Caratterizzazione del trascrittoma di PBMCs di pazienti affetti da aneurisma dell'aorta addominale e da ostruzione carotidea

Atherosclerosis (also known as Arteriosclerotic Vascular Disease or ASVD) is the condition in which an artery wall thickens as the result of a build-up of fatty materials such as cholesterol. It is a syndrome affecting arterial blood vessels, a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by Low-density lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL). It is commonly referred to as a hardening or furring of the arteries. It is caused by the formation of multiple plaques within the arteries. Other arteriosclerotic disease is aortic aneurysm usually affecting the abdominal tract of the vessel. The prevalence of abdominal aortic aneurysm (AAA) varies ranging from 4.1% to 11.5% in European men. This disorder is characterized by localized structural deterioration of the aortic wall, leading to progressive dilatation and eventual aortic rupture. Rupture of AAA is responsible for 1.5% of the total mortality in males over 55 years of age. Both disease seem to share common causes in alteration in lipid metabolism and immune response against host and oxidised lipids, leading to a condition of chronic inflammation affecting all the vascular system, especially the big diameter arteries. There are several factors such as smoking, hypertension, hypercholesterolemia and male sex, which are well known risk factors for the development of AAA and carotid stenosis. However, better understanding of molecular mechanisms is an important step toward clarification of the pathophysiology, identification of genetic and molecular biomarkers and development of new therapeutic strategies for AAA and atherosclerosis in general. Gene expression profiling studies by microarray technologies are particularly appropriate to investigate and create working hypotheses to understand the pathophysiology of complex genetic tracts such as arteriosclerotic diseases. Previous microarray studies focused on AAA utilized RNA derived from aortic tissue samples. The use of tissue samples has several disadvantages, including the difficulty of obtaining control samples and bias introduced by use of normal specimens from non-age-matched cadavers, organ-transplant donors or patients with different diseases. Peripheral blood is a complex fluid with a high cellular turnover rate that provides physiological connectivity between tissues. Environmental or disease perturbations in the body may leave molecular signatures detectable by analyzing blood-derived RNA. Most importantly, since blood samples can be obtained readily and with little discomfort to patients, biomarkers derived from blood RNA provide an easier integration to clinical and imaging data for the diagnosis and prognosis of AAA and carotid stenosis. In this study we investigate the gene expression profile of venous whole peripheral blood obtained from AAA patients by using microarray technology to provide insight into systemic pathophysiological processes involved in this disease. Using the data obtained from the microarray analysis from which we detected 91 genes differentially expressed from patients and positive controls, we selected 10 gene belonging to pathways involved in lipid metabolism and immune response to deep our analysis and eventually confirm the microarray data in order to set up new reliable parameters for arteriosclerosis diseases diagnosis and prognosis. The genes we chosen are Lipid Metabolism: 8. Monoglyceride lipase - MGLL - U67963 - 11343 9. Free fatty acid receptor 2 - FFAR2 - AF024690 – 2867 10. Adiponectin receptor 1 - ADIPOR1 - AK001484 – 51094 11. Phospholipase A2, group IB (pancreas) - PLA2G1B - M21054 – 5319 12. Hydroxysteroid (17-beta) dehydrogenase 14 - HSD17B14 - NM_016246 3738 13. Acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain - ACADS Z80345 – 682 14. Low density lipoprotein receptor-related protein 5 - LRP5 - AF077820 - 4041 Immune system: 4. Toll-like receptor adaptor molecule 1 - TICAM1 - AF070530 - 7746 5. Interleukin 1 receptor accessory protein-like 1 - IL1RAPL1 - AJ243874 11141 6. Tumor necrosis factor type 1 receptor-associated protein - TRAP1 NM_016292 – 10131 We designed 10 couples of primers to analyze the expression levels of the genes onto 3 groups of subjects: 1. Control: N=40 2. AAA patients: N=40 3. Patients affected from carotid artery stenosis: N=40 We first analyze the expression levels of the control gene GAPDH of each subject and than we check the expression levels of the all ten genes comparing the resulting CT values with the ones gotten from the housekeeping gene. The result is the so-called ∆CT and we got these values for all the 120 subjects for everyone of the 10 genes in duplicates for a total of 1200 ∆CT values. Than we compare the average ∆CT values of the 80 patients (40 AAA + 40 carotid stenosis) with the control values to get the ∆∆CT values from which calculate the fold number (2-∆∆CT =FOLD) that provides a numeric but qualitative value of the expression level of the target gene that allow to compare the values with others present in literature. Our analysis are only semiquantitative for the moment but we first needed to check the reliability of the experimental approach. For the future studies we are going to use Taqman® probes to rise the specificity and sensitivity of the analysis, introducing a quantitative method using titolation commercial kit that allow to quantify the exact numbers of copies of an m-RNA transcript calculating the exact amount of m-RNA by substituting the CT values in a linear equation. We confirmed the microarray data and extended the results to the carotid stenosis patients. We were able to confirm the genes upregulation and hypothesise that PBMCs could be a suitable base for the detection of novel risk and prognostic indicators for the arteriosclerotic diseases. In fact our aim is to identify new easy-to-detect markers to predict the risk of developing an artery stenosis or most important, an aneurismatic lesion that is symptomless until the very late phases and whose success in treating is proportional to well-timed diagnosis. In the end seems that the right direction to look into are the metabolism of lipids and phospholipids, that are involved in eicosanoids production and degradation (via fatty acid metabolism) and cell signalling in the immune system.