Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

Abstract

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...

Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

“…The results showed that exogenous HA had no protective effect upon UVB-induced or serum starvation-induced apoptosis. This contrasts with findings by Vigetti et al (37), in which the addition of exogenous HA was shown to protect human aortic smooth muscle cells from cell death caused by 4-MU treatment; major differences in cell type, culture conditions, and inhibitors used could easily explain the different outcomes.…”

“…In contrast, other studies showed that HA treatment induces cell death in cultured human gingival fibroblasts (47) and lymphoma cell lines (48). Thus, the regulatory effect of HA upon cell viability appears to depend upon cell context, even when controlling for the size of HA, because high molecular weight HA has been shown to either promote cellular survival (37) or induce cell death (49) in different cell types.…”

“…However, the role of HA in regulating cell death and survival has proven to be very complicated. HA is reported to protect against apoptosis and to promote survival in various types of cells, including granulosa cells (44), articular chondrocytes (45,46), and human aortic smooth muscle cells (37). In contrast, other studies showed that HA treatment induces cell death in cultured human gingival fibroblasts (47) and lymphoma cell lines (48).…”

“…The Addition of High Molecular Weight HA Failed to Abrogate the Apoptosis Sensitization Caused by Has2 RNAi-Vigetti et al (37) reported that HMW-HA effectively prevents the apoptosis of human aortic smooth muscle cells caused by 4-MU treatment. In order to test whether HMW-HA could protect skin fibroblasts against apoptosis, HA synthesis in fibroblasts was first inhibited by knockdown of Has2 gene expression with RNA interference, followed by the addition of HMW-HA to the culture media during serum starvation or immediately following UVB exposure.…”

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability

“…Certainly, versican cleavage can help loosen the HA strand network, thereby altering the interaction of HA with adhesive proteins and with cell surface receptors. Indeed, a growing body of evidence suggest that HA can also directly participate in sustaining the vitality of several cell types by binding to HA cell surface receptors, mainly to CD44 (72)(73)(74)(75). This molecular interaction seems also to take part in the control of cumulus survival.…”