Alessandra Pasquali

Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries

BackgroundAtherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.ResultsWe used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway.Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1alpha, MIP-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.ConclusionThe pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.

Impact of Insulin Receptor Substrate-1 Genotypes on Platelet Reactivity and Cardiovascular Outcomes in Patients With Type 2 Diabetes Mellitus and Coronary Artery Disease

OBJECTIVES The aim of this study was to assess the association between genetic variants of the insulin receptor substrate (IRS)-1 gene, platelet function, and long-term outcomes in patients with type 2 diabetes mellitus (DM) and stable coronary artery disease while on aspirin and clopidogrel therapy. BACKGROUND The effects of pharmacogenetic determinants on platelet function and cardiovascular outcomes in type DM patients are unknown. METHODS The association between IRS-1 genetic variants, platelet function, and the risk of major adverse cardiac events (MACE) at 2 years was assessed in 187 patients with type 2 DM and stable coronary artery disease on maintenance aspirin and clopidogrel therapy. RESULTS Seven tag single nucleotide polymorphisms were selected. Individuals with high platelet reactivity were more frequent among carriers of the C allele (GC and CC genotypes; approximately 20% of population) of the rs956115 marker (44.4% vs. 20.5%; odds ratio: 3.1, 95% confidence interval [CI]: 1.44 to 6.67; p = 0.006). These patients were at higher risk of MACE (28.0% vs. 10.9%; hazard ratio: 2.90, 95% CI: 1.38 to 6.11; p = 0.005). The C allele carriers of the rs956115 marker were more commonly associated with a hyperreactive platelet phenotype. This was confirmed in an external validation cohort of patients with type 2 DM but not in an external validation cohort of patients without DM. Carriers of the C allele of the rs956115 marker also had a significantly higher risk of MACE compared with noncarriers (30.6% vs. 11.4%; hazard ratio: 2.88, 95% CI: 1.35 to 6.14; p = 0.006). CONCLUSIONS Type 2 DM patients who are carriers of the C allele of the rs956115 marker of the IRS-1 gene have a hyperreactive platelet phenotype and increased risk of MACE.

The association of rs4307059 and rs35678 markers with autism spectrum disorders is replicated in Italian families

Objective The objective of this study was to replicate an association study on a newly collected Italian autism spectrum disorder (ASD) cohort by studying the genetic markers associated with ASDs from recent genome-wide and candidate gene association studies. Methods We have genotyped 746 individuals from 227 families of the Italian Autism Network using allelic discrimination TaqMan assays for seven common single-nucleotide polymorphisms: rs2292813 (SLC25A12 gene), rs35678 (ATP2B2 gene), rs4307059 (between CDH9 and CDH10 genes), rs10513025 (between SEMA5A and TAS2R1 genes), rs6872664 (PITX1 gene), rs1861972 (EN2 gene), and rs4141463 (MACROD2 gene). A family-based association study was conducted. Results A significant association was found for two of seven markers: rs4307059 T allele (odds ratio: 1.758, SE=0.236; P-value=0.017) and rs35678 TC genotype (odds ratio: 0.528, SE=0.199; P-value=0.0013). Conclusion A preferential allele transmission of two markers located at loci previously associated with social and verbal communication skill has been confirmed in patients of a new ASD family sample.

A Preliminary microRNA Analysis of Non Syndromic Thoracic Aortic Aneurysms.

ABSTRACT The development of thoracic aortic aneurysms (TAAs) involves a multifactorial process resulting in alterations of the structure and composition of the extracellular matrix (ECM). Recently, modifications in microRNA (miRNA) expression were implicated in the pathogenesis of TAA. This study presents a preliminary miRNA microarray analysis conducted on pooled ascending aorta RNAs obtained from non familial non syndromic TAA patients (five males and five females) compared to matched control pools. Ninety-nine differentially expressed miRNAs with >1.5-foldup- or down-regulation in TAAs compared to controls were identified, 16.0% of which were similarly regulated in the two sexes. Genes putatively targeted by differentially expressed miRNAs belonged preferentially to focal adhesion and adherens junction pathways. The results indicate an altered regulation of miRNA-mediated gene expression in the cellular interactions of aneurysmal aortic wall.

Detection of a large deletion in the P-selectin (SELP) gene

P-selectin is an adhesion molecule involved in the pathogenesis of inflammation, thrombosis, and oncogenesis. In this study of 51 polymorphisms in candidate genes for cardiovascular disease in 1561 individuals, we identified a new allelic variant of the SELP gene, g.18196_20704del, that determined the lack of genotyping for one polymorphism in one individual. It is a deletion of 2509 nucleotides which starts in intron 6 and ends in intron 8. Re-genotyping of 1023 apparent homozygotes indicated an overall allele frequency of 0.27%. The inclusion of this allelic variant in genetic association studies will avoid genotyping errors and marginally improve the sensitivity.

A novel synonymous substitution in the GCK gene causes aberrant splicing in an Italian patient with GCK-MODY phenotype

GCK gene analysis in an Italian MODY patient revealed a novel synonymous substitution in exon 4 (c.459T>G; p.Pro153Pro) resulting in an aberrant transcript lacking the last eight codons of the same exon. Our findings emphazise the importance of not underestimating synonymous variations when screening for disease-causing mutations.

Studies on sporadic non-syndromic thoracic aortic aneurysms: 1. Deregulation of Jagged/Notch 1 homeostasis and selection of synthetic/secretor phenotype smooth muscle cells

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. The study aimed at defining the peculiar morphologic and molecular changes occurring in the media layer of SNSTAAs. Design This study was based on a single centre design. Methods Media layer samples taken from seven carefully selected SNSTAAs and seven reference patients (controls) were investigated via quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, quantitative histology, and immunohistochemistry/immunofluorescence. Results In SNSTAAs media, aortic smooth muscle cells numbers were halved due to an apoptotic process coupled with a negligible cell proliferation. Cystathionine γ-lyase was diffusely up-regulated. Surviving aortic smooth muscle cells exhibited diverging phenotypes: in inner- and outer-media contractile cells prevailed, having higher contents of smooth-muscle-α-actin holoprotein (45-kDa) and of caspase-3-cleaved smooth-muscle-α-actin 25-kDa fragments; in mid-media, aortic smooth muscle cells exhibited a synthetic/secretor phenotype, down-regulating vimentin, but up-regulating glial fibrillary acidic protein, trans-Golgi network 46 protein, Jagged1 (172-kDa) holoprotein, and Jagged1’s receptor Notch1. Extracellular soluble Jagged1 (42-kDa) fragments accumulated. Conclusions In SNSTAAs, there is a relentless aortic smooth muscle cells attrition caused by the up-regulated cystathionine γ-lyase. In mid-media, synthetic/secretor aortic smooth muscle cells intensify Jagged1/NOTCH1 signalling in the attempt to counterbalance the weakened aortic wall, due to aortic smooth muscle cells net loss and mechanical stress. Synthetic/secretor aortic smooth muscle cells are apoptosis-prone, and the accruing thrombin-cleaved Jagged1 fragments counteract the otherwise useful effects of Jagged1/NOTCH1 signalling, thus hampering tissue homeostasis/remodelling, and aortic smooth muscle cells adhesion, differentiation, and migration.

Studies on sporadic non-syndromic thoracic aortic aneurysms: II. Alterations of extra-cellular matrix components and focal adhesion proteins

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.