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...

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.

UDP-glucose dehydrogenase (UGDH) supplies the cell with UDP-glucuronic acid (UDP-GlcUA), a precursor of glycosaminoglycan and proteoglycan synthesis. Here we reported the cloning and the characterization of the UGDH from the amphibian Xenopus laevis that is one of the model organisms for developmental biology. We found that X. laevis UGDH (xUGDH) maintained a very high degree of similarity with other known UGDH sequences both at the genomic and the protein levels. Also its kinetic parameters are similar to those of UGDH from other species. During X. laevis development, UDGH is always expressed but clearly increases its mRNA levels at the tail bud stage (i.e. 30 h post-fertilization). This result fits well with our previous observation that hyaluronan, a glycosaminoglycan that is synthesized using UDP-GlcUA and UDP-N-acetylglucosamine, is abundantly detected at this developmental stage. The expression of UGDH was found to be related to hyaluronan synthesis. In human smooth muscle cells the overexpression of xUGDH or endogenous abrogation of UGDH modulated hyaluronan synthesis specifically. Our findings were confirmed by in vivo experiments where the silencing of xUGDH in X. laevis embryos decreased glycosaminoglycan synthesis causing severe embryonic malformations because of a defective gastrulation process.

SUMMARY:Mixed endocrine-exocrine tumors of the gut are a heterogeneous group of neoplasms with uncertain histogenesis showing different morphologic and clinical features. The aim of this work is to clarify the histogenesis of these tumors by studying the genetic profile of both the endocrine and exocrine components. We performed an allelotyping analysis of five mixed endocrine-exocrine tumors (two gastric and three colonic) and one rectal collision tumor, using 35 polymorphic microsatellite markers covering a total of six chromosomes, including 3, 5q, 6, 11, 17, and 18. The loss of heterozygosity (LOH) analysis showed concurrent losses of the same allele in both the endocrine and exocrine components in all of the five mixed tumors composed by a poorly differentiated endocrine carcinoma or a well differentiated endocrine carcinoma associated with adenocarcinoma or adenoma. Among these tumors an identical LOH pattern was frequently found on chromosomes 17p, 18q, and 5q. Additional allelic losses restricted to the poorly differentiated endocrine carcinoma were often observed. On the contrary, in the only collision tumor composed by a well differentiated endocrine carcinoma associated with adenocarcinoma, completely different allelotypes between the two components were detected. These findings confirm a close genetic relationship between the two distinct histologic components within mixed endocrine-exocrine tumors, supporting the hypothesis that a monoclonal mechanism of tumorigenesis is the most frequent genetic event in mixed exocrine-endocrine tumors. The clonal divergence observed in the only collision tumor, composed by a well differentiated endocrine carcinoma associated with an adenocarcinoma, confirms the existence of double tumors growing next to each other coincidentally but showing different histogenesis and different tumorigenetic pathways. (Lab Invest 2003, 83:963-971).

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...