Investigation on the role of apoptosis in aneurysm rupture

Project location: ITALY
Project start date: September 2001 - Project end date: September 2002
Project number: 2001-13
Beneficiary: ATENA Onlus

Aneurysm: 2003 Project Description

Comparative Evaluation Through Microarray Technology of Gene Expression Profiles in Ruptured and Unruptured Human Intracranial Aneurysms. Preliminary Study for the Feasibility of a Genetic Screening for Intracranial Aneurysms in the General Population

Cerebrovascular diseases represent one of the most relevant causes of morbidity and mortality. About one-quarter of these diseases are reported to be caused by subarachnoid haemorrhage (SAH) from ruptured intracranial aneurysms (IAs). SAH has a great social relevance, among patients, 50% die at the time of aneurysm rupture or shortly thereafter, and 25% suffer permanent disability. Intracranial aneurysm growth and rupture represent the end-stage of a progressive alteration of intracranial arterial walls, depending on factors presently unknown, whose identification appears fundamental. Their preliminary investigations demonstrated how apoptosis, a genetic program of cell death, appears clearly enhanced in ruptured aneurysms, sustaining the hypothesis that apoptotic phenomena could play a role in aneurysm growth and rupture. It is here proposed a comparative evaluation of gene expression in ruptured and unruptured aneurysm specimens, in arteries obtained from the same patients as well as from control arteries, obtained from patients operated on for intracranial non vascular malformative disease. Such evaluation could clarify, depending upon the peculiar types of genes expressed, the possible genetic cause/s triggering the enlargement and rupture of aneurysmal wall. The management of aneurysms could be dramatically improved by identifying genes involved in aneurysm growth and rupture clarifying the real factors responsible of such pathology. This research project, financed by The Nando Peretti Foundation, represents an innovative and original approach in understanding the complex aspects of intracranial aneurysms aetiology and in improving their management. Few papers have been published about practical screening of intracranial aneurysms in familiar and/or general population. Presently the definitive diagnostic tool for intracranial aneurysms is cerebral angiography, although, Angio-RMN and Angio-TC have dramatically increased the possibility of a non invasive diagnosis for unruptured IAs. Nevertheless the prevalence of intracranial aneurysms in the general population is too low to justify the application of such examinations as screening methods. It is therefore accepted that screening for aneurysms in the general population is not practically feasible. An innovative, preliminary method of approaching this problem is here proposed. Presently the vast majority of intracranial aneurysms is still detected in the population only after their bleeding and the devastating event of a subarachnoid hemorrhage. If proven to be of efficacy, our study could bring a solution to one of the more challenging aspects of modern neurosurgical practice. The pre-rupture practical screening of intracranial aneurysms, and among them, the individuation of those at higher risk of rupture, should be also regarded as a major achievement in medical progress and of the greatest social utility.



Intracranial aneurysms appear as a focal dilatation of cerebral arteries, which could cause serious intracranial haemorrhage. The prevalence of intracranial aneurysms in the general population is 2-5%, while the incidence of SAH from intracranial aneurysms is only 10/100,000 population per year. From a simple calculation it appears obvious that the vast majority of subjects harbouring an unruptured intracranial aneurysm will never develop a SAH. With the remarkable progresses in diagnostic modalities, the number of unruptured aneurysm diagnoses is progressively increasing. Unfortunately, this means "surgery for unruptured aneurysms" even in people in which maybe, intracranial hemorrhage would have never occur. Therefore, the identification of patients harbouring unruptured aneurysms, and the individuation, among those cases, of aneurysms presenting an high risk of rupture, represents fundamental steps in the prevention of SAH.

The exact aetiology of intracranial aneurysms growth and rupture remains unclear. They appear the results from an interplay between haemodynamic and structural properties of intracranial arterial walls. Congenital medial defect and degenerative changes affected by shear stress and hypertension play an important role. Aneurysmal rupture could represent the end-stage of a progressive alteration of its walls, depending on factors which are presently unknown.
The association of various heritable disorders with intracranial aneurysms and their familiar aggregation, suggests a relevant role of genetic factors in their cause and pathogenesis. In order to structure a prevention of SAH from a ruptured intracranial aneurysm, it appears fundamental to identify possible genetic markers indicating the presence of an intracranial aneurysm, and its tendency toward rupture. A recent study based on SAGE (serial analysis of gene expression) performed on samples of intracranial aneurysm, showed that arterial dilatation is a highly dynamic cellular environment in which high levels of expression of genes previously associated with wound healing and tissue/extracellular matrix (ECM) remodelling take place. This tissue turnover process could be related with apoptosis, and needs to be investigated. The goal should be the identification of a pathway of programmed tissue degeneration. Recently it has been demonstrated by our group, that apoptosis, genetically programmed cell death, commonly observed in a wide number of physiological events and in several pathological scenarios, is involved and may play a relevant role in the process of aneurysm growth and rupture. There is evidence the pathogenesis of cerebral aneurysms growth and rupture has a significant genetic component. A number of heritable connective tissue disorders can be associated with formation of intracranial aneurysms. The most firm association has been established for autosomal dominant polycystic kidney disease (ADPKD); intracranial aneurysms are detected in approximately 25% of ADPKD patients at autopsy and, conversely, ADPKD accounts for between 2 and 7% of all cases of intracranial aneurysms.
Approximately 10% of asymptomatic adults with ADPKD after imaging analysis resulted to have intracranial aneurysms, and is estimated that aneurysms are cause of death in 20% of patients with ADPKD.
Mutations have been identified in two genes: PKD2 (locus 4q21) and, in particular, in the gene PKD1 (locus 16p13.3) which results linked to the more severe forms. PKD1 encodes for the membrane protein Polycystin that play a role in maintaining the structural integrity of the extracellular matrices (ECM) of connective tissues. Also involved in ECM remodelling is fibrillin-1, a glycoprotein whose encoding gene (FBN1, locus 15q21.1) is mutated in Marfan Syndrome. A deficiency of collagen type III associated with numerous mutations in the gene encoding the pro (III) chain of type III collagen (COL3A1, locus 2q31), is cause of Ehlers-Danlos syndrome type IV.
In the case of Neurofibromatosis type 1 the mutations that cause the pathology are located in the gene NF1 (locus 17q11.2) encoding the neurofibromin, a protein which may influence microtubular function and so may play a regulatory role in connective tissue development.
Basing on this variability and considering the dynamic nature of the pathogenesis of aneurysms, the propose research project will be oriented to a gene expression analysis strategy.
Gene expression analysis will be performed on tissue samples obtained from unruptured and ruptured aneurysms, from superficial temporal arteries and/or middle meningeal arteries obtained in the same patients and in control cases operated on for non vascular malformative pathology. In all cases, a study of gene expression obtained from blood samples will also be performed.
Aim of our study will be the analysis of gene expression patterns on all collected tissue and blood samples. Our expectation is to find different gene expression patterns between normal and pathological cases and between different moments of aneurysm pathogenesis, in order to identify genetic patterns indicating the enhanced risk for their rupture. Again it will be investigated the possibility that peculiar genetic expression in peripheral arteries may be indicative for the risk of rupture in a subject harbouring an unruptured intracranial aneurysm. Some critical moments of aneurysmal development and rupture, could be related to the up-regulation or down-regulation of some genes resulting in their over-expression or under-expression respectively. After the detection of a pathologic gene expression pattern a genomic approach will be developed in order to study the correlation with known mutations or new mutations of target genes linked with connective tissue disorders Mutation detection techniques could also be employed to study genes that showed to be specifically over-expressed or repressed in aneurysmal dilatations, in order to identify new mutations and functional polymorphisms.
Intracranial aneurysms are associated with a highly dynamic cellular environment, characterized by high levels of expression of gene involved in extracellular matrices structure and remodelling.
The ATENA group chaired by Professor Giulio Maira will focus the attention on the expression of genes encoding extracellular matrices components as Fibronectin, Collagen type III -1, Collagen type I 1 e -2, Collagen type VI 1 e -2, Collagen type IV -1 and elastin. In addition, other genes encoding proteins involved in extracellular matrices turnover and cell migration and adhesion like tissue inhibitor of metalloproteinase-3 (TIMP-3), SPARC (osteonectin), hevin, connective tissue growth factor (CTGF), -galactosidase-binding lectin, cdc-rel2a/PNUTL2, vinculin, tetraspanin-5, c-Abl, -ig-h3, osteoblast-specific factor-2 (OSF-2) and cathepsins B and D.
They will study genes encoding factors of immune/inflammatory response which may be associated with a cellular infiltration consistent with dramatic tissue remodelling. In detail, the levels of expression of mRNAs encoding human leukocyte antigens of class I (2-microglobulin, HLA-C, HLA-B44 and HLA-B) and class II (HLA-DP4, HLA-Dw12, HLA-DR) will be evaluated, and also the expression of genes encoding the Immunoglobulin chain (IgG heavy chain and Ig light chain) will be included.
As recently demonstrated by the ATENA group, apoptotic phenomena in aneurysm wall, are strongly associated with the "condition of ruptured aneurysm" and could remarkably contribute in reducing aneurysm wall resistance, playing a role in the genesis of its rupture. Another gene expression pattern to be studied is therefore, that undergoing cellular apoptotis and its relations with tissue remodelling and, in particular, the expression levels of fas gene and fas-ligand, myc, Bcl-2 and Bax. Furthermore we propose an investigation of gene expression in extra cerebral cranial arterial vessels in the same patients of the above mentioned study, so to verify the possibility that aneurysmal rupture may represent just the epiphenomena of a generalised sub-clinical alteration of all arterial vessels. The implications of such hypothesis are paramount: should it be correct, a simple study of apoptotic levels, performed on a peripheral arterial vessel in a patient harbouring an unruptured intracranial aneurysm incidentally diagnosed, could give indirect relevant information on the level of apoptosis in the aneurysmal dome, therefore influencing consequently surgical decisions, case by case.



Methods and Objectives
The study will be performed in co-work with the collegues of the laboratory of molecular genetics at the Institute of Human Anatomy and Cellular Biology of our University. The work will be targeted in a preliminary phase of evaluation of RNA gene expression in aneurysm, cranial artery of aneurysm patients and cranial artery in non aneurysm patients. Subsequently the detected alterations will be studied on peripheral blood samples in aneurysm and non aneurysm patients, in order to detect wether abnormal expression of specific genes may be a reliable factor in identifying in an otherwise healthy subject, the presence of an unruptured intracranial aneurysm and the genetically programmed risk of its rupture. The study appears very interesting and promising as a potential tool for screening unruptured intracranial aneurysms in the general population.
Pathological specimens from aneurysms will be collected at surgery. After the aneurysm clipping by standard neurosurgical techniques, and the verification of its complete exclusion from brain circulation, aneurysmal dome will be resected only in cases in which the operating neurosurgeon will feel it as a completely safe procedure. To study comparatively gene expression patterns between aneurysms and peripheral arterial vessels, during the surgical approach, specimens of temporal superficial or of middle meningeal arteries will be collected. For the study of arterial specimens, the same procedures used for aneurysms, and here described, will be adopted. Aneurysmal specimens will be immediately pre-treated in the operating room eliminating from their surface blood clots. After their collection, specimens will be immediately putted into liquid nitrogen (- 162° C) for their temporary storage during the transport to the laboratory and stored subsequently at - 80° C for the temporary storage before the analyses. RNA and DNA extraction will be performed within the following 24 hours. For DNA analysis, peripheral blood samples will be subjected to QIAamp DNA Blood MiniKit (QIAGEN, Hilden, Germany) standard extraction protocols.

Gene Expression Analyses
For gene expression analysis, aneurysmal and arterial samples will be collected at surgery. The samples will be immediately snap frozen in liquid nitrogen and then stored at - 80° C until nucleic acid extraction. RNA extraction will be performed by using TRIzol according to manufacturer protocol (Life Technologies inc, Gaithersburg M.D.). Total RNA will be reprecipitated using RNAmate (Biochain, San Leandro, CA), and mRNA will be isolated using oligo(deoxythymidine)-decorated latex beads (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. In order to synthesise double strand cDNA we will use Superscript II reverse transcriptase (Life Technologies, Inc.) with a primer encoding the T7 RNA polymerase promoter linked to oligo. After cDNA (QIAquick QIAGEN) purification, an in vitro transcription with T7 RNA polymerase (MEGAscript T7 kit, Ambion, Inc., Austin TX) incorporing biotin-UTP and biotin-CTP, will be performed to obtain the copy RNA (cRNA). The cRNA will be added to hybridization buffer after purification and fragmentation, and then hybridised to arrays. After washing, microarray will be stained and scanned with a dedicated instrument to capture a fluorescence image.
The DNA-chip set up will be demanded to Affymetrix service that will customize chips specific for our items. Data collection and bio-informatic data organization will be realised by chip manufacturer who will calculate index of gene expression for each probeset according to algorithms used by Affymetrix.

Mutation Analyses
Mutation detection will be performed by DNA amplification with specific primers and by denaturing high-performance liquid chromatography (DHPLC) technique. The DHPLC assay of PCR products is oriented to detect polymorphisms (heteroduplex) basing on the study of the melting profile by a fragment analysis system. Those samples which will show aberrant elution peaks will be re-amplified and subjected to direct sequencing with Big-Dye Terminator method.

Statistical Analysis
The ?2 test will be used for a statistical analysis of our results. Values of p< or equal to 0.05 will be considered as statistically significant.

Anticipated achievements or outcomes of the project
This study is focused on evaluating the role of gene expression component in intracranial aneurysms. To reach this purpose the ATENA Group will investigate the influence of gene mutation linked with heritable connective tissue disorders associated with intracranial aneurysms. In a second phase, ATENA will extract RNA from aneurysm tissue samples (ruptured and unruptured), from peripheral arteries in the same cases and from normal control arterial samples. Thereafter a retrotranscription reaction from mRNA to cDNA will be performed. The obtained cDNA will be subjected to a hybridisation on DNA chip according to microarray technology. The data from different samples will be matched with a bio-informatic support in order to detect a differential gene expression between ruptured and unruptured aneurysms and in normal and pathological arterial samples. If genes specifically up or down-regulated in certain moments of aneurysmal pathogenesis will be detected, the presence of mutations/polymorphisms in these genes using the DHPLC technology (denaturant high performance liquid chromatography) will be searched, and then passing to automated DNA sequencing to identify the type of mutation.
If ATENA Group can reach all these items it will be possible to evaluate the real role of genetic susceptibility to aneurysms, and it will be possible to know which genes are specifically over-expressed in intracranial aneurysms evaluating the role of programmed cellular degeneration versus extra-cellular environmental remodelling and immune/inflammatory response. An analysis of the same patterns on patient's and normal controls blood samples, will assess potential genetic parameters to screen the general population for unruptured intracranial aneurysms and possibly to predict which of these will be at higher risk for rupture.

The targets of our study could be summarized as follow:

Phase 1
Evaluation of gene mutation linked to heritable connective tissue disorders associated with intracranial aneurysms.

Phase 2
Gene expression analysis by DNA-chip of tissue samples from ruptured and unruptured aneurysms, and from peripheral arteries in the same cases and normal control arterial vessels. Evaluation of differential gene expression patterns between different samples basing on bio-informatic supports, and identification of gene target specifically up- or down-regulated.

Phase 3
Analysis of genes of interest (target) by PCR amplification followed by DHPLC analysis of amplicons. In those cases in which the chromatogram will show elution profiles characteristic of polymorphisms or unclassified variant, the subsequent step will be the sequencing of the fragment in order to identify the type of mutation. Analysis of the same genetic patterns on patient's and normal controls blood samples, to assess potential genetic parameters for a screening for the presence of unruptured intracranial aneurysms in the general population and, possibly to predict in which cases aneurysms will be potentially at high risk for rupture.

Conclusion
The management of aneurysms could be dramatically improved by identifying genetic factors involved in such pathology. The hypothesis that such factors may be investigated in the blood of normal populations, in an attempt to screen for unruptured intracranial aneurysms appears innovative and will be be adequately investigated. The ATENA group's study will be focused on the study of gene expression in intracranial aneurysms, by an initial evaluation of tissue RNA alterations. This will permit to clarify some aspects of their pathogenesis, differentiating gene alterations between ruptured / unruptured aneurysms, and in normal arteries, in an attempt to clarify whether peculiar tissue RNA alterations may match with the condition of ruptured/unruptured aneurysm and in trying to disclose gene alterations underlying their growth and rupture. The study could therefore bring remarkable information on genetic mechanisms related and possibly involved in aneurysm rupture. In a second future phase, this could permit, by simple genetic tests on DNA, performed on patient's blood samples, to detect in otherwise healthy subjects, the condition of high risk for harbouring an intracranial aneurysm, and possibly to disclose in this group those subjects with aneurysms at elevated risk for rupture.

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