GENETIC AND EXPRESSION ANALYSIS OF CANDIDATE GENES IN CAROTID PLAQUE AND PERIPHERAL BLOOD OF PATIENTS WITH ATHEROSCLEROSIS

L'aterosclerosi, un processo sistemico patologico, \ue8 la causa fondamentale della maggior parte degli eventi cardiovascolari, come ictus e infarto del miocardio. L'infiammazione gioca un ruolo centrale nello sviluppo della malattia aterosclerotica, dalle prime fasi di formazione della lesione alla rottura della placca, causa principale delle sindromi acute ischemiche. La prostaglandina E2 (PGE2) \ue8 un prostanoide implicato in varie patologie, la cui concentrazione risulta elevata nel processo infiammatorio. La ciclossigenasi (COX) e la prostaglandina E sintasi (PGES) sono gli enzimi responsabili della sintesi di PGE2. In questo lavoro di tesi abbiamo investigato se geni della via COX-2/PGE2 dipendente possono essere differenzialmente espressi nelle placche carotidee e nel sangue periferico di pazienti sintomatici rispetto a pazienti asintomatici. Al fine di valutare se i geni sovraespressi nel sangue periferico di pazienti con ictus siano specificamente associati all\u2019ictus ischemico aterotrombotico, abbiamo analizzato l'espressione di questi geni anche in pazienti con ictus cardioembolico. Abbiamo indagato, inoltre, se polimorfismi specifici nella regione del promotore dei geni up-regolati possano influenzarne l\u2019espressione. Mediante Real Time PCR \ue8 stata analizzata l\u2019espressione dei geni COX-2, TLR4, ACE, EP4, EP3, EPRAP e FACL4 e la loro correlazione \ue8 stata studiata mediante coefficiente di Pearson e modelli di regressione lineare. Da questo studio \ue8 emerso che i geni TLR4 e COX-2 sono sovra espressi nel sangue periferico di pazienti sintomatici e la loro espressione \ue8 risultata fortemente correlata. Entrambi i geni sono sovra regolati specificatamente nei pazienti con ictus aterotrombotico. Il polimorfismo -2604G>A \ue8 risultato associato ai livelli di espressione del gene TLR4 nel sangue periferico. In particolare, la presenza dell\u2019allele -2604A in omozigosi porta ad una diminuzione dell\u2019espressione genica. Analisi in silico hanno evidenziato che allele -2604A crea un sito di legame putativo per GATA-2, un regolatore negativo della trascrizione. Nonostante questi risultati debbano essere confermati in studi indipendenti, essi suggeriscono che il meccanismo periferico di danno infiammatorio dopo evento ischemico aterotrombotico pu\uf2 essere mediato da TLR4 attraverso una via COX-2-dipendente. Inoltre, la specificit\ue0 di entrambi i geni per l'ictus di origine aterotrombotico indica che l'analisi della loro espressione genica nel sangue periferico potrebbe essere usata come biomarker per la diagnosi del sottotipo di ictus. La deregolazione del gene TLR4 potrebbe essere influenzata dalla presenza del polimorfismo -2604G>A in pazienti aterosclerotici. Studi caso/controllo in popolazioni pi\uf9 grandi saranno necessari per determinare se questa variazione possa rappresentare un fattore di rischio per l'aterosclerosi.Atherosclerosis, a systemic pathological process, is the underlying cause of the majority of clinical cardiovascular events, such as stroke and myocardial infarction. Inflammation plays a central role in the development of atherosclerotic disease, from the early phases of lesion formation to plaque disruption, the main underlying cause of acute ischemic syndromes. Prostaglandin E2 (PGE2) is a prostanoid implicated in a variety of pathologies, whose concentration is elevated in inflammatory process. Cyclooxygenase (COX) and Prostaglandin E Synthase (PGES) are enzymes responsible for PGE2 synthesis. In this work we investigated if genes of COX-2/PGE2 pathway could be differentially expressed in carotid plaques and peripheral blood of symptomatic compared to asymptomatic patients. In order to evaluate if upregulated genes in peripheral blood of stroke patients may be specifically associated to atherothrombotic ischemic stroke, we analyzed expression of these genes also in cardioembolic stroke patients. Moreover, we investigated if specific polymorphisms in the promoter region of the upregulated genes could influence their expression. Expression of COX-2, TLR4, ACE, EP4, EP3, EPRAP and FACL4 genes was analyzed by Real Time PCR and their correlation was studied by Pearson coefficient and linear regression models. The results obtained by this study showed that TLR4 and COX-2 genes were upregulated and their expression was strongly correlated in peripheral blood of symptomatic patients. Both COX-2 and TLR4 genes are overexpressed only in atherothrombotic stroke. -2604G>A polimorphism is associated with the levels of TLR4 gene expression in peripheral blood. In particular, the presence of allele -2604A in homozygosis resulted in diminished gene expression. Analysis in silico evidenced that allele -2604A creates a putative binding site for GATA-2, a negative transcription regulator. Although these results would need to be confirmed by independent studies, they suggest that the peripheral mechanism of inflammatory injury after atherothrombotic stroke may be mediated by TLR4 through a COX-2-dependent pathway. Furthermore, the specificity of both genes for atherothrombotic stroke indicates that gene expression analysis in peripheral blood might be used as biomarkers for stroke subtype diagnosis. The deregulation of the gene TLR4 may be due by the presence of the polymorphism-2604G>A in atherosclerotic patients. Case/control studies in larger populations are warranted to determine if this variation can represent a risk factor for atherosclerosis

Molecular detection of bacterial RNA in atheromatous plaques of patients with stroke

Aim: Infections and chronic inflammatory conditions such as periodontitis, have been associated with the development and progression of atherosclerosic process and susceptibility to plaque rupture. The chronicity of periodontal disease provides a rich source of subgingival bacteria, mostly gram negative, which are frequently spread in the blood as a result of the periodontal lesion. DNA from periodontal pathogens has been detected in atherosclerotic lesions, but viable bacteria have not yet been isolated. This study was carried out to detect, by molecular techniques, the presence of RNA of bacteria in atheromatous plaques collected from both symptomatic and asymptomatic carotid stenosis patients. Methods: Total RNA extracts, derived from eighty-one atheromatous plaques collected during carotid endartrectomy (36 from symptomatic patients and 45 from asymptomatic patients) underwent reverse transcription to cDNA using random primers. All cDNA samples obtained were tested by PCR using universal primers for global bacterial detection (16S rRNA). Results: Of eighty-one sample tested, seven were found positive (8.6%), six out of the thirty-six (16.6%) of symptomatic patients and only one out forty-five (2.2%) from the asymptomatic patients group. Conclusion: This pilot study suggests the preferential presence of bacterial RNA in recently symptomatic atherosclerotic plaques. These results are of interest because the presence of viable bacteria may be presumed into the atheromatous plaques and, therefore, indicates a possible bacterial role in activation of several inflammatory factors and in atherosclerotic plaque vulnerability

High levels of COX-2 gene expression in peripheral blood of cardioembolic and atherothrombotic stroke patients

Aim: To determine if COX-2 and TLR4 gene expression in atherothrombotic strokes represents a marker of atherosclerotic plaque destabilization and rupture, or it rather is related to the inflammatory response caused by stroke itself. Secondly, to determine if COX-2 and TLR4 gene expression could be specific for stroke subtype. Methods: Total RNA was extracted from peripheral blood samples from atherosclerotic (N=30) and cardioembolic stroke (N=25) patients, and controls (N=27). Gene expression was analysed by real time RT-PCR. Results: Expression of COX-2 gene was increased in cardioembolic compared to atherothrombotic stroke patients or healthy controls (p<0.0001). TLR4 expression was higher cardioembolic stroke patients compared to controls (p=0.0001). Conclusions: Cardioembolic show higher levels of COX-2 expression compared to atherothrombotic stroke patients independent of clinical severity. Additional studies are warrant to establish if high level of COX-2 gene expression could be a marker of cardioembolic stroke

HDAC9, TWIST1 and FERD3L gene expression in asymptomatic stable and unstable carotid plaques

BACKGROUND AND OBJECTIVES: A variant located at the end of HDAC9 gene within clusters of DNAse I sensitivity zones and histone modification hotspots has been associated with large vessel stroke and could be linked to plaque instability. The aim of the study is to define if an altered expression of HDAC9, TWIST1 and FERD3L genes could be involved in plaque vulnerability.METHODS: Histological classification and gene expression analysis were performed in 6 stable and 16 unstable plaques obtained from asymptomatic patients undergoing endarterectomy. Gene expression was analysed by real-time PCR.RESULTS AND CONCLUSIONS: TWIST1 gene expression resulted higher in stable plaques (P &lt; 0.02). HDAC9 gene expression followed a similar trend (P = 0.11). These results highlighting the significant correlation between TWIST and HDAC9 gene expression suggest that both genes may contribute to plaque stability in a coordinated way

TLR4 -2604G>A variant confers differential DNA binding capacity to transcription factors of the GATA family and alters gene expression in peripheral blood of atherosclerotic patients.

Aim: Toll-like receptor-4 (TLR4) is a primary receptor of the innate immune reaction and compelling evidence demonstrate its envelopment in the pathogenesis of atherosclerosis. TLR4 is constitutively expressed on monocytes and endothelial cells, and it has been found to be highly expressed in atherosclerotic plaques, and in peripheral blood of patients after ischemic stroke. Polymorphisms in the promoter region of this gene may represent genetic risk factors involved in the predisposition to atherosclerotic disease. In this study we investigated the effect on TLR4 gene expression of three polymorphisms located in the 5\u2019 upstream region of the gene in peripheral blood of atherosclerotic patients. Methods: Polymorphisms -1607T>C (rs10759932) and -2026A>G (rs1927914) were genotyped by PCR-RFLP, and gene expression analysis was performed by Real Time PCR using Sybr Green in 62 atherosclerotic patients. Results: RNA from patients carrying the allele -2604A in homozygosis showed a lower expression of the gene when compared to patients carrying the counterparts GG + GA (P<0.02). EMSA assays demonstrated that allele -2604A confers a higher DNA binding capacity to transcription factors of the GATA family. Conclusions: The presence of allele -2604A is associated to a diminished level of gene expression in peripheral blood of patients with advanced carotid atherosclerotic plaques, possibly by creating a transcription factor binding site for a negative modulator(s) of transcription. Case/control studies in larger populations are worthy to determine if variants of polymorphism -2604A>G of TLR4 might represent a genetic risk factor for susceptibility to atherosclerotic plaque development and progression

Correlations between gene expression highlight a different activation of ACE/TLR4/PTGS2 signaling in symptomatic and asymptomatic plaques in atherosclerotic patients

Inflammation has a key role and translates the effects of many known risk factors for the disease in atherosclerotic vulnerable plaques. Aiming to look into the elements that induce the development of either a vulnerable or stable atherosclerotic plaque, and considering that inflammation has a central role in the progression of lesions, we analyzed the expression of genes involved in the ACE/TLR4/PTGS2 signaling in carotid plaques of symptomatic and asymptomatic patients. Patients with internal carotid artery stenosis undergoing carotid endarterectomy at Verona University Hospital were included in this study. A total of 71 patients was considered for gene expression analysis (29 atherothrombotic stroke patients and 42 asymptomatic patients). Total RNA was extracted from the excised plaques and expression of PTGS2, ACE, TLR4, PTGER4, PTGER3, EPRAP and ACSL4 genes was analyzed by real-time PCR. The correlation between the pair of genes was studied by Spearman coefficient. From the analyzed genes, we did not observe any individual difference in gene expression but the network of co-expressed genes suggests a different activation of pathways in the two groups of plaques

Expression of circulating miR-17-92 cluster and HDAC9 gene in atherosclerotic patients with unstable and stable carotid plaques

Aims: The miR-17-92 cluster and HDAC9 gene are involved in inflammatory, apoptotic, and angiogenic processes that are activated in the vulnerable carotid plaque. The aim of this research was to determine whether expression of one or more of the miRs of the cluster and/or HDAC9 expression could represent biomarkers for patients with unstable atherosclerotic carotid plaques. Materials and Methods: Plasma levels of miRs and HDAC9 expression in peripheral blood were analyzed by real-time PCR in patients with histologically classified stable or unstable plaques. Results: No differences were observed between the two groups. Discussion and Conclusions: Levels of the miR-17-92 cluster in plasma and HDAC9 gene expression in peripheral blood cannot be considered appropriate biomarkers to identify patients with unstable plaques at risk of rupture

AUMENTATA ESPRESSIONE DEL GENE COX-2 NEL SANGUE PERIFERICO DI PAZIENTI CON ICTUS ISCHEMICO

Obiettivo: Numerosi studi hanno riportato uno stato infiammatorio a seguito di un evento ischemico cerebrale. L’aumentata espressione di mediatori della cascata attivata dalla COX-2 è stata osservata in placche carotidee sintomatiche, ipotizzando quindi un coinvolgimento di tale via infiammatoria nell’evoluzione verso l’instabilità della placca. Scopo del presente studio è investigare se l’attivazione della cascata infiammatoria dopo un evento ischemico cerebrale sia collegato alla destabilizzazione del processo aterosclerotico sottostante o all’evento ischemico cerebrale in sé. Materiali e Metodi: E’ stato analizzato l’RNA estratto da sangue periferico, ottenuto da: 1) pazienti sottoposti ad endoarterectomia carotidea asintomatica (60 pazienti): 2) pazienti sottoposti ad endoarterectomia per stenosi carotidea recentemente sintomatica (40 pazienti); 3) pazienti con recente ictus cardioembolico (35 pazienti); 4) 27 controlli sani. E’ stata quindi studiata l’espressione di Toll-like Receptor 4 e COX-2 tramite real time PCR su sangue periferico a 24 h, 72 h e a 7 giorni dall’evento ischemico. La gravità clinica dei pazienti sintomatici è stata classificata secondo la scala NIHSS; l’entità della stenosi dei pazienti con stenosi carotidea secondo metodo NASCET.Risultati: L’espressione di COX-2 è risultata significativamente aumentata in entrambi i gruppi di pazienti con recente ictus a confronto con i gruppi di controllo (stenosi carotidea asintomatica e controlli sani) a 24 ore (p 0.02) e a 7 giorni (p 0.001). Si evidenzia inoltre una sovra espressione statisticamente significativa di COX-2 nei pazienti con ictus cardioembolico rispetto a quelli con stenosi sintomatica (p 1.847^10-6 a 7 giorni).Conclusioni: I livelli di COX-2 nei pazienti con recente ictus risultano elevati fino a 7 giorni dopo l’evento, indipendentemente da età, sesso, gravità clinica, e fattori di rischio. Rimane da chiarire la possibile causa della differente espressione tra i due gruppi affetti. Benché i pazienti con ictus cardioembolico abbiano mediamente gravità superiore a quelli con stenosi carotidea, non è stata verificata una correlazione fra espressione di COX2 e TLR4 con la gravità dell’evento. Si potrebbe ipotizzare un effetto sull’espressione di COX2 da parte di classi di terapia nei due gruppi di pazienti sintomatici, in particolare l’aspirina, che era significativamente più utilizzata nei pazienti con ictus aterotrombotico rispetto ai pazienti con ictus cardioembolico (p 0,00007)