Patrizia Uboldi

Long Pentraxin 3, a Key Component of Innate Immunity, Is Modulated by High-Density Lipoproteins in Endothelial Cells

Objective—High-density lipoproteins (HDL) are endowed with cardiovascular protective activities. In addition to their role in reverse cholesterol transport, HDL exert several beneficial effects on endothelial cells, including the induction of endothelial nitric oxide synthase and prostacyclin release, and the control of the immune and inflammatory response. Methods and Results—To identify possible mechanisms involved in these effects we investigated the modulation of the expression of acute phase proteins of the pentraxin superfamily, such as C-reactive protein (CRP), serum amyloid P component protein (SAP), and the long pentraxin 3 (PTX3) by HDL in human endothelial cells. HDL induced PTX3 mRNA expression and protein release, whereas no effect was observed on CRP and SAP expression. This effect was mainly dependent on the activation of the lysosphingolipids receptors-PI3K/Akt axis and was mimicked by sphingosine 1 phosphate and other S1P mimetics. This observation was confirmed in vivo; indeed an increased expression of PTX3 mRNA was detected in the aorta of transgenic mice overexpressing human apoA-I, compared to apoA-I knock-out mice. Furthermore, plasma levels of PTX3 significantly increased in C57BL/6 mice injected with HDL. Conclusions—These data suggest that part of the atheroprotective effects of HDL could result from the modulation of molecules that act as sensors of the immunoinflammatory balance in the vascular wall.

The Arachidonic Acid Metabolome Serves as a Conserved Regulator of Cholesterol Metabolism

Summary Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolome as conserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.

Increased atherosclerosis and vascular inflammation in APP transgenic mice with apolipoprotein E deficiency

OBJECTIVE Atherosclerosis is associated with Alzheimers disease (AD) in humans, but the nature of this link is still elusive. Aim of this study was to investigate aortic atherosclerosis development in a mouse model with central nervous system (CNS) restricted beta-amyloid precursor protein (APP) overexpression. METHODS AND RESULTS APP23 mice, overexpressing the Swedish mutated human APP selectively in the brain, were crossed with mice lacking apolipoprotein E (ApoE KO). Nine weeks old mice were fed a western type diet for eight weeks, then atherosclerotic lesions, aortic wall and cortical tissues gene expression and beta-amyloid (Abeta) deposition were evaluated. Compared with ApoE KO, APP23/ApoE KO mice developed larger aortic atherosclerotic lesions and showed significantly increased expression of MCP-1, IL-6, ICAM-1 and MTPase 6, a marker of oxidative stress in the vascular wall. Of note brain limited APP synthesis was associated with an increased microglia and brain endothelial cells activation, in spite of the absence of beta-amyloid deposits in the brain or alteration in the levels of oxidized metabolites of cholesterol such as 4-cholesten-3-one. CONCLUSION Our study suggests that the vascular pro-inflammatory effects of CNS-localised APP overexpression lead to atherogenesis before parenchymal Abeta deposition and neuronal dysfunction.

The 15-Lipoxygenase-Modified High Density Lipoproteins 3 Fail to Inhibit the TNF-α-Induced Inflammatory Response in Human Endothelial Cells

Endothelial dysfunction represents one of the earliest events in vascular atherogenesis. Proinflammatory stimuli activate endothelial cells, resulting in an increased expression of adhesion molecules and chemoattractants that mediate leukocyte and monocyte adhesion, migration, and homing. High density lipoproteins (HDL) inhibit endothelial cell expression of adhesion molecules in response to proinflammatory stimuli. In the present work, we demonstrate that the modification of HDL3 (the major and the most antiatherogenic HDL subfraction) by 15-lipoxygenase (15-LO), an enzyme overexpressed in the atherosclerotic lesions, impairs the anti-inflammatory activity of this lipoprotein. The 15-LO-modified HDL3 failed to inhibit TNF-α-mediated mRNA and protein induction of adhesion molecules and MCP-1 in several models of human endothelial cells, and promoted inflammatory response by up-regulating the expression of such mediators of inflammation and by increasing monocyte adhesion to endothelial cells. Moreover, 15-LO-modified HDL3 were unable to contrast the formation of reactive oxygen species in cells incubated with TNF-α, and increased the reactive oxygen species content in unstimulated cells. Activation of NF-κB and AP-1 was mainly involved in the expression of adhesion molecules and MCP-1 induced by 15-LO-HDL3. Altogether, these results demonstrate that enzymatic modification induced by 15-LO impaired the protective role of HDL3, generating a dysfunctional lipoprotein endowed with proinflammatory characteristics.

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG(1)), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1-1297) and in the middle region (mAb 6B: aa 1480-1693; mAbs 2A, 3B: aa 2152-2377; mAbs 9A, L2 and L4: aa 2657-3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG(1)-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).

Upregulation of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) expression in human endothelial cells by modified high density lipoproteins

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is the main endothelial receptor for oxidized low density lipoprotein (OxLDL). LOX-1 is highly expressed in endothelial cells of atherosclerotic lesions, but also in macrophages and smooth muscle cells. LOX-1 expression is upregulated by several inflammatory cytokines (such as TNF-α), by oxidative stress, and by pathological conditions, such as dyslipidemia, hypertension, and diabetes. High density lipoprotein (HDL) possess several atheroprotective properties; however under pathological conditions associated with inflammation and oxidative stress, HDL become dysfunctional and exhibit pro-inflammatory properties. In vitro, HDL can be modified by 15-lipoxygenase, an enzyme overexpressed in the atherosclerotic lesions. Here we report that, after modification with 15-lipoxygenase, HDL(3) lose their ability to inhibit TNFα-induced LOX-1 expression in endothelial cells; in addition, 15LO-modified HDL(3) induce LOX-1 mRNA and protein expression and bind to LOX-1 with increased affinity compared to native HDL(3). Altogether these findings confirm that 15LO-modified HDL(3) possess a pro-atherogenic role.

Immunoreactivity of apo B towards monoclonal antibodies that inhibit the LDL-receptor interaction: effects of LDL oxidation

We studied the immunochemical stability of the epitopes for six monoclonal antibodies to human apolipoprotein B-100 upon Cu(2+)-mediated (20 microM) oxidation of LDL. The antibodies used in this study, some of which are known to interfere with the interaction of LDL with their cellular receptors, recognize epitopes in the amino terminal region (Mb 19), in the middle part (6B, 2A, 7A, and 9A) and near aa 3500 (Mb 47) of native apo B. All antibodies except one (7A) recognized native and oxidized LDL (OxLDL) equally well; the immunoreactivity of the epitope for Ab 7A was markedly reduced upon LDL oxidation. Since antibodies 2A, 7A, 9A, and Mb 47 inhibit the LDL-receptor interaction and OxLDL poorly interact in vitro with the LDL receptor we conclude that: (1) various epitopes for monoclonal antibodies against native apo B are spared upon LDL oxidation; and (2) the epitopes for antibodies 2A, 9A, and Mb 47 do not define a unique domain of apo B directly involved in the binding of LDL to their receptor.

15-lipoxygenase-mediated modification of HDL3 impairs eNOS activation in human endothelial cells.

Caveolae are cholesterol and glycosphingolipids-enriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenase-mediated modification of HDL3 on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL3 failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL.

PCSK9 deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor

Aims PCSK9 loss of function genetic variants are associated with lower low-density lipoprotein cholesterol but also with higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Here, we investigated the molecular mechanisms underlying this association. Methods and results Pcsk9 KO, WT, Pcsk9/Ldlr double KO (DKO), Ldlr KO, albumin AlbCre+/Pcsk9LoxP/LoxP (liver-selective Pcsk9 knock-out mice), and AlbCre-/Pcsk9LoxP/LoxP mice were used. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Glucose clearance was significantly impaired in Pcsk9 KO mice fed with a standard or a high-fat diet for 20 weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying the low-density lipoprotein receptor (LDLR) as the proprotein convertase subtilisin/kexin Type 9 (PCSK9) target responsible for the phenotype observed. Further studies in albumin AlbCre+/Pcsk9LoxP/LoxP mice, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9, the principal target of monoclonal antibodies, does not impact beta-cell function and insulin secretion. Conclusion PCSK9 critically controls LDLR expression in pancreas perhaps contributing to the maintenance of a proper physiological balance to limit cholesterol overload in beta cells. This effect is independent of circulating PCSK9 and is probably related to locally produced PCSK9.

The 15-lipoxygenase-modified HDL3 fail to inhibit the TNF-α-induced inflammatory response in human endothelial cells

Pirillo, A., P. Uboldi, C. Bolego, H. Kuhn, A. L. Catapano. 2008. The 15-lipoxygenase-modified HDL3 fail to inhibit the TNF-α-induced inflammatory response in human endothelial cells. [ J. Immunol . 181: 2821–2830][1] . The revised legend for Figure 12 should read as follows: FIGURE 12. Effect