Ruggiero Mango

In Vivo and In Vitro Studies Support That a New Splicing Isoform of OLR1 Gene Is Protective Against Acute Myocardial Infarction

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the “non-risk” disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

Association of single nucleotide polymorphisms in the oxidised LDL receptor 1 (OLR1) gene in patients with acute myocardial infarction

Acute myocardial infarction (AMI) is a significant cause of mortality and morbidity. Substantial data support a plausible role for oxidised LDL (oxLDL) in the aetiology of this disease.1,2 The human OLR1 (or LOX 1) gene encodes the endothelium derived lectin-like oxidised low density lipoprotein (oxLDL) receptor, which is involved in the binding, internalisation, and proteolytic degradation of oxLDL, suggesting that it may play a significant role in atherogenesis.3 OLR1 is considered a good candidate for atherosclerosis and AMI since it is induced in vitro by inflammatory cytokines and in vivo by pro-atherogenic conditions like hypertension, hyperlipidaemia, and diabetes mellitus.4 Recently, upregulation of OLR1 has been shown in ischaemia reperfusion injury in the rat.5 OLR1 acts as a mediator of “endothelial dysfunction” favouring superoxide generation, inhibiting nitric oxide production, and enhancing endothelial adhesiveness for monocytes.6–8 It is noteworthy that the versatile activities of OLR1 also include the ability to bind not only oxLDL, but also aged red blood cells, apoptotic cells, and activated platelets.4 With this background, we sought to validate the hypothesis of OLR1 involvement in atherosclerosis and AMI by defining OLR1 genetic variation by an association study of intragenic SNPs. ### Study subjects The study included 150 individuals with AMI who were referred to the Centre of Atherosclerosis at the Medical School of the Tor Vergata University of Rome. All cases were clinically evaluated and all underwent coronary angiography and left ventriculography. The diagnosis of AMI was based on typical electrocardiographic changes and increased serum activities of at least two enzymes, such as creatine kinase, aspartate aminotransferase, and lactate dehydrogenase. The diagnosis was confirmed by the presence of wall motion abnormality on left ventriculography and attendant stenosis (>50%) in any of the major coronary arteries or in the left main coronary artery on coronary …

The splice variant LOXIN inhibits LOX-1 receptor function through hetero-oligomerization

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a central role in the pathogenesis of atherosclerosis. We have recently identified a truncated naturally occurring variant of the human receptor LOX-1, named LOXIN, which lacks part of the C-terminus lectin-like domain. In vivo and in vitro studies support that the new splicing isoform is protective against acute myocardial infarction. The mechanism by which LOXIN exerts its protective role is unknown. In this paper we report studies on the heterologous expression and functional characterization of LOXIN variant in mammalian fibroblasts and human endothelial cells. We found that LOXIN, when expressed in the absence of LOX-1, shows diminished plasma membrane localization and is deficient in ox-LDL ligand binding. When co-transfected with the full-length counterpart LOX-1, the two isoforms interact to form LOX-1 oligomers and their interaction leads to a decrease in the appearance of LOX-1 receptors in the plasma membrane and a marked impairment of ox-LDL binding and uptake. Co-immunoprecipitation studies confirmed the molecular LOX-1/LOXIN interaction and the formation of non-functional hetero-oligomers. Our studies suggest that hetero-oligomerization between naturally occurring isoforms of LOX-1 may represent a general paradigm for regulation of LOX-1 function by its variants.

Cholesterol-lowering drugs inhibit lectin-like oxidized low-density lipoprotein-1 receptor function by membrane raft disruption.

Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is up-regulated in atherosclerotic lesions. Statins are the principal therapeutic agents for cardiovascular diseases and are known to down-regulate LOX-1 expression. Whether the effect on the LOX-1 receptor is related to statin-mediated cholesterol-lowering activity is unknown. We investigate the requirement of cholesterol for LOX-1-mediated lipid particle internalization, trafficking, and processing and the role of statins as inhibitors of LOX-1 function. Disruption of cholesterol-rich membrane microdomains by acute exposure of cells to methyl-β-cyclodextrin or chronic exposure to different statins (lovastatin and atorvastatin) led to a spatial disorganization of LOX-1 in plasma membranes and a marked loss of specific LOX-1 function in terms of ox-LDL binding and internalization. Subcellular fractionation and immunochemical studies indicate that LOX-1 is naturally present in caveolae-enriched lipid rafts and, by cholesterol reduction, the amount of LOX-1 in this fraction is highly decreased (≥60%). In contrast, isoprenylation inhibition had no effect on the distribution and function of LOX-1 receptors. Furthermore, in primary cultures from atherosclerotic human aorta lesions, we confirm the presence of LOX-1 in caveolae-enriched lipid rafts and demonstrate that lovastatin treatment led to down-regulation of LOX-1 in lipid rafts and rescue of the ox-LDL-induced apoptotic phenotype. Taken together, our data reveal a previously unrecognized essential role of membrane cholesterol for LOX-1 receptor activity and suggest that statins protect vascular endothelium against the adverse effect of ox-LDL by disruption of membrane rafts and impairment of LOX-1 receptor function.

Functional Analysis and Molecular Dynamics Simulation of LOX-1 K167N Polymorphism Reveal Alteration of Receptor Activity

The human lectin-like oxidized low density lipoprotein receptor 1 LOX-1, encoded by the ORL1 gene, is the major scavenger receptor for oxidized low density lipoprotein in endothelial cells. Here we report on the functional effects of a coding SNP, c.501G>C, which produces a single amino acid change (K>N at codon 167). Our study was aimed at elucidating whether the c.501G>C polymorphism changes the binding affinity of LOX-1 receptor altering its function. The presence of p.K167N mutation reduces ox-LDL binding and uptake. Ox-LDL activated extracellular signal-regulated kinases 1 and 2 (ERK 1/2) is inhibited. Furthermore, ox-LDL induced biosynthesis of LOX-1 receptors is dependent on the p.K167N variation. In human macrophages, derived from c.501G>C heterozygous individuals, the ox-LDL induced LOX-1 46 kDa band is markedly lower than in induced macrophages derived from c.501G>C controls. Investigation of p.K167N mutation through molecular dynamics simulation and electrostatic analysis suggests that the ox-LDL binding may be attributed to the coupling between the electrostatic potential distribution and the asymmetric flexibility of the basic spine residues. The N/N-LOX-1 mutant has either interrupted electrostatic potential and asymmetric fluctuations of the basic spine arginines.

Influence of Pentraxin 3 (PTX3) Genetic Variants on Myocardial Infarction Risk and PTX3 Plasma Levels

PTX3 is a long pentraxin of the innate immune system produced by different cell types (mononuclear phagocytes, dendritic cells, fibroblasts and endothelial cells) at the inflammatory site. It appears to have a cardiovascular protective function by acting on the immune-inflammatory balance in the cardiovascular system. PTX3 plasma concentration is an independent predictor of mortality in patients with acute myocardial infarction (AMI) but the influence of PTX3 genetic variants on PTX3 plasma concentration has been investigated very little and there is no information on the association between PTX3 variations and AMI. Subjects of European origin (3245, 1751 AMI survivors and 1494 controls) were genotyped for three common PTX3 polymorphisms (SNPs) (rs2305619, rs3816527, rs1840680). Genotype and allele frequencies of the three SNPs and the haplotype frequencies were compared for the two groups. None of the genotypes, alleles or haplotypes were significantly associated with the risk of AMI. However, analysis adjusted for age and sex indicated that the three PTX3 SNPs and the corresponding haplotypes were significantly associated with different PTX3 plasma levels. There was also a significant association between PTX3 plasma concentrations and the risk of all-cause mortality at three years in AMI patients (OR 1.10, 95% CI: 1.01–1.20, p = 0.02). Our study showed that PTX3 plasma levels are influenced by three PTX3 polymorphisms. Genetically determined high PTX3 levels do not influence the risk of AMI, suggesting that the PTX3 concentration itself is unlikely to be even a modest causal factor for AMI. Analysis also confirmed that PTX3 is a prognostic marker after AMI.

Characterization of the loricrin (LOR) gene as a positional candidate for the PSORS4 psoriasis susceptibility locus

Psoriasis is a chronic inflammatory disease of the skin with both genetic and environmental risk factors. Non‐parametric linkage analyses have mapped many susceptibility loci on different chromosomes. We mapped one of these loci, PSORS4, on human chromosome 1q21. Using the linkage disequilibrium approach, we refined the critical region to a specific genomic interval of about 100 Kb which contains only the loricrin (LOR) gene. Here we report a genetic and functional study of this gene to verify its involvement in psoriasis pathogenesis. We document low expression of LOR in psoriatic skin of patients selected from families in which the disease was segregrating with the PSORS4 locus. Re‐sequencing of the entire gene in a subset of patients revealed the existence of novel polymorphisms able to influence the protein structure, as shown by molecular modelling studies. However, no evidence for genetic association was detected in a large cohort of Italian nuclear families. This rules out the LOR gene as a candidate for the PSORS4 locus.

Role of genetics in prevention of coronary atherosclerosis

Atherosclerosis is currently regarded as a complex disorder, triggered by gene–gene and gene–environment interactions. Despite the difficulties of multifactorial disease genetic analysis, significant progress has been achieved in the search for atherosclerosis susceptibility genes. Here, we review these advances with regard to genome scan results and candidate gene analysis.

LOX-1/LOXIN: the yin/yang of atheroscleorosis.

Atherosclerosis is the first cause of death in industrialized countries. Together with traditional risk factors (male gender, hypercholesterolemia, hypertension, diabetes, smoking and age), non-traditional risk factors have also been described as predisposing to this disease. Among these, oxidized low density lipoproteins (OxLDL) have been described in correlation to many proatherogenic processes. Many of the effects of OxLDL are mediated by the lectin like oxidized low density lipoprotein receptor 1 (LOX-1), expressed on endothelial cells, macrophages, SMCs and platelets. LOX-1 is encoded by the lectin like oxidized low density lipoprotein receptor 1 (OLR1) gene, located in the p12.3–p13.2 region of human chromosome 12. Variations on this gene have been studied extensively both at the functional and epidemiological level. Despite the fact that functional roles for two variants have been demonstrated, the epidemiological studies have provided inconsistent and inconclusive results. Of particular interest, it has been demonstrated that a linkage disequilibirum block of SNPs located in the intronic sequence of the OLR1 gene modulates the alternative splicing of OLR1 mRNA, leading to different ratios of LOX-1 full receptor and LOXIN, an isoform lacking part of the functional domain. As demonstrated, LOXIN acts by blocking the negative effective of LOX-1 activation. Here we review the state of the art regarding LOX-1, LOXIN, and the functional effects that are associated with the interaction of these molecules.

Regression and shift in composition of coronary atherosclerotic plaques by pioglitazone: insight from an intravascular ultrasound analysis.

Background Plaque reduction with the use of pioglitazone and statin combination therapy has been observed in carotid plaque. We sought to investigate the effect of combination therapy with statins and pioglitazone on coronary plaque regression and composition with the use of intravascular ultrasound (IVUS) and intravascular ultrasound-virtual histology (IVUS-VH). Methods We analysed 29 plaques in 25 diabetic patients with angiographic evidence of nonsignificant coronary lesions with IVUS-VH. Patients were treated with 80 mg of atorvastatin and 30 mg of pioglitazone daily for 6 months. After 6 months of therapy, IVUS-VH of each lesion was reacquired. Results Mean elastic external membrane volume was significantly reduced between baseline and follow-up (343.9 vs. 320.5 mm3; P < 0.05) as was mean total atheroma volume (179.3 vs. 166.6 mm3; P < 0.05). Change in total atheroma volume showed a 6.3% mean reduction. Areas of fibrous tissue, fibrolipidic tissue and calcium decreased over the 6 months of follow-up, although not significantly. On the other hand, the necrotic core increased from 9 to 14% (P < 0.05). Conclusion Our data demonstrated that atorvastatin/pioglitazone association is able to induce significant regression of coronary atherosclerosis, acting on plaque composition. Our findings are preliminary results and will be confirmed in an ongoing randomized placebo-controlled multicenter trial (PIPER; Pioglitazone for Prevention of Restenosis in Diabetics with Complex Lesion; trial registration: clinical trials.gov. Identifier: NCT 00376870).