Final Report Summary - FAD (Fighting Aneurysmal Diseases)
Executive summary:
Fighting Aneurysmal Disease (FAD, HEALTH-2008-200647) project aims to better understand the mechanism of dilation remodeling in the progression of aneurysm toward rupture, from molecules and cells, in order to develop new diagnostic and therapeutic tools in this disease, linked or not to aging. By nature, FAD is a translational project starting from objective interdisciplinary observations of aneurysms, using updated technological approaches, and applying to new tools for diagnosis and treatments. I order to progress in these ways, FAD limited its field of investigations to aneurysms of the aorta in human, including both aneurysms of the abdominal aorta (AAA) of atherothrombotic origin and aneurysms of the thoracic ascending aorta (TAA) of non-atheromatous origin. AAAs summarize some of the different components of atherothrombotic disease progression, including intraluminal thrombus formation and innate and adaptive immunity, whereas TAAs retain several aetiologies, including monogenic diseases, illustrated by Marfan syndrome in young patients, association with bicuspid aortic valves, and degenerative form with aging, leading to monomorphic pathologies: progressive dilation or acute intramural rupture (dissections). Whatever the location, aortic aneurysmal diseases are characterized by proteolytic injuries of the arterial wall, due to both, interactions between individual genetic background and environmental conditions, particularly local hemodynamic specificities, and circulating blood components and specific biological properties of the arterial wall itself.
These general functional genomic and pathophysiological concepts lead to the development of new diagnostic tools, including both new biological biomarkers of the diseases, and new functional and molecular imaging tools. FAD was constituted of fifteen partners distributed in twelve EU countries, and structured in five scientific workpackages (WP):
1) clinical database and biological biobanking,
2) genetics and functional genomics (from genes to phenotypes),
3) pathophysiology (from phenotypes to molecular determinants),
4) diagnosis, including biological genomic and circulating biomarkers, and in vivo imaging,
5) therapeutic developments.
Each partner and each WP contributed a lot to the success story of FAD. Partners participated to the constitution of diverse EU clinical subdatabases and rich biological collections involving DNA and plasma collections, but more originally, tissue and cell preparations and collections. These collections are hosted by some partners and shared at a EU level. Clinical database generated new epidemiological informations, including gender aspect (WP1); and Genetic approaches (WP2) lead to the discovery of new genes involved in monogenic forms of TAA and in genetic susceptibility to AAA. Functional genomic approaches lead to the discovery of a new epigenetic phenomenon specific to smooth muscle cells in TAAs. Pathophysiology (from phenotype to cell and molecular determinants, WP3) focused on the role of IntraLuminal Thrombus as an active determinant of the progression of AAAs, and of transmural convection of plasma zymogens and fibronectin modification of expression as active determinants of TAA progression. These determinants are directly related to biomechanics, including biomechanics of the arterial wall and hemodynamic in the aneurysmal sac. Important progresses were also achieved in diagnosis (WP4) including discovery of numerous new circulating biomarkers of ILT proteolytic, oxidant and pro-inflammatory activities in AAA and genomic biomarkers of TAA evolution. In parallel new contrast agents for molecular imaging of thrombi and proteolysis have been developed at a preclinical level, and original clinical investigations was performed using functional approaches of phagocytosis and adventitial inflammation and AAA, and of arterial wall motion in TAA. Lastly medical preventive approaches and new interventional tools have been proposed at a preclinical level as future prospective therapies in human (WP5).
Project Context and Objectives:
Aneurysms of the aorta, including abdominal (AAA) and thoracic (TAA) aortic aneurysms are important health question, in part due to the progressive aging of the EU population.
Epidemiological and clinical context: AAA occurs in up to 9% of adults older than 65 years of age, causing about 1-2% of male deaths in Western countries1. Early detection and elective AAA repair represent the main aspects to limit the mortality rate from AAA rupture. Although cost effectiveness of AAA screening was shown to be attractive2, the benefit of early detection of AAAs is limited because early repair of small AAA has been demonstrated to be inefficient and there is currently no established treatment for small AAAs3. On the other hand, the indication for elective repair is based upon the maximal diameter of AAA above 5 to 5.5 cm. The diameter of AAA is a surrogate marker of the growth rate that reflects the magnitude of the degenerative process in the vascular wall and infrarenal aortic diameter is an indicator of AAA disease, but is also an independent marker of all-cause mortality, mainly related to cardiovascular disease4. But AAA progression towards rupture is not linear, but usually presents points of acceleration which can appear at any time5, 6. Conversely, aortic dilatations can remain stable and asymptomatic for many years during which aged patients may die of other causes. In women, AAAs are rarer, and smaller, but represent a higher relative mortality than men7. Therefore, if AAA dimensions are canonically used as endpoints for AAA evaluation and treatment, numerous other biomarkers of risk, related to biological activities associated with AAA expansion remain to be discovered in order to ameliorate the prevention and the timing of interventional treatment.
Pathological context: AAA is a particular, specifically proteolytic and localized form of atherothrombotic disease, initiated by wall lipid retention, sharing the usual risk factors with occlusive atherothrombosis: male gender, aging, possible genetic susceptibility and dyslipidemia. Aging and smoking is the major risk factor in AAA. Among lipid markers, low HDL level is the most sensitive predictor of AAA13. This could be related to the impact of hypercholesterolemia on the initial step of atheroma in aorta, and to the low levels of 1-antitrypsin conveyed by HDL in human AAA14. The presence of AAA in a patient is a marker of atherothrombotic disease elsewhere15, and aortic diameter a predictor of total and cardiovascular mortality16. AAA is characterized by degradation of the media extracellular matrix, the smooth muscle cell disappearance, the presence of a chronic intraluminal thrombus (ILT), and the association with a significant adventitial reaction17. The ILT is a biologically active neo-tissue described as a laminated structure, containing several layers of fibrin clot, underlying a fresh, hematic and fibrin-rich luminal, and an actively fibrinolyzed abluminal layer. ILT is a dynamic biological balance between clotting at the luminal interface with circulating blood and outward progressive lysis, providing evidence of a spatial topology of temporal events (clotting and lysis). The ILT is traversed from the luminal to the abluminal surface by a continuous network of canaliculi, allowing unrestricted macro-molecular penetration18.
FAD objectives: The general translational, cognitive, technological and medical objectives of the FAD project, were to decipher new pathophysiological concepts in aortic aneurysmal remodeling, targeting cells and molecules, linking genetics with aneurysmal phenotypes, leading to the development of new tools for diagnosis, prognosis and therapeutics in aneurysmal diseases. The project consisted of a consortium integrating groups working on both AAA & TAA in a large scale European collaborative research project from Bedside to Bench and back.
The FAD project directly addressed the 3 aspects: pathophysiology, diagnostics and therapeutics, expressed in the EU proposal HEALTH-2007-A-1.2.4.2-2: Vascular remodeling in aneurysmal disease, through the 4 workpackages 2-3-4-5, using the WP-1 (human database) as the main translational tool for assessing the objectives. FAD responds to these three aspects by focusing specifically on expansive arterial remodeling, involving both cell and matrix functions and interactions; and exploring both genetic determinants and relevant pathophysiological phenotypes of this specific arterial wall remodeling, and then developing diagnostic and therapeutic applications.
The FAD project integrated the three epistemological dimensions of translational research for medical progress applied to aneurysmal pathology:
- The observation of human disease including the nosological definition of aneurysm, risk factors, clinical epidemiology, and public health concerns.
- The projection of the observed molecular diversity to application to human aneurysms including functional genomic approaches in vitro through molecular and cell biology, and in vivo through experimental models in murine.
- The search for new diagnostic and therapeutic targets through bedside to bench objective research on human pathology, including: genetic exploration of new determinants of susceptibility to aneurysms, human tissue collections, transcriptomic and proteomic methods applied to human tissue and cell biology derived from human tissue.
Translational objectives of FAD included:
- The genetic objectives to identify new susceptibility genes for TAA and AAA, and new mutations responsible for familial forms of TAAs (WP2)
- The genomic objectives to rely the observed mutations to the aneurysmal phenotype at a molecular, cellular and tissue levels (WP2).
- The first pathophysiological challenge was to resolve the question raised by the duality between the etiological molecular diversity of TAAs and the phenotypic monomorphism of aneurysm or dissections of the ascending aorta, including VSMC disappearance, areas of mucoid degeneration, and finally extracellular matrix breakdown leading to dilation and/or intraparietal rupture (WP3).
- The second one was to further decipher the spatiotemporal pathophysiology of AAA, including the rheological mechanism of formation of the Intra Luminal Thrombus (ILT) and the role of ILT in the degradation of the aortic media, and the impact of these phenomenons (ILT and media degradation) on the innate and adaptive immunity developed in the adventitia (WP3).
- The search, development and validation of new circulating biomarkers of TAA and AAA progression towards rupture, mainly by using proteomic approaches (WP4),
- and of new functional and molecular imaging tools of predictive value of TAA and AAA progression (WP4)
- The preclinical proof of concept of new medical therapeutic approaches in the prevention of aneurysm development and growth and preclinical search for new devices, biomaterials, cell and gene therapies for interventional treatments of aneurysm (WP5). This WP may open new avenue in original clinical trials in AAA and possibly in TAA (clinical trials are not included in FAD).
References
1. Sakalihasan N, Limet R, Defawe OD. Abdominal aortic aneurysm. Lancet. 2005;365:1577-1589
2. Lindholt JS, Sorensen J, Sogaard R, Henneberg EW. Long-term benefit and cost-effectiveness analysis of screening for abdominal aortic aneurysms from a randomized controlled trial. Br J Surg. 2010;97:826-834
3. Powell JT, Brown LC, Forbes JF, Fowkes FG, Greenhalgh RM, Ruckley CV, Thompson SG. Final 12-year follow-up of surgery versus surveillance in the uk small aneurysm trial. Br J Surg. 2007;94:702-708
4. Norman P, Le M, Pearce C, Jamrozik K. Infrarenal aortic diameter predicts all-cause mortality. Arterioscler Thromb Vasc Biol. 2004;24:1278-1282
5. Limet R, Sakalihassan N, Albert A. Determination of the expansion rate and incidence of rupture of abdominal aortic aneurysms. J Vasc Surg. 1991;14:540-548
6. Kurvers H, Veith FJ, Lipsitz EC, Ohki T, Gargiulo NJ, Cayne NS, Suggs WD, Timaran CH, Kwon GY, Rhee SJ, Santiago C. Discontinuous, staccato growth of abdominal aortic aneurysms. J Am Coll Surg. 2004;199:709-715
7. Hultgren R, Granath F, Swedenborg J. Different disease profiles for women and men with abdominal aortic aneurysms. Eur J Vasc Endovasc Surg. 2007;33:556-560
8. Olsson C, Thelin S, Stahle E, Ekbom A, Granath F. Thoracic aortic aneurysm and dissection: Increasing prevalence and improved outcomes reported in a nationwide population-based study of more than 14,000 cases from 1987 to 2002. Circulation. 2006;114:2611-2618
9. Golledge J, Eagle KA. Acute aortic dissection. Lancet. 2008;372:55-66
10. Elefteriades JA. Thoracic aortic aneurysm: Reading the enemy's playbook. Yale J Biol Med. 2008;81:175-186
11. Johansson G, Markstrom U, Swedenborg J. Ruptured thoracic aortic aneurysms: A study of incidence and mortality rates. J Vasc Surg. 1995;21:985-988
12. Hatzaras IS, Bible JE, Koullias GJ, Tranquilli M, Singh M, Elefteriades JA. Role of exertion or emotion as inciting events for acute aortic dissection. Am J Cardiol. 2007;100:1470-1472
13. Golledge J, van Bockxmeer F, Jamrozik K, McCann M, Norman PE. Association between serum lipoproteins and abdominal aortic aneurysm. Am J Cardiol. 2010;105:1480-1484
14. Ortiz-Munoz G, Houard X, Martin-Ventura JL, Ishida BY, Loyau S, Rossignol P, Moreno JA, Kane JP, Chalkley RJ, Burlingame AL, Michel JB, Meilhac O. Hdl antielastase activity prevents smooth muscle cell anoikis, a potential new antiatherogenic property. Faseb J. 2009;23:3129-3139
15. Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, Ballard DJ, Messina LM, Gordon IL, Chute EP, Krupski WC, Busuttil SJ, Barone GW, Sparks S, Graham LM, Rapp JH, Makaroun MS, Moneta GL, Cambria RA, Makhoul RG, Eton D, Ansel HJ, Freischlag JA, Bandyk D. Immediate repair compared with surveillance of small abdominal aortic aneurysms. N Engl J Med. 2002;346:1437-1444
16. Forsdahl SH, Solberg S, Singh K, Jacobsen BK. Abdominal aortic aneurysms, or a relatively large diameter of non-aneurysmal aortas, increase total and cardiovascular mortality: The tromso study. Int J Epidemiol. 2010;39:225-232
17. Michel JB. Contrasting outcomes of atheroma evolution: Intimal accumulation versus medial destruction. Arterioscler Thromb Vasc Biol. 2001;21:1389-1392.
18. Adolph R, Vorp DA, Steed DL, Webster MW, Kameneva MV, Watkins SC. Cellular content and permeability of intraluminal thrombus in abdominal aortic aneurysm. J Vasc Surg. 1997;25:916-926
19. de Figueiredo Borges L, Jaldin RG, Dias RR, Stolf NA, Michel JB, Gutierrez PS. Collagen is reduced and disrupted in human aneurysms and dissections of ascending aorta. Hum Pathol. 2008;39:437-443
20. Schlatmann TJ, Becker AE. Histologic changes in the normal aging aorta: Implications for dissecting aortic aneurysm. Am J Cardiol. 1977;39:13-20
21. Virmani R, Avolio AP, Mergner WJ, Robinowitz M, Herderick EE, Cornhill JF, Guo SY, Liu TH, Ou DY, O'Rourke M. Effect of aging on aortic morphology in populations with high and low prevalence of hypertension and atherosclerosis. Comparison between occidental and chinese communities. Am J Pathol. 1991;139:1119-1129
22. Michel JB, Thaunat O, Houard X, Meilhac O, Caligiuri G, Nicoletti A. Topological determinants and consequences of adventitial responses to arterial wall injury. Arterioscler Thromb Vasc Biol. 2007;27:1259-1268
Project Results:
Fighting Aneurysmal Disease (FAD, HEALTH-2008-200647) was a translational project, aiming to better understand the mechanism of dilation remodeling in the progression of aneurysm toward rupture, from molecules and cells, in order to develop new diagnostic and therapeutic tools in this disease, linked or not to aging.
WP1. Human clinical and biological databases
Objectives
1: To establish, a scientifically and ethically, robust EU web-based database for aortic aneurysms
2: To use the databases to describe for AAA and TAA the incidence and prognosis following diagnoses and operation of AAA and TAA.
3. To give support to clinical investigations in different aspects of aneurismal pathology including biomarkers in the follow-up of aneurysm progression and treatment, correlation to functional imaging, and therapeutic trials.
4: To standardize the European methodologies
5: To exchange data and biomaterials
6: To allow progress of the other WPs: 2, 3, 4, 5, through the use of these sample banks.
Achievements:
Creation of the FAD-database
The creation of the web based database was initially delayed due to summer holiday season in Denmark. In august 2008, an airport meeting in Copenhagen was held between JB Michel, Per Eriksson and Jes Lindholt, in order to prepare the plan and discussion of the database at the Kick off meeting in Liege in September. At that meeting, the variables of the database were decided and standards for some of the key variables as aortic diameter, systemic blood pressure measurement, and ankle brachial systolic blood pressure index (ABI) were agreed together with standards for biobanking. In addition, a none-planned additional sub-database was agreed upon, in order to supply the consortium with matched controls without aortic aneurysms.
The FAD Database Management Board.
A FAD Database Management Board consisting of participants from all partners involved in the database was created at the Liege meeting in order to secure ethical standards and individual partners properties are respected, when transfer of data among partners are requested. The board includes representation from project participants, a statistician and biologist, with appropriate representation of ethnic groups and women.
Each partner can get access to own data, but not other partners? data. Only the Work package 1-leader, Jes Lindholt, can get such access to all data from all participants. Requests of data from other partners are only executed, if accepted by this Board.
The Board members are responsible for their own data including security of the anonymity of cases, validity including that the FAD standards are fulfilled, as well as the administration of users to type in data. Board members and their users are given user names and passwords by the Work package 1-leader, Jes Lindholt, through an established additional consol attached to the database.
The FAD database
The FAD database consists of 5 subdatabases, which is one more than planned initially. This fifth subdatabase is for allowing controls to be entered. The other four is (i) to characterise the phenotype of rapid AAA expansion and need for repair in European of progressive aneurysmal disease as indicated by the late development of endoleaks after endografting; (iii) to characterise the phenotype of the ultimate termination of AAA - its rupture - (iiii) to characterise the phenotype of genetic causation, from European families with TAA and AAA (with WP2), and to allow age and sex matched controls, known to be with aneurysm. Finally, familiar disposition for AA is well known but the causality is mainly unknown. Consequently, information concerning familiar disposition is recorded in each subdatabase.
Consequently, the five FAD databases are now useable to support clinical investigations in different aspects of aneurismal pathology including biomarkers in the follow-up of aneurysm progression and treatment, correlation to functional imaging, and therapeutic trials.
Standardized European methodologies for contribution to the clinical database, and standardized sample banks of plasma, DNA, cells and tissue associated with the database have been essential to create in order to make it possible to merge data and biomaterials from several partners. Consequently, methodologies have been standardized concerning methods and intervals for AAA population screening, techniques for provision of DNA and criteria for diagnosis and analysis of endoleaks. Population screening is standardized according to current British and Danish methodologies and this will be applied to other EU and non-EU countries, which are starting population screening (eg Iceland). Standards for biomaterial has been formulated, and standards for reporting key variables as aneurismal size and endoleak has been proposed by relevant experts, and agreed upon at the kick off meeting in Liege. In presence of a localized dilatation, the right-angled maximal anteriorposterior diameter will be measured. Digital documentation or thermo-prints are made to demonstrate the visualised morphology and where the AAA was measured for later reproduction, when the patients attend follow up scans. Measurements will be performed in an inner to inner way, from plaque to plaque, if possible, in order to prevent variations in luminal-intramural-extramural measure points and sources of error. The luminal source of error will be the presence of a mural thrombus, and the extramural source will be the anterior longitudinal ligament or the vertebras themselves. All positive findings are being rescanned by a doctor responsible for the surveillance of the screening. In this way the numbers of observers will be kept at a minimum. Annual control scans will be performed of AAAs below 5 cm in maximal AP diameter, and biannually for AAA above 5 cm in maximal AP-diameter. If the AAA exceed 5.5 cm in maximal diameter, or becomes symptomatic, referral for surgical evaluation will be done. Performed operations including indication will be recorded.
AAA and TAA the incidence and prognosis following diagnoses and operation of AAA and TAA
One of the aims for WP1 were to use the data from the four subdatabases to describe the incidence and prognosis following diagnoses and operation of AAA and TAA. This has occurred in especially Sweden and Denmark with these publications so far:
P4. Karolinska:
1. Larsson E, Vishnevskaya L, Kalin B, Granath F, Swedenborg J, Hultgren R. High frequency of thoracic aneurysms in patients with abdominal aortic aneurysms. Ann Surg. 2011;253:180-4.
2. Hultgren R, Forsberg J, Alfredsson L, Swedenborg J, Leander K. Regional variation in the incidence of abdominal aortic aneurysm in Sweden. Br J Surg. 2012;99:647-
3. Villard C, Wågsäter D, Swedenborg J, Eriksson P, Hultgren R. 53 Biomarkers for Abdominal Aortic Aneurysms From a Sex Perspective. Gend Med. 2012 Jun 19. [Epub ahead of print]
P8. Viborg:
4.Lindholt JS, Norman PE. Meta-analysis of postoperative mortality after elective repair of abdominal aortic aneurysms detected by screening. Br J Surg. 2011 May;98(5):619-22.
5.Lindholt JS, Sørensen J, Søgaard R, Henneberg EW. Long-term benefit and cost-effectiveness analysis of screening for abdominal aortic aneurysms from a randomized controlled trial. Br J Surg. 2010 Jun;97(6):826-34.
6.Lindholt JS, Norman P. Screening for abdominal aortic aneurysm reduces overall mortality in men. A meta-analysis of the mid- and long-term effects of screening for abdominal aortic aneurysms. Eur J Vasc Endovasc Surg. 2008 Aug;36(2):167-71.
7. Lindholt JS, Sogaard, Laustsen J. The prognosis of ruptured abdominal aortic aneurysms in Denmark 1994-2008. Epidemiology 2012;4:111-3
8. Grondal N, Sogaard R, Henneberg EW, Lindholt JS. The Viborg Vascular (VIVA) screening trial of 65-74 year old men in the central region of Denmark: study protocol. Trials. 2010 May 27;11:67.
Assessing family history and present medication as a risk factor
In Sweden (P4), the nationwide registries of all in-hospital care (IPR=in patient registry) and the cause of death registries will be used to identify all persons diagnosed with, operated for, or having died from AAA in Sweden. For each of these cases, controls are randomly selected from the population; the controls are matched according to birth year, sex and region of residence. This data set will be linked to the Swedish Multigeneration registry in order to identify first-degree relatives of cases and controls. Subsequently, these families will be linked to IPR and the cause of death registry to identify family history of AAA.
Results during Jun 2008 to December 2009: All persons (3183) born after 1932, diagnosed with AAA between 2001 and 2005, and a random selection of 15,943 age-, gender-, and region-matched controls were included. First-degree relatives of cases and controls were identified via the Multigeneration Register. Family history of AAA for cases and controls was assessed by linking the relatives to the Hospital Discharge Register and Cause of Death Register. The data were analyzed by conditional logistic regression. The overall relative risk of AAA associated with family history compared to no family history was 1.9 (95% confidence interval [CI] 1.6-2.2). Comorbidities were more common among the cases than the controls (P < .0001) but the relative risks remained unchanged after adjustment for comorbidities. Stratification for absence or presence of comorbidities showed no significant difference between the two groups (P = .29). The relative risk of AAA for first-degree relatives was similar for women and men (P = .22 for gender differences), ie, the relative risk of AAA was not dependent on the gender of the index person. In conclusion, in this nationwide survey, the relative risk of developing AAA for first-degree relatives to persons diagnosed with AAA was approximately doubled compared to persons with no family history. Neither the gender of the index person nor the first-degree relative influenced the risk of AAA (partner 4, KI, submitted).
In a second study, the role of hereditary and environmental factors to development of AAA in a large population-based sample of twins was studied. The Swedish Twin Registry, containing data on twins born in the country since 1886, was cross-linked with the Inpatient Registry, providing national coverage of discharge diagnoses coded according to the International Classification of Diseases (ICD). All twins with an infrarenal AAA were identified. Concordance rates and tetrachoric correlations were calculated for monozygotic (MZ) and dizygotic (DZ) twins. Tetrachoric correlations were calculated assuming an underlying normal distribution of liability, with multiple factors contributing additively and a threshold value that discriminates between AAA and no AAA. Higher concordance rates and correlations of liability in MZ twins than in DZ twins suggest that genetic factors influence disease development. Structural equation modeling techniques, Mx-analyses, were used to estimate the contributions of genetic effects as well as shared and non-shared environmental factors for development of AAA. There were 172890 twins registered at the time of the study including 266 twins (81% men; mean age 72 years, range 48-94) with AAA. There were 7 MZ and 5 DZ concordant pairs as well as 44 MZ and 197 DZ discordant pairs with AAA. The probandwise concordance rates for MZ and DZ pairs were 24% and 4.8%, respectively. The tetrachoric correlations were 0.71 in MZ pairs and 0.31 in DZ pairs. In the structural equation models, genetic effects accounted for 70% (95% CI: 0.33-0.83) shared environmental effects for 0% (95% CI: 0-0.27) and non-shared environmental effects for 30% (95% CI: 0.17-0.46) of the phenotypic variance among twins. In conclusion, robust epidemiological evidence that heritability contributes to aneurysm formation is provided. Concordances and correlations were higher in MZ compared with DZ twins, indicating genetic effects. A heritability of 70% of the total trait variance was estimated. The remaining variance was explained by non-shared environmental factors with no support for a role of shared environmental influences.
Individual data were collated from 15 475 people under follow-up for a small aneurysm in 18 studies. The influence of co-variables (including demographics, medical and drug history) on aneurysm growth and rupture rates (analysed using longitudinal random-effects modelling and survival analysis with adjustment for aneurysm diameter) were summarized in an individual patient meta-analysis.
Available biosamples associated or not with clinical databases
As described in the grant agreement, for practical and ethical reasons, the standardized biological collections performed by each partner were prepared and conserved by each partner. The table represents all the biological samples available through FAD partnership.
These biobanking activities have included:
- Preparation of the samples involving dissection of the arterial tissue, separation between diseased parts and non or less diseased parts, separation of thrombi, intima, media, and adventitia; direct freezing and secondary cryo-pulverization, preparation of conditioned medium, and enzymatic digestion for preparation of primary culture of human endothelial cells, medial smooth muscle cells, and adventitial fibroblasts, and isolation of leukocytes. This step includes quality controls of the initial samples.
- Storage and Conservation of the samples at -80°C, in a specific area (Biological Resource Center) devoted to this activity
- Cession of the samples for experimental researches within the laboratory and between different laboratories through the FAD partnership and outside. These procedures include the signature of a Material Transfert Agreement and a declaration to the national regulatory authorities.
WP2. Genetics and functional genomics [from genes to phenotype]
Objectives:
To explore the molecular determinants of aortic dilation in humans through genetic and functional genomics in both humans and mice. Specifically WP2 includes:
- identification of new susceptibility genes for AAA and TAA
- identification of new mutations responsible for familial forms of TAA
- exploring the signalopathy associated with aneurysms
- development of animal models to study candidate genes for TAA and AAA.
Genome-wide/candidate gene case-control study to identify new susceptibility genes for AAA.
Genetic susceptibility studies in humans necessitate large cohorts of patients versus controls. So far, two large genome wide association studies (GWAS) have been performed on AAA, both lead by the FAD consortium. A meta analysis using the results of the different GWAS is currently conducted.
One of the main achievements of FAD in identifying genetic susceptibility to AAA was made by DeCODE (partner 15, ref. 17). Using a GWAS approach, a sequence variant was identified within intron 1 of the DAB2IP gene as a powerful genetic marker of AAA risk. DAB2 Interacting Protein is a member of the RAS-GTPase-activating protein family. DAB2IP suppresses cell survival and proliferation and is expressed by vascular cells, particularly by VSMCs. The identified variant could probably influence directly or indirectly the survival of VSMCs in response to proteolytic injury. Partners 4 (Karolinska), 6 (Liege) and 8 (Viborg) all contributed to this discovery.
- In addition, partner 15, identified two new AAA risk variants located within the LPA gene associate with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism. These variants were identified through genotyping and testing of a set of CAD associated SNPs on a large sample set of European AAA case-control samples (Ref Helgadottir A et al. 2012).
A second GWAS was recently conducted by partner 2 (London) and the AAA Consortium. Partners 4 (Karolinska), 8 (Viborg) and 15 (DeCODE) contributed to this discovery (ref AJHG 2011). The study identified the low-density-lipoprotein receptor-related protein 1 (LRP1) as a susceptibility gene for AAA. Interestingly, LRP1 has been shown to have a role in the regulation of MMP9 expression and murine models have demonstrated that LRP1 is essential for the maintenance of vascular wall integrity and that this effect is mediated via PDGF receptor beta and Smad signaling.
Identification of new mutations responsible for familial forms of TAA
In contrast to AAAs, a significant fraction of pathologies of the ascending aorta, including aneurysms (TAA) and dissections (TAD), are determined by single mutations in one specific gene. Several mutated genes have been identified as being directly responsible for TAAD, including both syndromic forms, of which Marfan syndrome is the most frequent, and familial forms. This approach of monogenic determinants necessitates the recruitment of informative families by clinicians. Numerous genes have now been identified: fibrillin (FBN1), SMC myosin (MYH11), actin (ACTA2), TGFBReceptors, SMAD3, a glucose transporter (GLUT10), etc. FAD partners 3 (Gent) and 1 (Inserm) actively participated in the discovery of these genes prior to FAD (background work). New genes have been identified by FAD participants during the last four years. In particular, partner 1 (Inserm) recently identified a mutation in TGF- β 2 as responsible for a mild form of Marfan syndrome (ref. 150). New informative families have been identified (ref 40), leading to the identification of new genes responsible for FTAA. Also partner 16 has collected several interesting FTAA families suitable for gene identification. (ref 125). At this moment whole exome sequencing is performed using the powerful Next Generation Sequencing approach, on many FTAA families collected during the FAD project. We can assume that this will reveal in the near future many new genes. The important added value of all these findings clearly shows that the extracellular matrix exert not only a structural function but that besides its mechanical role in providing strength and support to the tissues, it also acts as a reservoir for cytokines and growth factors implicated in cellular proliferation, differentiation, migration and survival and therefore has an important regulatory function in the development and homeostasis of body organs and tissues. It is clear that these new insights open perspective to plausible therapeutic approaches for this group of aorta related disorders as it is proposed that treatments which decrease TGF- β expression, as angiotensin II antagonist, can attenuate of prevent the phenotypes.
TGF β signalopathy in TAA (functional genomics)
One major question arising from genetic studies in TAA is how does the molecular diversity in aetiology lead to aortic aneurysms and/or dissections which are all very similar histopathologically. Rapidly it appeared that although diverse mutations in TGF- β pathways were important genetic determinants of monogenic forms of TAA, mutations were not limited to this pathway (MYH11, GlUT10, ACTA2), and that changes in expression of TGF- β molecules, mainly Smad2, were common to all forms of TAA, including forms not genetically detemined.
Alternatively spliced extra domain A (EDA) of fibronectin plays an essential role in tissue repair under the control of the TGF- β signaling pathway. Partner 4 (KI) has analyzed the expression of fibronectin spliceforms in dilated and non-dilated ascending aorta of tricuspid (TAV) and BAV patients. EDA expression was increased in dilated aorta from TAV patients compared with non-dilated aorta. In contrast, EDA expression was not increased in dilated aorta from BAV patients. The expression of EDA correlated with maximum aortic diameter in TAV but not in BAV patients. TGF β treatment influenced the splicing of FN and enhanced the formation of EDA-containing FN in cultured medial VSMCs from TAV patients but not in VSMCs derived from BAV patients. Gene set enrichment analysis suggested that differences in TGF β signaling may explain the impaired EDA splicing in BAV patients (Partner 4).
- Functional genomics in transgenic mice
Work involving transgenic mice and zebrafish has also been initiated by different FAD partners. The studies of animal models with aortic abonormalities suggested an important role for TGFBeta in the pathogenesis of those disorders. Fbn1 deficient mice as well as the Slc2a10 deficient zebrafish, revealing the arterial tortuosity phenotype, showed each a clear aberrant TGFBeta signaling. To further explore the relationship between the up or down regulation of TGFBeta and the phenotypical characteristics of the disorders partner 3 developed a tgfbr1 knock-in mouse model as well as a transgenic mice for the vascular type of EDS. This latter model spontaneously developed at two months open wounds starting in the shoulder area and penetrating skin and subdermal tissue. These skin lesions evolve fast and also fragility of the arterial vessels is noticed. Partner 3 also knocked down the slc2a10 gene in zebrafish to further investigated the pathogenic mechanisms causing arterial tortuosity. (Ref. Willaert et al. HMG, 2011).Partner 1 developed a different Col3a1 transgenic knock-out mice model (Ehler-Danlos) in order to study the vascular phenotype. At the same time partner 1 developed a myh11 mouse model. These transgenesis approaches were initiated during FAD, and mechanical, functional and clinical studies are currently underway.
WP3. Pathophysiology [from phenotype to molecular determinants]
Objectives:
To explore molecular and biomechanical determinants of aortic dilation and rupture in order to identify new targets for diagnosis and treatment of TAA and AAA. WP 3 includes:
1) Identification of new molecular and cellular targets beyond proteases in TAA and AAA, focusing on the involvement of mucoid degeneration in TAA and on the luminal thrombus as a conveyor of protease activities, pro-inflammatory mediators and neo-antigens in AAA.
2) The inflammatory response to proteolytic injury in AAA, in impeding cellular mechanisms of aneurysmal wall repair, will be explored in this context.
3) The role of cell signaling in the susceptibility for AAA development and ultimate rupture, and the role of elastin derived peptides in inflammation, angiogenesis and calcification, will be elucidated by transgenic approaches in mice.
- Outward convection and activation of blood-borne proteases in aneurysmal wall.
Since the contention of the pressurized blood within the arterial conduct is the fact of the insoluble extracellular matrix, synthetized by the VSMC, dilation and finally rupture of the wall is always the fact of proteolytic degradation of the insoluble extracellular matrix. In parallel, molecular transport within the arterial wall is mainly the fact of the outward radial hydraulic conductance, generated by blood pressure through the wall (background). The main achievements of FAD in the pathophysiology of aortic wall dilation and finally rupture focused on the spatio-temporal mechanisms of plasma-borne zymogens, transported through and activated within the aortic wall and consequences for the matrix, the VSMC, and the adventitial inflammatory response. This pathophysiological concept of aneurysmal pathology, whatever the localization and the aetiology (atheromatous versus non-atheroma, aneurysm versus dissection, thoracic aorta versus abdominal aorta, or other localizations) open new avenues for better understanding vascular pathology.
- Medial and adventitial responses
In TAA, partner 4 established the differential pattern of gene expression in the media associated with bicuspid or tricuspid aortic wall. A set of immune response genes was specifically identified in TAA of tricuspid valves, but not in TAA with bicuspidy, suggesting a more important stimulation of inflammatory response in the former. Similarly the pattern of MMPs expression was established in TAA wall, comparing bicuspidy and tricuspidy, and showing an over-expression of MMP-14 and MMP-19 in dilated aortas. MMP-3 and MMP-7 mRNA were not detected, confirming plasma as their main source. In parallel partner 1 showed an epigenetic modification of expression pattern in VSMC of TAA (see WP2). The consequences of this epigenetic regulation of Smad2 expression on target genes, including proteolytic and antiproteolytic (serpins) effectors, extracellular matrix proteins, are explored in aneurysms and dissection of the ascending aorta.
- Biomechanics of aortic aneurysm
In-vitro biomechanical testing of aneurysm tissue from open surgical repair provided novel biomechanical information of the aneurysm wall as well as the intra-luminal thrombus. This is essential data for reliable organ-level biomechanical simulations with specific importance for the biomechanical AAA rupture risk assessment. The large pool of CT-A data available through the FAD consortium allowed a direct clinical interpretation of biomechanical computed indices, as it is important to integrate biomechanical simulations in the clinical decision making process. Polarized light microscopy of wall samples from open aneurysm repair provided novel data of the collagen organization in the AAA wall. This information is not only important for a qualitative understanding of the AAA wall but also critical input for multi-scale histomechanical constitutive models. Here, the integration of histology and biomechanics provided a basic understanding of load carrying mechanisms of the AAA wall. Hemodynamic simulations of the blood flow through the normal and the aneurysmal aorta showed significant differences, which motivated a novel hypothesis for intra-luminal thrombus development in the AAA. Integrating the coagulation cascade in the analysis further reinforced this hypothesis. The outcome could potentially help to design therapies for a conservative treatment of AAAs. Based on follow-up CT-A data of patients with small AAAs their three-dimensional development was studied. The collected information suggested that AAAs should be monitored all over the aneurysmatic sac in order to identify the site of faster diameter growth. This information could essentially improve monitoring protocols for small aneurysms and towards avoiding expansion-related ruptures. In addition, the collected information is the basis for the development of aneurysm expansion models, which could further improve the surveillance of small AAAs.
WP4. Diagnosis
Objectives of this WP4 are to translate WP2 and 3 to diagnostic tools for human aneurysms, using specific new technologies, including proteomics and in vivo imaging technologies involving functional and molecular imaging as intermediary phenotypes in TAA and AAA pathologies. WP4 also involve discovery of new tracers for molecular imaging, establishment of proof of concept at a preclinical level, and initiation of clinical investigations.
- Plasma biomarkers in AAA.
Numerous circulating biomarkers of AAA were identified by the partners as background of FAD: MMP-9 (partner 6), fibrinolyic signal (partner 8) platelet activation (partner 1), interleukins (partner 10). In particular, partner 7 identified IGFBP-1 as a circulating biomarker, released by ILT, and associated with AAA size. Studies exploring the value of these biomarkers were performed in larger cohorts. Other pathophysiological biomarkers were discovered in FAD, such as circulating free DNA (partner 1) or NGAL (partner 7) as markers of neutrophil activation, and anti-Pg antibody titration as marker of periodontal infection associated with AAA evolution (partner 1).
- Circulating cell biomarkers, progenitors and leukocytes
Partners 7 (UAM) and 1 (INSERM) discovered differential expression of a number of redox proteins in circulating neutrophils and in the membrane of red blood cells of AAA patients, and are currently pursuing validation of these in larger cohorts. Among these proteins, catalase and peroxiredoxin were decreased in both cell types, with a concomitant increase in prooxidant factors suggesting the involvement of oxidative stress in AAA pathogenesis. Similarly, partner 1bis performed a profiling of macrophages from AAA (Lamblin N, Ratajczak P, Hot D, Dubois E, Chwastyniak M, Beseme O, Drobecq H, Lemoine Y, Koussa M, Amouyel P, Pinet F. Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study. J Proteome Res. 2010 Jul 2;9(7):3720-9). In parallel circulating progenitors were identified and characterized in association with AAA (partner 2, St Georges Hospital, and partner 9, CNRS). Circulating microparticles of platelet and neutrophil origin were also identified in association with AAA.
- Functional hemodynamic imaging in TAA.
Beside the usual morphological measurements of aneurysmal dimension (maximal diameter, length, etc.) and the presence of ILT in AAA, FAD developed several new approaches including measurement of aortic rigidity in TAA, using ultrasound and now Magnetic Resonance Imaging (MRI). In particular partner 12 (AMC) developed this imaging technology. MRI also can image non-laminar flow in aneurysm, allowing a more precise analysis of recirculation and vortex phenomenon associated with initiation and progression of aortic dilation. This technology is not yet proposed in TAA due to the complexity of the software and the time of calculation. These approaches link the diagnosis (WP4) to biomechanics (WP3).
- Iron traffic in AAA
Since iron gives a powerful negative signal in MRI, this technology allowed us to explore the spontaneous presence of iron in AAA, in ILT and wall, and the ability of phagocytes present in ILT to uptake exogenous, intravenously injected, Small Paramagnetic Iron Oxide (SPIO) particles (partner 6 Ulg and 1 Inserm). This data are important since they demonstrate the ability of MRI to image in vivo in human (WP4) the involvement of iron, and downstream of oxidation in the pathophysiology of AAA (WP3). Nevertheless the data also raised the question of the sensitivity of MRI to detect molecular events.
- PETscan and aneurysm progression.
Partner 6, ULg, was the first to show that 18F-DeoxyGlucose uptake, detected by Positron Emission Tomography coupled with Computed Tomography scan (Rx) for morphology (18FDG PET/CT) could be used as prognosis marker in aneurysmal disease (background). During FAD, ULg constituted an important series of 711 18FDG PET/CTscan in in 428 patients presenting aortic diseases, including AAA (n= 301) with or without EVAR, dissections (n=25) of the descending aorta, TAA (n=66) and Aortitis (n=36). The results concerning TAA are mainly negative, except rare exceptions, probably, due to the absence of adventitial inflammatory granuloma in association with TAA. These negative data suggest that TAA do not generate important adventitial immune response. In contrast, data concerning AAA and dissection are particularly rich and interesting. In parallel to PET a biological database (plasma and DNA samples) was constituted and morphology was accessible by the CT data. This triple approach: PET showing localized spots of high 18FDG uptake (high cellular metabolism), associated with CT allowing dimensions measurements and morphology classification, and biology offer an unique opportunity to link the evolution of aneurysmal dilation (CT) to cell function imaging (PET) and biology. Usually the hot spots of 18FDG take place in the external part of the aortic wall. The exploitation of this triple database is now in progress.
- Development of new molecular tracers targeting Intra luminal thrombi (partner1)
Preclinical proof of concept that 99mTc-Fucoidan images intraluminal thrombi
P-selectin expression is involved in the pathophysiology of biologically active arterial thrombus, and endothelial activation following transient ischemic event. Fucoidan is a polysaccharidic ligand of P-selectin with a nanomolar affinity (background). In FAD we have proposed a new approach of P-selectin molecular imaging based on radiolabeled fucoidan. Two kinds of experimental models were selected to evaluate the ability of radiolabeled fucoidan to detect P-selectin expression: platelet-rich arterial thrombi (arterial mural thrombus in experimental aneurysm), and myocardial ischemia-reperfusion. These two settings were chosen because they were clinically relevant, and both were associated with an important overexpression of platelet and endothelial P-selectin, respectively. Radiolabeled fucoidan SPECT was able (a) to detect the presence of platelet-rich arterial thrombi in all animals, with a median target to background ratio of 3.6 in mural aneurysmal thrombus (b) to detect a persistent endothelial activation 2 hours after reperfusion; in this latter model the magnitude of the signal was correlated with the extent of myocardium that underwent transient ischemia. The sensitivity of selectivity of the uptake and retention of 99mTc-fucoidan in both settings was excellent. This study supports 99mTc-fucoidan as a relevant imaging agent for in vivo detection of biological activities associated with P-selectin overexpression, such as arterial thrombus and ischemic memory. Given the previously reported wide availability at a low cost, and its low toxicity, fucoidan seems to overcome some of the limitations of previous P-selectin-targeted imaging agents. This approach has been patented by partner 1: WO 2010/116209 A1: Fucoidans as ligands for the diagnosis of degenerative pathologies, published 14 october 2010, extended to USA 02970-24565US02, January 9, 2012; and published (J Nucl Med 2011).
- Development of new molecular tracers targeting protease activities (partner 13 and partner1)
Serine proteases hold a central role in the pathophysiology of degenerative arterial diseases, but to date there is no suitable agent for molecular imaging. The project was designed to (1) develop a radiolabeled peptide probe for serine proteases imaging, including plasmin and leukocyte elastase, and (2) evaluate these new agents in vivo in a rodent model of arterial thrombus, including experimental aneurysms.
WP5. Therapeutics
Objectives.
To the validate current treatment and new therapeutics in human aneurismal diseases. WP5 will develop clinical investigations in patients and preclinical testing in experimental models.
This program includes:
1- an evaluation of medical prevention of aneurysm development and progression by existing medications,
2- the development of curative cell and bio-engineered therapeutics at a preclinical level and further extension to human,
3- further development of new therapeutic strategies.
- Medical prevention of aneurysm development and progression (preclinical)
Since there is no specific treatment able to completely prevent or inhibit progression of aneurysm, the FAD WP5 explored new medical therapeutic approaches at a preclinical level. First, since WP3 demonstrated that ILT is one of the main driving forces for AAA progression, the ability of inhibition of platelet aggregation to limit experimental and clinical progression of AAA was tested. Experimentally platelet inhibition prevented AAA development in an experimental model in rats. This result was retrospectively confirmed in human. Therefore, inhibition of platelet aggregation could be recommended in patients with small aneurysms, in prevention of growth (Partner 1 and 8). In parallel treatments able to impair the retention of neutrophils in ILT or antibiotics, which treated bacterial contamination, were also tested with success in experimental models. Evaluation of AZD 9668, a powerful inhibitor of leukocyte elastase is under progress.
- Novel interventional curative therapeutics through cell seeding, gene therapy and biomaterials
Several new models of aneurysms have been developed during FAD for evaluating new interventional therapeutics. These models are transposition of classical model (elastase, decellularized xenograt) to large animals than mice and rats, including rabbit and pigs (partner 1, 9 and 10). A model of EVAR is developed in rats (partner 1). These models were used for testing cell seeding as interventional therapeutic approach in aneurysm, and for developing new hydrogels avec vector of local cell or molecular therapy.
9). In particular sulphated hydrogel was used to heparin binding mediators such VEGF or SDF-1.
- New therapeutic strategies
These different approaches allowed us to initiate or to participate to clinical trials, including trial of angiotensin II antagonists in Marfan syndrome, inhibitor of mast cell activation (Cardoz), and cyclosporin in human AAA. Others are in discussion with pharmaceutical industry (leukocyte elastase inhibition with Astra-Zeneca).
Scientific and medical conclusions.
FAD was a highly translational and transversal large-scale program, mobilizing numerous energy in order to better understand what are the biological determinants of aneurysm development in order to propose new diagnosis and therapeutic tools. FAD achievements are diverse, due to the number of partner, each developing its own project. Nevertheless the majority of the works were complementary through the different WPs and the different partners. Two examples are presented in the table beyond. The works performed on TGF-beta signaling pathway covered all the WPs, mainly WP1, 2, 3, and 5, and involved height partners among fifteen. Similarly the works performed on the Intraluminal thrombus as the predominant driving force in AAA evolution, covered all the WPs and involved eleven partners among fifteen. Similarly the number of common publications, 81 among 292 (28%), co-signed by 2 partners or more, also provide evidences of the transversality of FAD.
Major achievements have been performed in each WP opening new avenue for future Research and Development (R&D) in the field of aneurismal disease of the aorta:
- Clinical and genetic database and tissue and cell collections were constituted through FAD, These database and collections, shared inside FAD by several partners, but also opened to other teams outside of FAD consortium, allow important translational work in the future.
- Discovery of new gene mutations in aneurismal disease raises the important question of genetic diversity leading to common pathological features of arterial wall and rupture.
- Pathophysiology leads to the general concept of blood-borne injuries of the arterial wall, sensitized by intrinsic defect of the wall components, leading to medial and adventitial response.
- Diagnostic applications are in progress, including circulating biomarkers, but also important progress in functional and molecular imaging of aneurismal pathology. This progress include clinical applications, but also new tracers for which the preclinical proof of concept has been now established. Further developments as first injection in human are programmed.
- Similarly, new therapeutic approaches are in progress through clinical investigations in human patient.
Potential Impact:
The potential impacts, including the socio-economic impact and the wider societal implications.
FAD will have medical and socio-economic impacts in health, medicine, and economic development of new tools.
The development of screening programs will have, with time, consequences on health in aging EU population. Screening for AAA will have important consequences on the care of aging male without symptom. This beneficial effect will extend more than AAA alone. It will be extended to the detection of atherothrombotic disease whatever the localization and the prevention of periodontal diseases in aging population. The relationship between periodontal disease in aging population and the cardiovascular risk is now well established by epidemiological studies. Through translational research, FAD provided evidences of the biological relationship between periodontal disease, blood weak pathogen transitory passage, ILT retention of these weak pathogens, leading to AAA progression. Such health programs are now progressing slowly, but must be developed with the support of EU for Research and Development. We proposed this transversal project, including cardiologists, neurologists, biologists, odontologists, twice to EU (ERC 2010 and COST), but we didn't succeed to be selected.
The concept developed in FAD will also impact medicine of other vascular pathologies, including:
-Genetics of vascular diseases: the effort performed with success in genetics during FAD will be pursued including discovery of new genes through informative family approaches and genetic sensitivity trough GWAS approach in large cohort.
- Other localization of aneurysmal diseases: In particular cerebral aneurysms. At the beginning of FAD specific links was established with ANEURIST (see http://www.aneurist.org/ online). @neurIST -Integrated Biomedical Informatics for the Management of Cerebral Aneurysms was a European initiative within the Sixth Framework Programme of the Information Society Technologies IST. Alejandro Frangi (Aneurist coordinator) and Juahana Frozen, a Finnish neurosurgeaon, specialist of cerebral aneurysms, were member of the FAD scientific advisory board, and Jean-Baptiste Michel, FAD coordinatore participated to @neurIST meetings in Barcelonna. In particular FAD proposed that the pathogenicity of ILT is common to AAA and cerebral aneurysm.
- New stenting technology: In the context of cerebral aneurysm, a new type of multilayer not covered stent, Flow Diverter (see http://www.cardiatis.com/ online) has been implanted in human patients, particularly in treatment of cerebral aneurysm. The principle was that flow diversion of the aneurysmal sac could provoke sac thrombosis and healing. Unfortunately this technology was not associated with total success. Several secondary aneurysmal ruptures of cerebral aneurysms, but also of aortic and other peripheral aneurysms, impair the results. Since flow diversion is not ILT exclusion, the ILT associated with this type of stents continued to be biologically active leading to continuous proteolytic degradation of the arterial wall.
- Atherothrombotic diseases: Since AAA is an atherothrombotic disease, works performed on AAA also impact directly what we know about occlusive atherothombosis. In particular, works performed in genetics of sensitivity to AAA also impact genetics of coronary and carotid artery diseases in humans.
One other important point is that the studies made on IntraLuminal Thrombus (ILT) during FAD directly impact the pathophysiology of IntraPlaque Hemorrhage (IPH) in occlusive atherothrombosis. IPHs are now well established as the driving force leading to plaque rupture and clinical expression. We wrote important reviews in this field and gave several international conferences on this important subject (EVBO meeting in Krakow, ESC 2012 Munich). It is time now to develop something at the EU level concerning this most important feature in human atherothrombotic diseases. We proposed twice a COST and an ERC project in this field at the EU level (IPH-COST action 2011, ERC Spa-Athero 2012) in order to initiate an EU network in this field. We didn?t succeed. We will try again. We wish and hope that this important field of heme/iron metabolism in atherothrombotic diseases will be the object of EU calls at the beginning of FP-8!
- Aortic valve disease: Similarly to the preceding fields, the works performed on TAA particularly on genetics, genomics and biomechanics, allowed to FAD partners to continue collaborations in this field focusing on the relationship between TAA and bicuspid aortic valves. MIBAVA- Mechanistic Interrogation of Bicuspid Aortic Valve associated Aortopathy (see http://www.fondationleducq.org/ online), was selected Under the Transatlantic Networks of Excellence in Cardiovascular Research Program awarded by Fondation Leducq U.S. $6,000,000 over five years for internationally collaborative research. Bart Loeys EU coordinator (partber 3, ex UG), and Karolinska Institue (KI, partner 4) a partner.
- Economic Impact: The most important economic potential impact will be directly linked to the development of applications initiated within FAD. Among the patents generated through FAD the most important and numerous concern diagnostic applications, genetic markers, circulating biomarkers and molecular imaging. The Inserm patent and the corresponding published article on the use of fucoidan as a new pharmacophore for the diagnosis of vascular disease is probably the more advanced ones. The preclinical proof of concept has been established. The program is now to go to the first injection in human. This must be achieved through several steps including the definitive choice of the fucoidan preparation, the preparation in GMP conditions for first injection in human, minimal toxicologic studies, the writing of the regulatory files, the definition and the writing of the clinical investigation protocole, and finally the financial support to do that. This development is now in progress with a financial support at a national level.
Main dissemination activities and exploitation of results (WP6)
Objectives
- Ensure regular training of Junior Scientists and Technical Staff participating in FAD project to acquire new skills and techniques.
- In-house training is expected to promote personnel exchanges and amplify excellence and scientific links within the Network.
- To disseminate the results generated in FAD in medical science.
- To reach all the potential audiences, in particular, pharma companies, industrial partner for diagnostic tools, patient organisations and public health authorities.
A website has been created (see http://www.fightin-aneurysm.org online) to be a showcase of the project, and present the project to the public. The success of this website as a tool to promote FAD can be shown by the statistic of visits of the website, which regularly increase since its launching.
3 training sessions have been organized during the 4 years of the project: Proteomics in aneurismal disease: from sampling to analysis (April 2009), Animal models of AAA: rational to practice (March 2010) and CV molecular imaging (March 2012). The purpose of this training was decided after consultation of the partners on their needs linked to FAD works and centralization of the requests. Each partner has been involved as speakers or as attendees. The participants to these training sessions were French, Danish, English, Sweden, Belgian, Spanish and Czech. Behind the exchange of knowledge between the partners, these courses were a real opportunity for the partners to exchange knowledge and open the door to new collaborations.
The proof of the existence of this expertise resides in the fact that one hundred articles were reviewed by FAD leaders between July 2008 and July 2012. As an example, Jes Lindholt (WP1 team leader, partner 8, Viborg Hospital) is a member of the board at The European Journal of Vascular and Endovascular Surgery and Jean-Baptiste Michel has reviewed 19 articles of the 30 articles on the aneurysms published in ATVB and 10 on 35 in Cardiovascular Research.
Project website: http://www.fighting-aneurysm.org/
Contact: Jean-Baptiste.Michel@inserm.fr
Fighting Aneurysmal Disease (FAD, HEALTH-2008-200647) project aims to better understand the mechanism of dilation remodeling in the progression of aneurysm toward rupture, from molecules and cells, in order to develop new diagnostic and therapeutic tools in this disease, linked or not to aging. By nature, FAD is a translational project starting from objective interdisciplinary observations of aneurysms, using updated technological approaches, and applying to new tools for diagnosis and treatments. I order to progress in these ways, FAD limited its field of investigations to aneurysms of the aorta in human, including both aneurysms of the abdominal aorta (AAA) of atherothrombotic origin and aneurysms of the thoracic ascending aorta (TAA) of non-atheromatous origin. AAAs summarize some of the different components of atherothrombotic disease progression, including intraluminal thrombus formation and innate and adaptive immunity, whereas TAAs retain several aetiologies, including monogenic diseases, illustrated by Marfan syndrome in young patients, association with bicuspid aortic valves, and degenerative form with aging, leading to monomorphic pathologies: progressive dilation or acute intramural rupture (dissections). Whatever the location, aortic aneurysmal diseases are characterized by proteolytic injuries of the arterial wall, due to both, interactions between individual genetic background and environmental conditions, particularly local hemodynamic specificities, and circulating blood components and specific biological properties of the arterial wall itself.
These general functional genomic and pathophysiological concepts lead to the development of new diagnostic tools, including both new biological biomarkers of the diseases, and new functional and molecular imaging tools. FAD was constituted of fifteen partners distributed in twelve EU countries, and structured in five scientific workpackages (WP):
1) clinical database and biological biobanking,
2) genetics and functional genomics (from genes to phenotypes),
3) pathophysiology (from phenotypes to molecular determinants),
4) diagnosis, including biological genomic and circulating biomarkers, and in vivo imaging,
5) therapeutic developments.
Each partner and each WP contributed a lot to the success story of FAD. Partners participated to the constitution of diverse EU clinical subdatabases and rich biological collections involving DNA and plasma collections, but more originally, tissue and cell preparations and collections. These collections are hosted by some partners and shared at a EU level. Clinical database generated new epidemiological informations, including gender aspect (WP1); and Genetic approaches (WP2) lead to the discovery of new genes involved in monogenic forms of TAA and in genetic susceptibility to AAA. Functional genomic approaches lead to the discovery of a new epigenetic phenomenon specific to smooth muscle cells in TAAs. Pathophysiology (from phenotype to cell and molecular determinants, WP3) focused on the role of IntraLuminal Thrombus as an active determinant of the progression of AAAs, and of transmural convection of plasma zymogens and fibronectin modification of expression as active determinants of TAA progression. These determinants are directly related to biomechanics, including biomechanics of the arterial wall and hemodynamic in the aneurysmal sac. Important progresses were also achieved in diagnosis (WP4) including discovery of numerous new circulating biomarkers of ILT proteolytic, oxidant and pro-inflammatory activities in AAA and genomic biomarkers of TAA evolution. In parallel new contrast agents for molecular imaging of thrombi and proteolysis have been developed at a preclinical level, and original clinical investigations was performed using functional approaches of phagocytosis and adventitial inflammation and AAA, and of arterial wall motion in TAA. Lastly medical preventive approaches and new interventional tools have been proposed at a preclinical level as future prospective therapies in human (WP5).
Project Context and Objectives:
Aneurysms of the aorta, including abdominal (AAA) and thoracic (TAA) aortic aneurysms are important health question, in part due to the progressive aging of the EU population.
Epidemiological and clinical context: AAA occurs in up to 9% of adults older than 65 years of age, causing about 1-2% of male deaths in Western countries1. Early detection and elective AAA repair represent the main aspects to limit the mortality rate from AAA rupture. Although cost effectiveness of AAA screening was shown to be attractive2, the benefit of early detection of AAAs is limited because early repair of small AAA has been demonstrated to be inefficient and there is currently no established treatment for small AAAs3. On the other hand, the indication for elective repair is based upon the maximal diameter of AAA above 5 to 5.5 cm. The diameter of AAA is a surrogate marker of the growth rate that reflects the magnitude of the degenerative process in the vascular wall and infrarenal aortic diameter is an indicator of AAA disease, but is also an independent marker of all-cause mortality, mainly related to cardiovascular disease4. But AAA progression towards rupture is not linear, but usually presents points of acceleration which can appear at any time5, 6. Conversely, aortic dilatations can remain stable and asymptomatic for many years during which aged patients may die of other causes. In women, AAAs are rarer, and smaller, but represent a higher relative mortality than men7. Therefore, if AAA dimensions are canonically used as endpoints for AAA evaluation and treatment, numerous other biomarkers of risk, related to biological activities associated with AAA expansion remain to be discovered in order to ameliorate the prevention and the timing of interventional treatment.
Pathological context: AAA is a particular, specifically proteolytic and localized form of atherothrombotic disease, initiated by wall lipid retention, sharing the usual risk factors with occlusive atherothrombosis: male gender, aging, possible genetic susceptibility and dyslipidemia. Aging and smoking is the major risk factor in AAA. Among lipid markers, low HDL level is the most sensitive predictor of AAA13. This could be related to the impact of hypercholesterolemia on the initial step of atheroma in aorta, and to the low levels of 1-antitrypsin conveyed by HDL in human AAA14. The presence of AAA in a patient is a marker of atherothrombotic disease elsewhere15, and aortic diameter a predictor of total and cardiovascular mortality16. AAA is characterized by degradation of the media extracellular matrix, the smooth muscle cell disappearance, the presence of a chronic intraluminal thrombus (ILT), and the association with a significant adventitial reaction17. The ILT is a biologically active neo-tissue described as a laminated structure, containing several layers of fibrin clot, underlying a fresh, hematic and fibrin-rich luminal, and an actively fibrinolyzed abluminal layer. ILT is a dynamic biological balance between clotting at the luminal interface with circulating blood and outward progressive lysis, providing evidence of a spatial topology of temporal events (clotting and lysis). The ILT is traversed from the luminal to the abluminal surface by a continuous network of canaliculi, allowing unrestricted macro-molecular penetration18.
FAD objectives: The general translational, cognitive, technological and medical objectives of the FAD project, were to decipher new pathophysiological concepts in aortic aneurysmal remodeling, targeting cells and molecules, linking genetics with aneurysmal phenotypes, leading to the development of new tools for diagnosis, prognosis and therapeutics in aneurysmal diseases. The project consisted of a consortium integrating groups working on both AAA & TAA in a large scale European collaborative research project from Bedside to Bench and back.
The FAD project directly addressed the 3 aspects: pathophysiology, diagnostics and therapeutics, expressed in the EU proposal HEALTH-2007-A-1.2.4.2-2: Vascular remodeling in aneurysmal disease, through the 4 workpackages 2-3-4-5, using the WP-1 (human database) as the main translational tool for assessing the objectives. FAD responds to these three aspects by focusing specifically on expansive arterial remodeling, involving both cell and matrix functions and interactions; and exploring both genetic determinants and relevant pathophysiological phenotypes of this specific arterial wall remodeling, and then developing diagnostic and therapeutic applications.
The FAD project integrated the three epistemological dimensions of translational research for medical progress applied to aneurysmal pathology:
- The observation of human disease including the nosological definition of aneurysm, risk factors, clinical epidemiology, and public health concerns.
- The projection of the observed molecular diversity to application to human aneurysms including functional genomic approaches in vitro through molecular and cell biology, and in vivo through experimental models in murine.
- The search for new diagnostic and therapeutic targets through bedside to bench objective research on human pathology, including: genetic exploration of new determinants of susceptibility to aneurysms, human tissue collections, transcriptomic and proteomic methods applied to human tissue and cell biology derived from human tissue.
Translational objectives of FAD included:
- The genetic objectives to identify new susceptibility genes for TAA and AAA, and new mutations responsible for familial forms of TAAs (WP2)
- The genomic objectives to rely the observed mutations to the aneurysmal phenotype at a molecular, cellular and tissue levels (WP2).
- The first pathophysiological challenge was to resolve the question raised by the duality between the etiological molecular diversity of TAAs and the phenotypic monomorphism of aneurysm or dissections of the ascending aorta, including VSMC disappearance, areas of mucoid degeneration, and finally extracellular matrix breakdown leading to dilation and/or intraparietal rupture (WP3).
- The second one was to further decipher the spatiotemporal pathophysiology of AAA, including the rheological mechanism of formation of the Intra Luminal Thrombus (ILT) and the role of ILT in the degradation of the aortic media, and the impact of these phenomenons (ILT and media degradation) on the innate and adaptive immunity developed in the adventitia (WP3).
- The search, development and validation of new circulating biomarkers of TAA and AAA progression towards rupture, mainly by using proteomic approaches (WP4),
- and of new functional and molecular imaging tools of predictive value of TAA and AAA progression (WP4)
- The preclinical proof of concept of new medical therapeutic approaches in the prevention of aneurysm development and growth and preclinical search for new devices, biomaterials, cell and gene therapies for interventional treatments of aneurysm (WP5). This WP may open new avenue in original clinical trials in AAA and possibly in TAA (clinical trials are not included in FAD).
References
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12. Hatzaras IS, Bible JE, Koullias GJ, Tranquilli M, Singh M, Elefteriades JA. Role of exertion or emotion as inciting events for acute aortic dissection. Am J Cardiol. 2007;100:1470-1472
13. Golledge J, van Bockxmeer F, Jamrozik K, McCann M, Norman PE. Association between serum lipoproteins and abdominal aortic aneurysm. Am J Cardiol. 2010;105:1480-1484
14. Ortiz-Munoz G, Houard X, Martin-Ventura JL, Ishida BY, Loyau S, Rossignol P, Moreno JA, Kane JP, Chalkley RJ, Burlingame AL, Michel JB, Meilhac O. Hdl antielastase activity prevents smooth muscle cell anoikis, a potential new antiatherogenic property. Faseb J. 2009;23:3129-3139
15. Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, Ballard DJ, Messina LM, Gordon IL, Chute EP, Krupski WC, Busuttil SJ, Barone GW, Sparks S, Graham LM, Rapp JH, Makaroun MS, Moneta GL, Cambria RA, Makhoul RG, Eton D, Ansel HJ, Freischlag JA, Bandyk D. Immediate repair compared with surveillance of small abdominal aortic aneurysms. N Engl J Med. 2002;346:1437-1444
16. Forsdahl SH, Solberg S, Singh K, Jacobsen BK. Abdominal aortic aneurysms, or a relatively large diameter of non-aneurysmal aortas, increase total and cardiovascular mortality: The tromso study. Int J Epidemiol. 2010;39:225-232
17. Michel JB. Contrasting outcomes of atheroma evolution: Intimal accumulation versus medial destruction. Arterioscler Thromb Vasc Biol. 2001;21:1389-1392.
18. Adolph R, Vorp DA, Steed DL, Webster MW, Kameneva MV, Watkins SC. Cellular content and permeability of intraluminal thrombus in abdominal aortic aneurysm. J Vasc Surg. 1997;25:916-926
19. de Figueiredo Borges L, Jaldin RG, Dias RR, Stolf NA, Michel JB, Gutierrez PS. Collagen is reduced and disrupted in human aneurysms and dissections of ascending aorta. Hum Pathol. 2008;39:437-443
20. Schlatmann TJ, Becker AE. Histologic changes in the normal aging aorta: Implications for dissecting aortic aneurysm. Am J Cardiol. 1977;39:13-20
21. Virmani R, Avolio AP, Mergner WJ, Robinowitz M, Herderick EE, Cornhill JF, Guo SY, Liu TH, Ou DY, O'Rourke M. Effect of aging on aortic morphology in populations with high and low prevalence of hypertension and atherosclerosis. Comparison between occidental and chinese communities. Am J Pathol. 1991;139:1119-1129
22. Michel JB, Thaunat O, Houard X, Meilhac O, Caligiuri G, Nicoletti A. Topological determinants and consequences of adventitial responses to arterial wall injury. Arterioscler Thromb Vasc Biol. 2007;27:1259-1268
Project Results:
Fighting Aneurysmal Disease (FAD, HEALTH-2008-200647) was a translational project, aiming to better understand the mechanism of dilation remodeling in the progression of aneurysm toward rupture, from molecules and cells, in order to develop new diagnostic and therapeutic tools in this disease, linked or not to aging.
WP1. Human clinical and biological databases
Objectives
1: To establish, a scientifically and ethically, robust EU web-based database for aortic aneurysms
2: To use the databases to describe for AAA and TAA the incidence and prognosis following diagnoses and operation of AAA and TAA.
3. To give support to clinical investigations in different aspects of aneurismal pathology including biomarkers in the follow-up of aneurysm progression and treatment, correlation to functional imaging, and therapeutic trials.
4: To standardize the European methodologies
5: To exchange data and biomaterials
6: To allow progress of the other WPs: 2, 3, 4, 5, through the use of these sample banks.
Achievements:
Creation of the FAD-database
The creation of the web based database was initially delayed due to summer holiday season in Denmark. In august 2008, an airport meeting in Copenhagen was held between JB Michel, Per Eriksson and Jes Lindholt, in order to prepare the plan and discussion of the database at the Kick off meeting in Liege in September. At that meeting, the variables of the database were decided and standards for some of the key variables as aortic diameter, systemic blood pressure measurement, and ankle brachial systolic blood pressure index (ABI) were agreed together with standards for biobanking. In addition, a none-planned additional sub-database was agreed upon, in order to supply the consortium with matched controls without aortic aneurysms.
The FAD Database Management Board.
A FAD Database Management Board consisting of participants from all partners involved in the database was created at the Liege meeting in order to secure ethical standards and individual partners properties are respected, when transfer of data among partners are requested. The board includes representation from project participants, a statistician and biologist, with appropriate representation of ethnic groups and women.
Each partner can get access to own data, but not other partners? data. Only the Work package 1-leader, Jes Lindholt, can get such access to all data from all participants. Requests of data from other partners are only executed, if accepted by this Board.
The Board members are responsible for their own data including security of the anonymity of cases, validity including that the FAD standards are fulfilled, as well as the administration of users to type in data. Board members and their users are given user names and passwords by the Work package 1-leader, Jes Lindholt, through an established additional consol attached to the database.
The FAD database
The FAD database consists of 5 subdatabases, which is one more than planned initially. This fifth subdatabase is for allowing controls to be entered. The other four is (i) to characterise the phenotype of rapid AAA expansion and need for repair in European of progressive aneurysmal disease as indicated by the late development of endoleaks after endografting; (iii) to characterise the phenotype of the ultimate termination of AAA - its rupture - (iiii) to characterise the phenotype of genetic causation, from European families with TAA and AAA (with WP2), and to allow age and sex matched controls, known to be with aneurysm. Finally, familiar disposition for AA is well known but the causality is mainly unknown. Consequently, information concerning familiar disposition is recorded in each subdatabase.
Consequently, the five FAD databases are now useable to support clinical investigations in different aspects of aneurismal pathology including biomarkers in the follow-up of aneurysm progression and treatment, correlation to functional imaging, and therapeutic trials.
Standardized European methodologies for contribution to the clinical database, and standardized sample banks of plasma, DNA, cells and tissue associated with the database have been essential to create in order to make it possible to merge data and biomaterials from several partners. Consequently, methodologies have been standardized concerning methods and intervals for AAA population screening, techniques for provision of DNA and criteria for diagnosis and analysis of endoleaks. Population screening is standardized according to current British and Danish methodologies and this will be applied to other EU and non-EU countries, which are starting population screening (eg Iceland). Standards for biomaterial has been formulated, and standards for reporting key variables as aneurismal size and endoleak has been proposed by relevant experts, and agreed upon at the kick off meeting in Liege. In presence of a localized dilatation, the right-angled maximal anteriorposterior diameter will be measured. Digital documentation or thermo-prints are made to demonstrate the visualised morphology and where the AAA was measured for later reproduction, when the patients attend follow up scans. Measurements will be performed in an inner to inner way, from plaque to plaque, if possible, in order to prevent variations in luminal-intramural-extramural measure points and sources of error. The luminal source of error will be the presence of a mural thrombus, and the extramural source will be the anterior longitudinal ligament or the vertebras themselves. All positive findings are being rescanned by a doctor responsible for the surveillance of the screening. In this way the numbers of observers will be kept at a minimum. Annual control scans will be performed of AAAs below 5 cm in maximal AP diameter, and biannually for AAA above 5 cm in maximal AP-diameter. If the AAA exceed 5.5 cm in maximal diameter, or becomes symptomatic, referral for surgical evaluation will be done. Performed operations including indication will be recorded.
AAA and TAA the incidence and prognosis following diagnoses and operation of AAA and TAA
One of the aims for WP1 were to use the data from the four subdatabases to describe the incidence and prognosis following diagnoses and operation of AAA and TAA. This has occurred in especially Sweden and Denmark with these publications so far:
P4. Karolinska:
1. Larsson E, Vishnevskaya L, Kalin B, Granath F, Swedenborg J, Hultgren R. High frequency of thoracic aneurysms in patients with abdominal aortic aneurysms. Ann Surg. 2011;253:180-4.
2. Hultgren R, Forsberg J, Alfredsson L, Swedenborg J, Leander K. Regional variation in the incidence of abdominal aortic aneurysm in Sweden. Br J Surg. 2012;99:647-
3. Villard C, Wågsäter D, Swedenborg J, Eriksson P, Hultgren R. 53 Biomarkers for Abdominal Aortic Aneurysms From a Sex Perspective. Gend Med. 2012 Jun 19. [Epub ahead of print]
P8. Viborg:
4.Lindholt JS, Norman PE. Meta-analysis of postoperative mortality after elective repair of abdominal aortic aneurysms detected by screening. Br J Surg. 2011 May;98(5):619-22.
5.Lindholt JS, Sørensen J, Søgaard R, Henneberg EW. Long-term benefit and cost-effectiveness analysis of screening for abdominal aortic aneurysms from a randomized controlled trial. Br J Surg. 2010 Jun;97(6):826-34.
6.Lindholt JS, Norman P. Screening for abdominal aortic aneurysm reduces overall mortality in men. A meta-analysis of the mid- and long-term effects of screening for abdominal aortic aneurysms. Eur J Vasc Endovasc Surg. 2008 Aug;36(2):167-71.
7. Lindholt JS, Sogaard, Laustsen J. The prognosis of ruptured abdominal aortic aneurysms in Denmark 1994-2008. Epidemiology 2012;4:111-3
8. Grondal N, Sogaard R, Henneberg EW, Lindholt JS. The Viborg Vascular (VIVA) screening trial of 65-74 year old men in the central region of Denmark: study protocol. Trials. 2010 May 27;11:67.
Assessing family history and present medication as a risk factor
In Sweden (P4), the nationwide registries of all in-hospital care (IPR=in patient registry) and the cause of death registries will be used to identify all persons diagnosed with, operated for, or having died from AAA in Sweden. For each of these cases, controls are randomly selected from the population; the controls are matched according to birth year, sex and region of residence. This data set will be linked to the Swedish Multigeneration registry in order to identify first-degree relatives of cases and controls. Subsequently, these families will be linked to IPR and the cause of death registry to identify family history of AAA.
Results during Jun 2008 to December 2009: All persons (3183) born after 1932, diagnosed with AAA between 2001 and 2005, and a random selection of 15,943 age-, gender-, and region-matched controls were included. First-degree relatives of cases and controls were identified via the Multigeneration Register. Family history of AAA for cases and controls was assessed by linking the relatives to the Hospital Discharge Register and Cause of Death Register. The data were analyzed by conditional logistic regression. The overall relative risk of AAA associated with family history compared to no family history was 1.9 (95% confidence interval [CI] 1.6-2.2). Comorbidities were more common among the cases than the controls (P < .0001) but the relative risks remained unchanged after adjustment for comorbidities. Stratification for absence or presence of comorbidities showed no significant difference between the two groups (P = .29). The relative risk of AAA for first-degree relatives was similar for women and men (P = .22 for gender differences), ie, the relative risk of AAA was not dependent on the gender of the index person. In conclusion, in this nationwide survey, the relative risk of developing AAA for first-degree relatives to persons diagnosed with AAA was approximately doubled compared to persons with no family history. Neither the gender of the index person nor the first-degree relative influenced the risk of AAA (partner 4, KI, submitted).
In a second study, the role of hereditary and environmental factors to development of AAA in a large population-based sample of twins was studied. The Swedish Twin Registry, containing data on twins born in the country since 1886, was cross-linked with the Inpatient Registry, providing national coverage of discharge diagnoses coded according to the International Classification of Diseases (ICD). All twins with an infrarenal AAA were identified. Concordance rates and tetrachoric correlations were calculated for monozygotic (MZ) and dizygotic (DZ) twins. Tetrachoric correlations were calculated assuming an underlying normal distribution of liability, with multiple factors contributing additively and a threshold value that discriminates between AAA and no AAA. Higher concordance rates and correlations of liability in MZ twins than in DZ twins suggest that genetic factors influence disease development. Structural equation modeling techniques, Mx-analyses, were used to estimate the contributions of genetic effects as well as shared and non-shared environmental factors for development of AAA. There were 172890 twins registered at the time of the study including 266 twins (81% men; mean age 72 years, range 48-94) with AAA. There were 7 MZ and 5 DZ concordant pairs as well as 44 MZ and 197 DZ discordant pairs with AAA. The probandwise concordance rates for MZ and DZ pairs were 24% and 4.8%, respectively. The tetrachoric correlations were 0.71 in MZ pairs and 0.31 in DZ pairs. In the structural equation models, genetic effects accounted for 70% (95% CI: 0.33-0.83) shared environmental effects for 0% (95% CI: 0-0.27) and non-shared environmental effects for 30% (95% CI: 0.17-0.46) of the phenotypic variance among twins. In conclusion, robust epidemiological evidence that heritability contributes to aneurysm formation is provided. Concordances and correlations were higher in MZ compared with DZ twins, indicating genetic effects. A heritability of 70% of the total trait variance was estimated. The remaining variance was explained by non-shared environmental factors with no support for a role of shared environmental influences.
Individual data were collated from 15 475 people under follow-up for a small aneurysm in 18 studies. The influence of co-variables (including demographics, medical and drug history) on aneurysm growth and rupture rates (analysed using longitudinal random-effects modelling and survival analysis with adjustment for aneurysm diameter) were summarized in an individual patient meta-analysis.
Available biosamples associated or not with clinical databases
As described in the grant agreement, for practical and ethical reasons, the standardized biological collections performed by each partner were prepared and conserved by each partner. The table represents all the biological samples available through FAD partnership.
These biobanking activities have included:
- Preparation of the samples involving dissection of the arterial tissue, separation between diseased parts and non or less diseased parts, separation of thrombi, intima, media, and adventitia; direct freezing and secondary cryo-pulverization, preparation of conditioned medium, and enzymatic digestion for preparation of primary culture of human endothelial cells, medial smooth muscle cells, and adventitial fibroblasts, and isolation of leukocytes. This step includes quality controls of the initial samples.
- Storage and Conservation of the samples at -80°C, in a specific area (Biological Resource Center) devoted to this activity
- Cession of the samples for experimental researches within the laboratory and between different laboratories through the FAD partnership and outside. These procedures include the signature of a Material Transfert Agreement and a declaration to the national regulatory authorities.
WP2. Genetics and functional genomics [from genes to phenotype]
Objectives:
To explore the molecular determinants of aortic dilation in humans through genetic and functional genomics in both humans and mice. Specifically WP2 includes:
- identification of new susceptibility genes for AAA and TAA
- identification of new mutations responsible for familial forms of TAA
- exploring the signalopathy associated with aneurysms
- development of animal models to study candidate genes for TAA and AAA.
Genome-wide/candidate gene case-control study to identify new susceptibility genes for AAA.
Genetic susceptibility studies in humans necessitate large cohorts of patients versus controls. So far, two large genome wide association studies (GWAS) have been performed on AAA, both lead by the FAD consortium. A meta analysis using the results of the different GWAS is currently conducted.
One of the main achievements of FAD in identifying genetic susceptibility to AAA was made by DeCODE (partner 15, ref. 17). Using a GWAS approach, a sequence variant was identified within intron 1 of the DAB2IP gene as a powerful genetic marker of AAA risk. DAB2 Interacting Protein is a member of the RAS-GTPase-activating protein family. DAB2IP suppresses cell survival and proliferation and is expressed by vascular cells, particularly by VSMCs. The identified variant could probably influence directly or indirectly the survival of VSMCs in response to proteolytic injury. Partners 4 (Karolinska), 6 (Liege) and 8 (Viborg) all contributed to this discovery.
- In addition, partner 15, identified two new AAA risk variants located within the LPA gene associate with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism. These variants were identified through genotyping and testing of a set of CAD associated SNPs on a large sample set of European AAA case-control samples (Ref Helgadottir A et al. 2012).
A second GWAS was recently conducted by partner 2 (London) and the AAA Consortium. Partners 4 (Karolinska), 8 (Viborg) and 15 (DeCODE) contributed to this discovery (ref AJHG 2011). The study identified the low-density-lipoprotein receptor-related protein 1 (LRP1) as a susceptibility gene for AAA. Interestingly, LRP1 has been shown to have a role in the regulation of MMP9 expression and murine models have demonstrated that LRP1 is essential for the maintenance of vascular wall integrity and that this effect is mediated via PDGF receptor beta and Smad signaling.
Identification of new mutations responsible for familial forms of TAA
In contrast to AAAs, a significant fraction of pathologies of the ascending aorta, including aneurysms (TAA) and dissections (TAD), are determined by single mutations in one specific gene. Several mutated genes have been identified as being directly responsible for TAAD, including both syndromic forms, of which Marfan syndrome is the most frequent, and familial forms. This approach of monogenic determinants necessitates the recruitment of informative families by clinicians. Numerous genes have now been identified: fibrillin (FBN1), SMC myosin (MYH11), actin (ACTA2), TGFBReceptors, SMAD3, a glucose transporter (GLUT10), etc. FAD partners 3 (Gent) and 1 (Inserm) actively participated in the discovery of these genes prior to FAD (background work). New genes have been identified by FAD participants during the last four years. In particular, partner 1 (Inserm) recently identified a mutation in TGF- β 2 as responsible for a mild form of Marfan syndrome (ref. 150). New informative families have been identified (ref 40), leading to the identification of new genes responsible for FTAA. Also partner 16 has collected several interesting FTAA families suitable for gene identification. (ref 125). At this moment whole exome sequencing is performed using the powerful Next Generation Sequencing approach, on many FTAA families collected during the FAD project. We can assume that this will reveal in the near future many new genes. The important added value of all these findings clearly shows that the extracellular matrix exert not only a structural function but that besides its mechanical role in providing strength and support to the tissues, it also acts as a reservoir for cytokines and growth factors implicated in cellular proliferation, differentiation, migration and survival and therefore has an important regulatory function in the development and homeostasis of body organs and tissues. It is clear that these new insights open perspective to plausible therapeutic approaches for this group of aorta related disorders as it is proposed that treatments which decrease TGF- β expression, as angiotensin II antagonist, can attenuate of prevent the phenotypes.
TGF β signalopathy in TAA (functional genomics)
One major question arising from genetic studies in TAA is how does the molecular diversity in aetiology lead to aortic aneurysms and/or dissections which are all very similar histopathologically. Rapidly it appeared that although diverse mutations in TGF- β pathways were important genetic determinants of monogenic forms of TAA, mutations were not limited to this pathway (MYH11, GlUT10, ACTA2), and that changes in expression of TGF- β molecules, mainly Smad2, were common to all forms of TAA, including forms not genetically detemined.
Alternatively spliced extra domain A (EDA) of fibronectin plays an essential role in tissue repair under the control of the TGF- β signaling pathway. Partner 4 (KI) has analyzed the expression of fibronectin spliceforms in dilated and non-dilated ascending aorta of tricuspid (TAV) and BAV patients. EDA expression was increased in dilated aorta from TAV patients compared with non-dilated aorta. In contrast, EDA expression was not increased in dilated aorta from BAV patients. The expression of EDA correlated with maximum aortic diameter in TAV but not in BAV patients. TGF β treatment influenced the splicing of FN and enhanced the formation of EDA-containing FN in cultured medial VSMCs from TAV patients but not in VSMCs derived from BAV patients. Gene set enrichment analysis suggested that differences in TGF β signaling may explain the impaired EDA splicing in BAV patients (Partner 4).
- Functional genomics in transgenic mice
Work involving transgenic mice and zebrafish has also been initiated by different FAD partners. The studies of animal models with aortic abonormalities suggested an important role for TGFBeta in the pathogenesis of those disorders. Fbn1 deficient mice as well as the Slc2a10 deficient zebrafish, revealing the arterial tortuosity phenotype, showed each a clear aberrant TGFBeta signaling. To further explore the relationship between the up or down regulation of TGFBeta and the phenotypical characteristics of the disorders partner 3 developed a tgfbr1 knock-in mouse model as well as a transgenic mice for the vascular type of EDS. This latter model spontaneously developed at two months open wounds starting in the shoulder area and penetrating skin and subdermal tissue. These skin lesions evolve fast and also fragility of the arterial vessels is noticed. Partner 3 also knocked down the slc2a10 gene in zebrafish to further investigated the pathogenic mechanisms causing arterial tortuosity. (Ref. Willaert et al. HMG, 2011).Partner 1 developed a different Col3a1 transgenic knock-out mice model (Ehler-Danlos) in order to study the vascular phenotype. At the same time partner 1 developed a myh11 mouse model. These transgenesis approaches were initiated during FAD, and mechanical, functional and clinical studies are currently underway.
WP3. Pathophysiology [from phenotype to molecular determinants]
Objectives:
To explore molecular and biomechanical determinants of aortic dilation and rupture in order to identify new targets for diagnosis and treatment of TAA and AAA. WP 3 includes:
1) Identification of new molecular and cellular targets beyond proteases in TAA and AAA, focusing on the involvement of mucoid degeneration in TAA and on the luminal thrombus as a conveyor of protease activities, pro-inflammatory mediators and neo-antigens in AAA.
2) The inflammatory response to proteolytic injury in AAA, in impeding cellular mechanisms of aneurysmal wall repair, will be explored in this context.
3) The role of cell signaling in the susceptibility for AAA development and ultimate rupture, and the role of elastin derived peptides in inflammation, angiogenesis and calcification, will be elucidated by transgenic approaches in mice.
- Outward convection and activation of blood-borne proteases in aneurysmal wall.
Since the contention of the pressurized blood within the arterial conduct is the fact of the insoluble extracellular matrix, synthetized by the VSMC, dilation and finally rupture of the wall is always the fact of proteolytic degradation of the insoluble extracellular matrix. In parallel, molecular transport within the arterial wall is mainly the fact of the outward radial hydraulic conductance, generated by blood pressure through the wall (background). The main achievements of FAD in the pathophysiology of aortic wall dilation and finally rupture focused on the spatio-temporal mechanisms of plasma-borne zymogens, transported through and activated within the aortic wall and consequences for the matrix, the VSMC, and the adventitial inflammatory response. This pathophysiological concept of aneurysmal pathology, whatever the localization and the aetiology (atheromatous versus non-atheroma, aneurysm versus dissection, thoracic aorta versus abdominal aorta, or other localizations) open new avenues for better understanding vascular pathology.
- Medial and adventitial responses
In TAA, partner 4 established the differential pattern of gene expression in the media associated with bicuspid or tricuspid aortic wall. A set of immune response genes was specifically identified in TAA of tricuspid valves, but not in TAA with bicuspidy, suggesting a more important stimulation of inflammatory response in the former. Similarly the pattern of MMPs expression was established in TAA wall, comparing bicuspidy and tricuspidy, and showing an over-expression of MMP-14 and MMP-19 in dilated aortas. MMP-3 and MMP-7 mRNA were not detected, confirming plasma as their main source. In parallel partner 1 showed an epigenetic modification of expression pattern in VSMC of TAA (see WP2). The consequences of this epigenetic regulation of Smad2 expression on target genes, including proteolytic and antiproteolytic (serpins) effectors, extracellular matrix proteins, are explored in aneurysms and dissection of the ascending aorta.
- Biomechanics of aortic aneurysm
In-vitro biomechanical testing of aneurysm tissue from open surgical repair provided novel biomechanical information of the aneurysm wall as well as the intra-luminal thrombus. This is essential data for reliable organ-level biomechanical simulations with specific importance for the biomechanical AAA rupture risk assessment. The large pool of CT-A data available through the FAD consortium allowed a direct clinical interpretation of biomechanical computed indices, as it is important to integrate biomechanical simulations in the clinical decision making process. Polarized light microscopy of wall samples from open aneurysm repair provided novel data of the collagen organization in the AAA wall. This information is not only important for a qualitative understanding of the AAA wall but also critical input for multi-scale histomechanical constitutive models. Here, the integration of histology and biomechanics provided a basic understanding of load carrying mechanisms of the AAA wall. Hemodynamic simulations of the blood flow through the normal and the aneurysmal aorta showed significant differences, which motivated a novel hypothesis for intra-luminal thrombus development in the AAA. Integrating the coagulation cascade in the analysis further reinforced this hypothesis. The outcome could potentially help to design therapies for a conservative treatment of AAAs. Based on follow-up CT-A data of patients with small AAAs their three-dimensional development was studied. The collected information suggested that AAAs should be monitored all over the aneurysmatic sac in order to identify the site of faster diameter growth. This information could essentially improve monitoring protocols for small aneurysms and towards avoiding expansion-related ruptures. In addition, the collected information is the basis for the development of aneurysm expansion models, which could further improve the surveillance of small AAAs.
WP4. Diagnosis
Objectives of this WP4 are to translate WP2 and 3 to diagnostic tools for human aneurysms, using specific new technologies, including proteomics and in vivo imaging technologies involving functional and molecular imaging as intermediary phenotypes in TAA and AAA pathologies. WP4 also involve discovery of new tracers for molecular imaging, establishment of proof of concept at a preclinical level, and initiation of clinical investigations.
- Plasma biomarkers in AAA.
Numerous circulating biomarkers of AAA were identified by the partners as background of FAD: MMP-9 (partner 6), fibrinolyic signal (partner 8) platelet activation (partner 1), interleukins (partner 10). In particular, partner 7 identified IGFBP-1 as a circulating biomarker, released by ILT, and associated with AAA size. Studies exploring the value of these biomarkers were performed in larger cohorts. Other pathophysiological biomarkers were discovered in FAD, such as circulating free DNA (partner 1) or NGAL (partner 7) as markers of neutrophil activation, and anti-Pg antibody titration as marker of periodontal infection associated with AAA evolution (partner 1).
- Circulating cell biomarkers, progenitors and leukocytes
Partners 7 (UAM) and 1 (INSERM) discovered differential expression of a number of redox proteins in circulating neutrophils and in the membrane of red blood cells of AAA patients, and are currently pursuing validation of these in larger cohorts. Among these proteins, catalase and peroxiredoxin were decreased in both cell types, with a concomitant increase in prooxidant factors suggesting the involvement of oxidative stress in AAA pathogenesis. Similarly, partner 1bis performed a profiling of macrophages from AAA (Lamblin N, Ratajczak P, Hot D, Dubois E, Chwastyniak M, Beseme O, Drobecq H, Lemoine Y, Koussa M, Amouyel P, Pinet F. Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study. J Proteome Res. 2010 Jul 2;9(7):3720-9). In parallel circulating progenitors were identified and characterized in association with AAA (partner 2, St Georges Hospital, and partner 9, CNRS). Circulating microparticles of platelet and neutrophil origin were also identified in association with AAA.
- Functional hemodynamic imaging in TAA.
Beside the usual morphological measurements of aneurysmal dimension (maximal diameter, length, etc.) and the presence of ILT in AAA, FAD developed several new approaches including measurement of aortic rigidity in TAA, using ultrasound and now Magnetic Resonance Imaging (MRI). In particular partner 12 (AMC) developed this imaging technology. MRI also can image non-laminar flow in aneurysm, allowing a more precise analysis of recirculation and vortex phenomenon associated with initiation and progression of aortic dilation. This technology is not yet proposed in TAA due to the complexity of the software and the time of calculation. These approaches link the diagnosis (WP4) to biomechanics (WP3).
- Iron traffic in AAA
Since iron gives a powerful negative signal in MRI, this technology allowed us to explore the spontaneous presence of iron in AAA, in ILT and wall, and the ability of phagocytes present in ILT to uptake exogenous, intravenously injected, Small Paramagnetic Iron Oxide (SPIO) particles (partner 6 Ulg and 1 Inserm). This data are important since they demonstrate the ability of MRI to image in vivo in human (WP4) the involvement of iron, and downstream of oxidation in the pathophysiology of AAA (WP3). Nevertheless the data also raised the question of the sensitivity of MRI to detect molecular events.
- PETscan and aneurysm progression.
Partner 6, ULg, was the first to show that 18F-DeoxyGlucose uptake, detected by Positron Emission Tomography coupled with Computed Tomography scan (Rx) for morphology (18FDG PET/CT) could be used as prognosis marker in aneurysmal disease (background). During FAD, ULg constituted an important series of 711 18FDG PET/CTscan in in 428 patients presenting aortic diseases, including AAA (n= 301) with or without EVAR, dissections (n=25) of the descending aorta, TAA (n=66) and Aortitis (n=36). The results concerning TAA are mainly negative, except rare exceptions, probably, due to the absence of adventitial inflammatory granuloma in association with TAA. These negative data suggest that TAA do not generate important adventitial immune response. In contrast, data concerning AAA and dissection are particularly rich and interesting. In parallel to PET a biological database (plasma and DNA samples) was constituted and morphology was accessible by the CT data. This triple approach: PET showing localized spots of high 18FDG uptake (high cellular metabolism), associated with CT allowing dimensions measurements and morphology classification, and biology offer an unique opportunity to link the evolution of aneurysmal dilation (CT) to cell function imaging (PET) and biology. Usually the hot spots of 18FDG take place in the external part of the aortic wall. The exploitation of this triple database is now in progress.
- Development of new molecular tracers targeting Intra luminal thrombi (partner1)
Preclinical proof of concept that 99mTc-Fucoidan images intraluminal thrombi
P-selectin expression is involved in the pathophysiology of biologically active arterial thrombus, and endothelial activation following transient ischemic event. Fucoidan is a polysaccharidic ligand of P-selectin with a nanomolar affinity (background). In FAD we have proposed a new approach of P-selectin molecular imaging based on radiolabeled fucoidan. Two kinds of experimental models were selected to evaluate the ability of radiolabeled fucoidan to detect P-selectin expression: platelet-rich arterial thrombi (arterial mural thrombus in experimental aneurysm), and myocardial ischemia-reperfusion. These two settings were chosen because they were clinically relevant, and both were associated with an important overexpression of platelet and endothelial P-selectin, respectively. Radiolabeled fucoidan SPECT was able (a) to detect the presence of platelet-rich arterial thrombi in all animals, with a median target to background ratio of 3.6 in mural aneurysmal thrombus (b) to detect a persistent endothelial activation 2 hours after reperfusion; in this latter model the magnitude of the signal was correlated with the extent of myocardium that underwent transient ischemia. The sensitivity of selectivity of the uptake and retention of 99mTc-fucoidan in both settings was excellent. This study supports 99mTc-fucoidan as a relevant imaging agent for in vivo detection of biological activities associated with P-selectin overexpression, such as arterial thrombus and ischemic memory. Given the previously reported wide availability at a low cost, and its low toxicity, fucoidan seems to overcome some of the limitations of previous P-selectin-targeted imaging agents. This approach has been patented by partner 1: WO 2010/116209 A1: Fucoidans as ligands for the diagnosis of degenerative pathologies, published 14 october 2010, extended to USA 02970-24565US02, January 9, 2012; and published (J Nucl Med 2011).
- Development of new molecular tracers targeting protease activities (partner 13 and partner1)
Serine proteases hold a central role in the pathophysiology of degenerative arterial diseases, but to date there is no suitable agent for molecular imaging. The project was designed to (1) develop a radiolabeled peptide probe for serine proteases imaging, including plasmin and leukocyte elastase, and (2) evaluate these new agents in vivo in a rodent model of arterial thrombus, including experimental aneurysms.
WP5. Therapeutics
Objectives.
To the validate current treatment and new therapeutics in human aneurismal diseases. WP5 will develop clinical investigations in patients and preclinical testing in experimental models.
This program includes:
1- an evaluation of medical prevention of aneurysm development and progression by existing medications,
2- the development of curative cell and bio-engineered therapeutics at a preclinical level and further extension to human,
3- further development of new therapeutic strategies.
- Medical prevention of aneurysm development and progression (preclinical)
Since there is no specific treatment able to completely prevent or inhibit progression of aneurysm, the FAD WP5 explored new medical therapeutic approaches at a preclinical level. First, since WP3 demonstrated that ILT is one of the main driving forces for AAA progression, the ability of inhibition of platelet aggregation to limit experimental and clinical progression of AAA was tested. Experimentally platelet inhibition prevented AAA development in an experimental model in rats. This result was retrospectively confirmed in human. Therefore, inhibition of platelet aggregation could be recommended in patients with small aneurysms, in prevention of growth (Partner 1 and 8). In parallel treatments able to impair the retention of neutrophils in ILT or antibiotics, which treated bacterial contamination, were also tested with success in experimental models. Evaluation of AZD 9668, a powerful inhibitor of leukocyte elastase is under progress.
- Novel interventional curative therapeutics through cell seeding, gene therapy and biomaterials
Several new models of aneurysms have been developed during FAD for evaluating new interventional therapeutics. These models are transposition of classical model (elastase, decellularized xenograt) to large animals than mice and rats, including rabbit and pigs (partner 1, 9 and 10). A model of EVAR is developed in rats (partner 1). These models were used for testing cell seeding as interventional therapeutic approach in aneurysm, and for developing new hydrogels avec vector of local cell or molecular therapy.
9). In particular sulphated hydrogel was used to heparin binding mediators such VEGF or SDF-1.
- New therapeutic strategies
These different approaches allowed us to initiate or to participate to clinical trials, including trial of angiotensin II antagonists in Marfan syndrome, inhibitor of mast cell activation (Cardoz), and cyclosporin in human AAA. Others are in discussion with pharmaceutical industry (leukocyte elastase inhibition with Astra-Zeneca).
Scientific and medical conclusions.
FAD was a highly translational and transversal large-scale program, mobilizing numerous energy in order to better understand what are the biological determinants of aneurysm development in order to propose new diagnosis and therapeutic tools. FAD achievements are diverse, due to the number of partner, each developing its own project. Nevertheless the majority of the works were complementary through the different WPs and the different partners. Two examples are presented in the table beyond. The works performed on TGF-beta signaling pathway covered all the WPs, mainly WP1, 2, 3, and 5, and involved height partners among fifteen. Similarly the works performed on the Intraluminal thrombus as the predominant driving force in AAA evolution, covered all the WPs and involved eleven partners among fifteen. Similarly the number of common publications, 81 among 292 (28%), co-signed by 2 partners or more, also provide evidences of the transversality of FAD.
Major achievements have been performed in each WP opening new avenue for future Research and Development (R&D) in the field of aneurismal disease of the aorta:
- Clinical and genetic database and tissue and cell collections were constituted through FAD, These database and collections, shared inside FAD by several partners, but also opened to other teams outside of FAD consortium, allow important translational work in the future.
- Discovery of new gene mutations in aneurismal disease raises the important question of genetic diversity leading to common pathological features of arterial wall and rupture.
- Pathophysiology leads to the general concept of blood-borne injuries of the arterial wall, sensitized by intrinsic defect of the wall components, leading to medial and adventitial response.
- Diagnostic applications are in progress, including circulating biomarkers, but also important progress in functional and molecular imaging of aneurismal pathology. This progress include clinical applications, but also new tracers for which the preclinical proof of concept has been now established. Further developments as first injection in human are programmed.
- Similarly, new therapeutic approaches are in progress through clinical investigations in human patient.
Potential Impact:
The potential impacts, including the socio-economic impact and the wider societal implications.
FAD will have medical and socio-economic impacts in health, medicine, and economic development of new tools.
The development of screening programs will have, with time, consequences on health in aging EU population. Screening for AAA will have important consequences on the care of aging male without symptom. This beneficial effect will extend more than AAA alone. It will be extended to the detection of atherothrombotic disease whatever the localization and the prevention of periodontal diseases in aging population. The relationship between periodontal disease in aging population and the cardiovascular risk is now well established by epidemiological studies. Through translational research, FAD provided evidences of the biological relationship between periodontal disease, blood weak pathogen transitory passage, ILT retention of these weak pathogens, leading to AAA progression. Such health programs are now progressing slowly, but must be developed with the support of EU for Research and Development. We proposed this transversal project, including cardiologists, neurologists, biologists, odontologists, twice to EU (ERC 2010 and COST), but we didn't succeed to be selected.
The concept developed in FAD will also impact medicine of other vascular pathologies, including:
-Genetics of vascular diseases: the effort performed with success in genetics during FAD will be pursued including discovery of new genes through informative family approaches and genetic sensitivity trough GWAS approach in large cohort.
- Other localization of aneurysmal diseases: In particular cerebral aneurysms. At the beginning of FAD specific links was established with ANEURIST (see http://www.aneurist.org/ online). @neurIST -Integrated Biomedical Informatics for the Management of Cerebral Aneurysms was a European initiative within the Sixth Framework Programme of the Information Society Technologies IST. Alejandro Frangi (Aneurist coordinator) and Juahana Frozen, a Finnish neurosurgeaon, specialist of cerebral aneurysms, were member of the FAD scientific advisory board, and Jean-Baptiste Michel, FAD coordinatore participated to @neurIST meetings in Barcelonna. In particular FAD proposed that the pathogenicity of ILT is common to AAA and cerebral aneurysm.
- New stenting technology: In the context of cerebral aneurysm, a new type of multilayer not covered stent, Flow Diverter (see http://www.cardiatis.com/ online) has been implanted in human patients, particularly in treatment of cerebral aneurysm. The principle was that flow diversion of the aneurysmal sac could provoke sac thrombosis and healing. Unfortunately this technology was not associated with total success. Several secondary aneurysmal ruptures of cerebral aneurysms, but also of aortic and other peripheral aneurysms, impair the results. Since flow diversion is not ILT exclusion, the ILT associated with this type of stents continued to be biologically active leading to continuous proteolytic degradation of the arterial wall.
- Atherothrombotic diseases: Since AAA is an atherothrombotic disease, works performed on AAA also impact directly what we know about occlusive atherothombosis. In particular, works performed in genetics of sensitivity to AAA also impact genetics of coronary and carotid artery diseases in humans.
One other important point is that the studies made on IntraLuminal Thrombus (ILT) during FAD directly impact the pathophysiology of IntraPlaque Hemorrhage (IPH) in occlusive atherothrombosis. IPHs are now well established as the driving force leading to plaque rupture and clinical expression. We wrote important reviews in this field and gave several international conferences on this important subject (EVBO meeting in Krakow, ESC 2012 Munich). It is time now to develop something at the EU level concerning this most important feature in human atherothrombotic diseases. We proposed twice a COST and an ERC project in this field at the EU level (IPH-COST action 2011, ERC Spa-Athero 2012) in order to initiate an EU network in this field. We didn?t succeed. We will try again. We wish and hope that this important field of heme/iron metabolism in atherothrombotic diseases will be the object of EU calls at the beginning of FP-8!
- Aortic valve disease: Similarly to the preceding fields, the works performed on TAA particularly on genetics, genomics and biomechanics, allowed to FAD partners to continue collaborations in this field focusing on the relationship between TAA and bicuspid aortic valves. MIBAVA- Mechanistic Interrogation of Bicuspid Aortic Valve associated Aortopathy (see http://www.fondationleducq.org/ online), was selected Under the Transatlantic Networks of Excellence in Cardiovascular Research Program awarded by Fondation Leducq U.S. $6,000,000 over five years for internationally collaborative research. Bart Loeys EU coordinator (partber 3, ex UG), and Karolinska Institue (KI, partner 4) a partner.
- Economic Impact: The most important economic potential impact will be directly linked to the development of applications initiated within FAD. Among the patents generated through FAD the most important and numerous concern diagnostic applications, genetic markers, circulating biomarkers and molecular imaging. The Inserm patent and the corresponding published article on the use of fucoidan as a new pharmacophore for the diagnosis of vascular disease is probably the more advanced ones. The preclinical proof of concept has been established. The program is now to go to the first injection in human. This must be achieved through several steps including the definitive choice of the fucoidan preparation, the preparation in GMP conditions for first injection in human, minimal toxicologic studies, the writing of the regulatory files, the definition and the writing of the clinical investigation protocole, and finally the financial support to do that. This development is now in progress with a financial support at a national level.
Main dissemination activities and exploitation of results (WP6)
Objectives
- Ensure regular training of Junior Scientists and Technical Staff participating in FAD project to acquire new skills and techniques.
- In-house training is expected to promote personnel exchanges and amplify excellence and scientific links within the Network.
- To disseminate the results generated in FAD in medical science.
- To reach all the potential audiences, in particular, pharma companies, industrial partner for diagnostic tools, patient organisations and public health authorities.
A website has been created (see http://www.fightin-aneurysm.org online) to be a showcase of the project, and present the project to the public. The success of this website as a tool to promote FAD can be shown by the statistic of visits of the website, which regularly increase since its launching.
3 training sessions have been organized during the 4 years of the project: Proteomics in aneurismal disease: from sampling to analysis (April 2009), Animal models of AAA: rational to practice (March 2010) and CV molecular imaging (March 2012). The purpose of this training was decided after consultation of the partners on their needs linked to FAD works and centralization of the requests. Each partner has been involved as speakers or as attendees. The participants to these training sessions were French, Danish, English, Sweden, Belgian, Spanish and Czech. Behind the exchange of knowledge between the partners, these courses were a real opportunity for the partners to exchange knowledge and open the door to new collaborations.
The proof of the existence of this expertise resides in the fact that one hundred articles were reviewed by FAD leaders between July 2008 and July 2012. As an example, Jes Lindholt (WP1 team leader, partner 8, Viborg Hospital) is a member of the board at The European Journal of Vascular and Endovascular Surgery and Jean-Baptiste Michel has reviewed 19 articles of the 30 articles on the aneurysms published in ATVB and 10 on 35 in Cardiovascular Research.
Project website: http://www.fighting-aneurysm.org/
Contact: Jean-Baptiste.Michel@inserm.fr
