Heparan sulfates, the carbohydrate chains of heparan sulfate proteoglycans, play an important role in basement membrane organization and endothelial barrier function. We explored whether endothelial cells secrete a heparan sulfate degrading heparanase under inflammatory conditions and what pathways were responsible for heparanase expression. Heparanase mRNA and protein by Western blot were induced when cultured endothelial cells were treated with cytokines, oxidized low-density lipoprotein (LDL) or fatty acids. Heparanase protein in the cell media was induced 2-10-fold when cells were treated with tumor necrosis factor alpha (TNFalpha) or interleukin 1beta (IL-1beta). Vascular endothelial growth factor (VEGF), in contrast, decreased heparanase secretion. Inhibitors to nuclear factor-kappaB (NFkappaB), PI3-kinase, MAP kinase, or c-jun kinase (JNK) did not affect TNFalpha-induced heparanase secretion. Interestingly, inhibition of caspase-8 completely abolished heparanase secretion induced by TNFalpha. Fatty acids also induced heparanase, and this required an Sp1 site in the heparanase promoter. Immunohistochemical analyses of cross sections of aorta showed intense staining for heparanase in the endothelium of apoE-null mice but not wild-type mice. Thus, heparanase is an inducible inflammatory gene product that may play an important role in vascular biology.

Sirt1 (member of the sirtuin family) is a nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase that removes acetyl groups from various proteins. A wide variety of proteins are Sirt1 substrates; the list includes many transcription factors and cofactors. Deacetylation of these factors may lead to activation or inactivation of the factor, thus impacting downstream gene expression. In addition to direct deacetylation, Sirt1 can modulate protein activity by other mechanisms. Although initial research focused on sirtuin's role in life span extension especially in lower organisms more recent studies show that Sirt1 activity can impact a wide array of proteins implicated in cardiovascular (CV) and metabolic diseases. Several patents have been published in the last 5 years describing the application of sirtuin compounds in the treatment of metabolic diseases. This review will focus on those Sirt1-modifiable proteins that have an impact on CV and metabolic diseases. Pharmacological agents that activate Sirt1 and thus impact the disease process will also be reviewed.

Vessel wall subendothelial extracellular matrix, a dense mesh formed of collagens, fibronectin, laminin, and proteoglycans, has important roles in lipid and lipoprotein retention and cell adhesion. In atherosclerosis, vessel wall heparan sulfate proteoglycans (

Lipoprotein lipase (LPL)1 is a 120-kDa dimeric protein that associates with the luminal surface of endothelial cells in multiple organs but especially in cardiac and skeletal muscle and in adipose tissue (1). This enzyme hydrolyzes the triglyceride in circulating lipoproteins such as chylomicrons and VLDL and produces free fatty acids that are used for metabolic energy or for fat storage. Endothelial cells do not synthesize LPL; rather myocytes and adipocytes produce it. Thus, it is a protein that requires transcytosis across the endothelial cell barrier, in this case from the interstitial fluid to the luminal side of the cells.There are several possible ways that LPL could cross the endothelial barrier. Nonspecific transport of molecules across endothelial monolayers occurs either via paracellular routes between the cells or via vesicular transit through cells (2). Alternatively, a specific transcytosis pathway could exist which requires LPL to associate with a cell surface receptor and then transports LPL through the cells. This process would be analogous to that which transfers IgA across epithelial cells (3). The first step in a specific LPL transcytosis pathway would involve LPL interaction with the basolateral side of endothelial cells. LPL binds to a number of cell surface molecules including heparan sulfate proteoglycans (HSPGs) and members of the LDL receptor family (4). In bovine endothelial cells the most highly expressed of these receptors is the VLDL receptor (VLDLr) (5). A previous study suggested that HSPGs are required for LPL transcytosis (6). It is, however, unclear whether HSPGs are sufficient for transport or whether HSPGs must operate in concert with receptors. The binding of LPL to several members of the LDL receptor family leads to uptake and degradation of LPL by cells. There are no data on whether these receptors participate in transendothelial movement of LPL or other ligands.In this report, we present data showing that LPL transcytosis across endothelial monolayers requires both HSPGs and the VLDLr. LPL transcytosis was diminished by removal of HSPGs and inhibition of receptors by RAP, a 39-kDa protein that was copurified with the LDL receptor-related protein (LRP) (7). This protein binds to members of the LDL receptor family and inhibits ligand binding and uptake by those receptors (8, 9). Furthermore, antibodies against the VLDLr blocked LPL translocation and increased expression of this receptor-increased transcytosis. Thus, LPL requires both HSPGs and receptors for translocation across endothelial cells. EXPERIMENTAL PROCEDURESPurification and Radioiodination of LPL-LPL was purified from unpasteurized bovine milk according to the method of Socorro et al. (10)

OBJECTIVE-Lipoic acid synthase (LASY) is the enzyme that is involved in the endogenous synthesis of lipoic acid, a potent mitochondrial antioxidant. The aim of this study was to study the role of LASY in type 2 diabetes.RESEARCH DESIGN AND METHODS-We studied expression of LASY in animal models of type 2 diabetes. We also looked at regulation of LASY in vitro under conditions that exist in diabetes. Additionally, we looked at effects of LASY knockdown on cellular antioxidant status, inflammation, mitochondrial function, and insulin-stimulated glucose uptake.RESULTS-LASY expression is significantly reduced in tissues from animal models of diabetes and obesity compared with ageand sex-matched controls. In vitro, LASY mRNA levels were decreased by the proinflammatory cytokine tumor necrosis factor (TNF)-␣ and high glucose. Downregulation of the LASY gene by RNA interference (RNAi) reduced endogenous levels of lipoic acid, and the activities of critical components of the antioxidant defense network, increasing oxidative stress. Treatment with exogenous lipoic acid compensated for some of these defects. RNAi-mediated downregulation of LASY induced a significant loss of mitochondrial membrane potential and decreased insulinstimulated glucose uptake in skeletal muscle cells. In endothelial cells, downregulation of LASY aggravated the inflammatory response that manifested as an increase in both basal and TNF-␣-induced expression of the proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Overexpression of the LASY gene ameliorated the inflammatory response.CONCLUSIONS-Deficiency of LASY results in an overall disturbance in the antioxidant defense network, leading to increased inflammation, insulin resistance, and mitochondrial dysfunction. Diabetes 58:600-608, 2009 T ype 2 diabetes is the most prevalent chronic metabolic disease in the world. In the past decade, considerable evidence has accumulated implicating oxidative stress as a key factor that accelerates the onset and progression of type 2 diabetes. Chronic oxidative stress causes inflammation and mitochondrial dysfunction and culminates in insulin resistance, which ultimately progresses to diabetes. Oxidative stress also promotes cellular dysfunction and damage, leading to the development of secondary complications of diabetes. The underlying cause of redox imbalance is a deficiency in the endogenous antioxidant network. This deficiency would result in an inability to combat excessive amounts of reactive oxygen species (ROS) and tip the balance in favor of oxidative stress.Redox balance is maintained by an antioxidant defense network within mitochondria, consisting of stress-responsive enzymes such as superoxide dismutase (SOD), catalase and reduced glutathione (GSH), and antioxidants. The antioxidant defense network is activated in response to excessive production of ROS in the mitochondria, thereby neutralizing the ROS before they inflict damage on cellular molecules. Lipoic acid is a potent mitochondrial antioxidant that plays a central role in...