AMP-activated protein kinase regulates hyaluronan biosynthesis.

HA is an atypical GAG because, although it is composed by GlcUA and GlcNAc, it does not contain sulfatation and it is not linked to any PG core protein. Moreover, HA synthesis is on plasma membrane by HASs, instead of Golgi. HA, as hydrophilic and viscous polysaccharide, increases the volume of extracellular space, thus contributing to tissue remodeling and facilitating cell mobility. This classical view of HA as a space filler and swelling agent has been complemented recently by interesting findings indicating that it mediates homing of stem cells, leukocyte adherence to endothelial cells, monocyte binding to virally infected or stressed mesenchymal cells and bacterial adherence. Furthermore, high molecular mass HA and its oligosaccharide degradation products create specific intracellular signals that promote cell locomotion, influence cell division, block apoptosis and induce membrane transporters (Rahmanian et al., 1997; Takahashi et al., 2005). Moreover, HA has a proatherosclerotic role in vascular diseases, because it promotes cellular migration and proliferation and because it’s involved in immune cells recruitment. As the synthesis of HA precursors requires ATP, UTP and other critical metabolic molecules (as glucose, glutamine and acetyl-CoA) making the HA production an energy consuming process, we hypnotized that in condition of low energy charge this anabolic process could be controlled by AMPK, a serine/threonine kinase which works as an energy sensor in all eukaryotic cells. In condition of low ATP content, this enzyme is activated and subsequently it switches off several anabolic processes (i.e.: gluconeogenesis, fatty acids synthesis, lipolysis, glycogen synthesis) and switches on some catabolic events (i.e.: glycolisis, fatty acids -oxidation) inside the cell. Moreover AMPK activation can be regulated not only by a decrement of ATP/AMP ratio, but also by physiological stimuli independent of the energy status of the cell, including hormones, nutrient depletion, heat shock and some drugs as metformin and AICAR. In the present study, we have used human aortic smooth muscle cells (hAoSMCs) because they produce an elevate amount of HA, essential for their proliferation and migration and for immune cells recruitment. All these processes are the most critical events involved in the onset of neointima formation, which is a pivotal step for the development and progression of all vascular diseases (including atherosclerosis), a main complication in diabetes. In fact, physiologically, SMCs resides in the tunica media in a contractile quiescent phenotype determining the vessel tone, whereas the neointima-forming SMCs posses a synthetic phenotype that induces cell proliferation and migration. Moreover, synthetic SMCs produce many ECM molecules, including HA, and ECM modifying enzymes, as metalloproteinases (Vigetti et al., 2006). The increment of HA in the media induces SMC motility through the interaction with CD44 and RHAMM (Savani et al., 1995). SMCs produced HA enhances immune cell recruitment and SMC dedifferentiation (Cuff et al., 2001). Therefore the ability to control and regulate HA biosynthesis by AMP-activated protein kinase could be a new good strategy to arrest atherosclerotic progression. In this work, AMPK-activation has been induced by AICAR, metformin and 2-deoxyglucose (2-DG) treatments, observing a significant and dose-dependent reduction of HA biosynthesis and an unchanged production of the other GAGs. These observations has been confirmed using different AICAR inhibitors (AMDA and Dipyridamole) and specific AMPK inhibitor (Compound C), and by transient transfections with constitutive active form of AMPK (CAAMPK) in AMPK-/- Mef-t cells. As the reduction of HA synthesis induced by AMPK activation was not due to an alteration of expression of genes involved in HA metabolism (as verified by quantitative RT-PCR analyses), we hypothesized that AMPK could act at post-transcriptional level, modifying the activity of HA synthetic enzymes (i.e.: Has1, Has2 and Has3). Cotransfecting each HAS isoform with (CA)AMPK in COS1 cells we found that AMPK specifically inhibits HAS2 activity. As AMPK is a kinase, we assayed whether a phosphorylative event could control HAS2 activity: by treatments in COS1 membrane fractions with SAP and CIP we observed that the removal of the hypothetic phosphorylation site fully restores HAS2 activity. For these reasons we hypothesized that AMPK could downregulate HA-synthesis inhibiting HAS2 activity by phosphorylation. In order to confirm this results, we have performed an immunoprecipitation assay in transfected COS1 cells, following by mass spectrometry analysis (MS-analysis). As HA has a pivotal role in some processes involved in the onset of atherosclerotic plaque as cell proliferation, migration and immune cells recruitment, we assayed the involvement of AMPK in those. Using hAoSMCs, we have demonstrated the ability of AMPK (activated by transfection as well as by treatments with AICAR and metformin) to arrest cell-cycle in G0/G1 phase, without causing apoptosis, to inhibit cell migration and to reduce monocytes adhesion. Taken together these results demonstrate that AICAR and metformin could have a new inhibitory effect on HA synthesis in hAoSMCs and an additional vasoprotective effect. Moreover, the increasing number of processes in which HA is involved suggest that to know the way to control and regulate its metabolism could be an important finding for a large number of medical applications as cancer, diabetes and atherosclerosis

AMP-activated protein kinase regulates hyaluronan biosynthesis.

HA is an atypical GAG because, although it is composed by GlcUA and GlcNAc, it does not contain sulfatation and it is not linked to any PG core protein. Moreover, HA synthesis is on plasma membrane by HASs, instead of Golgi. HA, as hydrophilic and viscous polysaccharide, increases the volume of extracellular space, thus contributing to tissue remodeling and facilitating cell mobility. This classical view of HA as a space filler and swelling agent has been complemented recently by interesting findings indicating that it mediates homing of stem cells, leukocyte adherence to endothelial cells, monocyte binding to virally infected or stressed mesenchymal cells and bacterial adherence. Furthermore, high molecular mass HA and its oligosaccharide degradation products create specific intracellular signals that promote cell locomotion, influence cell division, block apoptosis and induce membrane transporters (Rahmanian et al., 1997; Takahashi et al., 2005). Moreover, HA has a proatherosclerotic role in vascular diseases, because it promotes cellular migration and proliferation and because it’s involved in immune cells recruitment. As the synthesis of HA precursors requires ATP, UTP and other critical metabolic molecules (as glucose, glutamine and acetyl-CoA) making the HA production an energy consuming process, we hypnotized that in condition of low energy charge this anabolic process could be controlled by AMPK, a serine/threonine kinase which works as an energy sensor in all eukaryotic cells. In condition of low ATP content, this enzyme is activated and subsequently it switches off several anabolic processes (i.e.: gluconeogenesis, fatty acids synthesis, lipolysis, glycogen synthesis) and switches on some catabolic events (i.e.: glycolisis, fatty acids -oxidation) inside the cell. Moreover AMPK activation can be regulated not only by a decrement of ATP/AMP ratio, but also by physiological stimuli independent of the energy status of the cell, including hormones, nutrient depletion, heat shock and some drugs as metformin and AICAR. In the present study, we have used human aortic smooth muscle cells (hAoSMCs) because they produce an elevate amount of HA, essential for their proliferation and migration and for immune cells recruitment. All these processes are the most critical events involved in the onset of neointima formation, which is a pivotal step for the development and progression of all vascular diseases (including atherosclerosis), a main complication in diabetes. In fact, physiologically, SMCs resides in the tunica media in a contractile quiescent phenotype determining the vessel tone, whereas the neointima-forming SMCs posses a synthetic phenotype that induces cell proliferation and migration. Moreover, synthetic SMCs produce many ECM molecules, including HA, and ECM modifying enzymes, as metalloproteinases (Vigetti et al., 2006). The increment of HA in the media induces SMC motility through the interaction with CD44 and RHAMM (Savani et al., 1995). SMCs produced HA enhances immune cell recruitment and SMC dedifferentiation (Cuff et al., 2001). Therefore the ability to control and regulate HA biosynthesis by AMP-activated protein kinase could be a new good strategy to arrest atherosclerotic progression. In this work, AMPK-activation has been induced by AICAR, metformin and 2-deoxyglucose (2-DG) treatments, observing a significant and dose-dependent reduction of HA biosynthesis and an unchanged production of the other GAGs. These observations has been confirmed using different AICAR inhibitors (AMDA and Dipyridamole) and specific AMPK inhibitor (Compound C), and by transient transfections with constitutive active form of AMPK (CAAMPK) in AMPK-/- Mef-t cells. As the reduction of HA synthesis induced by AMPK activation was not due to an alteration of expression of genes involved in HA metabolism (as verified by quantitative RT-PCR analyses), we hypothesized that AMPK could act at post-transcriptional level, modifying the activity of HA synthetic enzymes (i.e.: Has1, Has2 and Has3). Cotransfecting each HAS isoform with (CA)AMPK in COS1 cells we found that AMPK specifically inhibits HAS2 activity. As AMPK is a kinase, we assayed whether a phosphorylative event could control HAS2 activity: by treatments in COS1 membrane fractions with SAP and CIP we observed that the removal of the hypothetic phosphorylation site fully restores HAS2 activity. For these reasons we hypothesized that AMPK could downregulate HA-synthesis inhibiting HAS2 activity by phosphorylation. In order to confirm this results, we have performed an immunoprecipitation assay in transfected COS1 cells, following by mass spectrometry analysis (MS-analysis). As HA has a pivotal role in some processes involved in the onset of atherosclerotic plaque as cell proliferation, migration and immune cells recruitment, we assayed the involvement of AMPK in those. Using hAoSMCs, we have demonstrated the ability of AMPK (activated by transfection as well as by treatments with AICAR and metformin) to arrest cell-cycle in G0/G1 phase, without causing apoptosis, to inhibit cell migration and to reduce monocytes adhesion. Taken together these results demonstrate that AICAR and metformin could have a new inhibitory effect on HA synthesis in hAoSMCs and an additional vasoprotective effect. Moreover, the increasing number of processes in which HA is involved suggest that to know the way to control and regulate its metabolism could be an important finding for a large number of medical applications as cancer, diabetes and atherosclerosis

Analysis of glycosaminoglycans by electrophoretic approach

Glycosaminoglycans (GAGs) are non branched polysaccharides which are raising a great interest among the scientists for their biological roles. In fact GAGs play a pivotal role in several biological events, since they participate in and regulate cell adhesion, migration and proliferation. The quantification and analysis of the fine structure of GAGs are increasingly important not only for understanding many biological processes, but also for elucidate many critical aspects in human pathology development. Chondroitin sulfate (CS), heparan sulfate (HS) and keratan sulfate (KS) are commonly described as sulfated GAGs and these molecules are linked to a core protein forming proteoglycans; the sulfation pattern shows a high level of complexity and it is associated with specific function in the tissues. The only GAG without protein core is hyaluronan (HA), which is produced in almost all tissues, often with a molecular weight of 10^6 Daltons. Several human tissues contain high amount of GAGs and the change of the quantity and the structure of these macromolecules are described in tissue development and it is commonly associated with diseases. Electrophoretic methods based on the gel separation of 2-aminoacridone labelled HA and CS sulfate delta-disaccharides, derived from GAG digestion with specific eliminases, have been recently proposed. These new techniques represent a suitable method for GAG fast and sensitive analysis. In this review we will describe the recently achieved methods on the GAG analysis based on the electrophoretic approach in comparison with the more standard chromatographic techniques (HPLC)

Vascular pathology and the role of hyaluronan

The development of vascular pathology is often coupled to dramatic alterations of the extracellular matrix (ECM), which provides critical support for vascular tissue as a scaffold for maintaining the organization of vascular cells into blood vessels, for blood vessel stabilization, morphogenesis, and for cell proliferation, migration, and survival. Hyaluronan (HA) is an important component of the ECM that has generated increasing interest because of its multitude of functions. HA is a linear polymer belonging to the family of glycosaminoglycans (GAGs), which comprises the major fraction of carbohydrates in ECM. Evidence supports the hypothesis that HA is an important contributor to human aortic smooth muscle cell (AoSMC) migration which represents a crucial point in the onset of pathology. By reducing HA synthesis and therefore the AoSMC motility, 4-Metyllumbelliferone (4-MU) could represent a new molecule with additional beneficial pharmacological effects in vivo

Localization of Carboxyl Ester Lipase in Human Pituitary Gland and Pituitary Adenomas

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells. (J Histochem Cytochem 58:881–889, 2010

The effects of 4-methylumbelliferone on hyaluronan synthesis, MMP2 activity, proliferation and motility of human aortic smooth muscle cells

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

Hyaluronan-CD44-ERK1/2 regulate human aortic smooth muscle cell motility during aging

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