ECM-modifying metalloproteinases (4) and by increased rates of proliferation and migration. Therefore, SMCs acquire the capability to invade the vascular tunica intima, thereby contributing to vessel wall thickening. HMW-HA is involved in the modulation of SMC migration and proliferation through interaction with CD44 (5-7), which can mediate a signaling cascade inside the cell that activates different pathways, including PI3K, AKT, and ERK1/2 (8).We demonstrated previously that human aortic SMC (AoSMC) migration is strictly dependent on HA-CD44 signaling and recently reported that the HA synthesis inhibitor 4-methylumbelliferone (4-MU) reduced proatherosclerotic properties of AoSMCs by decreasing cell migration and proliferation and by inhibiting monocyte binding to the HA-rich ECM that contributes to inflammation (9, 10). Moreover, we found that the simultaneous addition of HMW-HA to 4-MUtreated AoSMCs restored cell proliferation to the levels of controls. Therefore, the aim of this study was to investigate at the molecular level the effects of HMW-HA after 4-MU treatment of AoSMCs and the pathway...