Highlights of the Recent Literature on Abdominal Aortic Aneurysm Research

Editor’s Commentary Highlights of the Recent Literature Abdominal Aortic Aneurysm Research

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Significant advances have been made since January 2004 when Annals of Vascular Surgery published an Editor’s Commentary entitled ‘‘Are There Genes for Aneurysms in the Blueprint of The Human Genome?’’1 Here, we highlight some of the most interesting results obtained using genetic, genomic, and proteomic approaches to study abdominal aortic aneurysms (AAAs). We selected these studies (Table I) from over 100 published articles. Genetic association studies with biologically plausible candidate genes. The ‘‘genetic association study’’ approach has been used in several publications to try to identify genetic factors involved in the development of AAA. The rationale of these studies is to select genes for investigation based on educated guesses and what is known about their role in vascular biology. The hypothesis is that genetic variations in these genes contribute to greater or lesser activity of the gene products and thereby to susceptibility for the development of AAAs. Fatini et al.2 investigated the role of genetic variations called polymorphisms in the genes encoding the angiotensin converting enzyme (ACE) and angiotensin II type I receptor (AT1R) in the development of AAA in 250 Italian patients. These are excellent candidate genes for AAA because (1) they are expressed in human aneurysmal aorta3 and (2) their polymorphisms have been associated with other cardiovascular diseases.4 There was a significant difference in the frequency of the genotypes (p = 0.0002) and alleles (p < 0.0001) of the ACE polymorphism between patients and controls. No significant association was found between AT1R polymorphism and AAA. As far as hypertension is considered, they also found a significant difference between hypertensive AAA patients and hypertensive controls, as well as between normotensive AAA and normotensive controls. The so-called D allele of the ACE gene (D stands for deletion) has been associated with increased serum levels of circulating ACE and can be used as a marker of atherosclerotic cardiovascular complica-

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tions. A possible role for the renin-angiotensin system in AAA is supported by the finding that angiotensin II promotes hypertension and alters shear stress. Another mechanism by which angiotensin II may affect vascular tissue remodeling is the activation of growth factors and inflammation. In another genetic association study, Massart et al.5 investigated the polymorphisms in several other genes, including elastin (ELN), estrogen receptor-a (ESR1), estrogen receptor-b (ESR2), progesterone receptor (PGR), and transforming growth factor-b1 (TGFB1) in 99 Italian AAA patients. The selection of ELN as a candidate gene can be justified since it is a major protein of the aortic tunica media and it confers strength and elasticity to the aortic wall. Estrogen (but not estrogen plus progestin) treatment is known to affect connective tissue metabolism.6 There was a significant difference in the frequency of genotypes for the ESR2 polymorphism between AAA cases and controls (p < 0.05). Although the importance of this particular ESR2 polymorphism in the expression and function of ESR2 has not been established, this observation suggests that estrogens contribute to the development of AAA. It is also of interest that the authors did not find an association between AAA and polymorphisms in ELN, ESR1, PGR, or TGFB1. We carried out a genetic association study for 14 polymorphisms from 13 genes encoding for proteins considered to be important structural molecules of the aortic wall or involved in extracellular matrix remodeling.7 Altogether, 387 AAA patients and 425 controls were studied. A statistical analysis showed an association with two polymorphisms in the gene for tissue inhibitor of metalloproteinases 1 (TIMP1) in males without a family history of AAA. These findings suggest that genetic variations in TIMP1 may contribute to the pathogenesis of AAAs. It is noteworthy that, contrary to a previous study by Jones et al.,8 we did not find an association between a polymorphism in the gene for matrix metalloproteinase-9 (MMP9) and AAA, suggesting genetic heterogeneity of the disease. In summary, the studies described above suggested that the list of candidate genes for AAA includes ACE, ESR2, and TIMP1. Obviously, these findings need to be confirmed in other sets of samples before we can make recommendations for clinical testing. 1

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Annals of Vascular Surgery

Table I. Characteristics of the studies discussed here Study

Type of study

Candidate genes or pathways investigateda

Fatini et al.2 Massart et al.5 Ogata et al.7 Shibamura et al.9 Feezor et al.10 Zhao et al.11 Shimizu et al.12 Kaschina et al.13 Sigala et al.14 Sho et al.15

Genetic association study Genetic association study Genetic association study Genomewide linkage study Transcriptional profiling Animal model Animal model Animal model Animal model Animal model

ACE, AT1R ELN, ESR1, ESR2, PGR, TGFB1 MMPs, TIMPs, ELN, TGFB1, COL3A1 Entire genome All expressed genes ALOX5 pathway INFG pathway, IL4, MMP9, MMP12 KNG1 NOS2A NFKB1, CFS2, MIF, HBEGF, NOS2A

a

Gene symbols used are approved by the Human Genome Organization Gene Nomenclature Committee and obtained from www.gene.ucl.ac.uk/nomenclature.

Genomewide DNA linkage study: the unbiased and comprehensive approach for identifying genetic risk factors. Another approach is to study the entire genome in a statistical method called DNA linkage study to identify genetic factors contributing to the diseases. We used this approach to find chromosomal regions harboring susceptibility genes for AAA in families in which at least two blood relatives were diagnosed with an AAA.9 We used the so-called affected relative pair linkage analysis approach, with sex and family history as covariates. We found strong evidence of linkage [logarithm of odds (LOD) score = 4.64] to a region on chromosome 19 with 36 families when including sex and number of affected firstdegree relatives of the proband (Naff) as covariates. We then genotyped 83 additional families for the same genetic markers, typed additional genetic markers for all families, and obtained a LOD score of 4.75 (p = 0.00014), with sex, Naff, and their interaction as covariates. We also identified a region on chromosome 4q31 with a LOD score of 3.73 (p = 0.0012) using the same covariate model as for chromosome 19. These findings provide evidence for the presence of susceptibility genes for AAA on chromosomes 19q13 and 4q31. These regions can now be considered ‘‘candidate regions’’ for AAA susceptibility genes. The task ahead of us is to find the gene(s) and the specific variations in it leading to increased risk for AAA. Transcriptional profiling provides expression patterns for aneurysms. Microarray expression analysis was used to find out which genes had altered level of expression in peripheral blood leukocytes from patients with thoracoabdominal aortic aneurysms.10 Changes in the expression of 146 genes were observed (p < 0.001). The expression pattern of

138 genes (p < 0.001) and the concentrations of seven plasma proteins discriminated between patients who developed multiorgan dysfunction syndrome (MODS) and those who did not, and many of these differences were evident even before surgery. These findings suggest that changes in blood leukocyte gene expression and plasma protein concentrations can illuminate pathophysiological processes that are subsequently associated with the clinical sequelae of systemic inflammatory response syndrome and MODS. The fascinating part of this technology is that it can provide information on thousands of expressed genes in the same experiment and that it can be automated and run in a high-throughput genomics facility. Results from studies with animal models. Animal models of AAA are an important and very active area of research. Zhao et al.11 suggested the 5-lipoxygenase (ALOX5) pathway as a new pathway involved in the development of aortic aneurysms. They showed that ALOX5-positive macrophages localize to the adventitia of mice with aneurysms and of human arteries in areas of neoangiogenesis and that these cells constitute a main cellular component in aortic aneurysms induced by an atherogenic diet in mice deficient in apolipoprotein E. ALOX5 deficiency in mice markedly attenuates the formation of aortic aneurysms and is associated with reduced MMP2 activity and diminished chemokine (C-C motif) ligand 3 (CCL3) but only minimally affects the formation of lipid-rich lesions. The leukotriene LTD4 strongly stimulates expression of CCL3 in macrophages and chemokine (C-X-C motif) ligand 2 (CXCL2) in endothelial cells. These data link the ALOX5 pathway to hyperlipidemia-dependent inflammation of the arterial wall and to pathogenesis of aortic aneurysms through a potential chemokine intermediary route.

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Fig. 1. Four pillars of abdominal aortic aneurysm (AAA) research. Unraveling the pathophysiology of AAA and identifying the specific risk factors for AAA will require interdisciplinary approaches. Specific examples from each of these approaches applied to AAA research are presented in Table I.

Using an allograft model, Shimizu et al.12 studied the interferon-c (IFNG) pathway in mice lacking the receptors for IFNG. Allografts in wildtype recipients developed intimal hyperplasia, whereas allografts in IFNG receptor—deficient (GRKO) hosts developed severe AAAs associated with markedly increased levels of MMP9 and MMP12. Allografts in GRKO recipients treated with anti-interleukin-4 (IL4) antibody to block the function of IL4 or allografts in GRKO hosts congenitally deficient in IL4 did not develop AAA and likewise exhibited attenuated collagenolytic and elastolytic activities. These findings suggested new insights into the mechanisms of aneurysmal disease and that IL4 blockade or IFNG activation may inhibit the development and growth of arterial aneurysms. In another study, Kaschina and coworkers13 showed that genetic kininogen (KNG1) deficiency renders vascular tissue prone to aneurysm formation but not to atherosclerosis. The authors compared KNG1-deficient brown Norway Katholiek rats with wild-type brown Norway rats after feeding them a high-fat (atherogenic) diet. Aneurysms were associated with increased degradation of elastin, increased expression of MMP2 and MMP3, downregulation of TIMP4, and FasL- and caspase-3mediated apoptosis. KNG1-deficient animals also featured changes in plasma cytokine levels compatible with apoptotic vascular damage, i.e., upregulation of IFNG and downregulation of CSF2 and IL1B. Finally, in response to an atherogenic diet, KNG1-deficient animals developed an increase in high-density lipoprotein/total cholesterol index, pronounced fatty liver and heart degeneration, and lipid depositions in aortic media without atherosclerotic plaque formation.

Editor’s commentary 3

Yet another animal model for AAA is the socalled elastase infusion model in rats. Using this model system, Sigala et al.14 examined the expression of inducible nitric oxide synthase (NOS2A) in the aortic wall and its relation to cellular growth parameters. They showed that NOS2Aderived nitric oxide is associated with the cellular growth parameters of the vessel cells, predominantly smooth muscle cells (SMCs), and selective NOS2A blockage ameliorates the cellular remodeling seen in AAAs. Sho et al.15 also used this model to investigate cell type-specific expression of aneurysmal wall by isolating SMC and predominantly macrophage-containing mural cell populations. They showed that flow differentially regulates cell-specific gene expression in AAAs. The authors pointed out that whole-organ analysis of AAA tissue lysates might obscure important cellular responses to inflammation and flow. Cassis et al.16 investigated the effect of aldosterone in angiotensin II-induced arteriosclerosis and AAA in mice that were deficient in apolipoprotein E. Mice were fed a hyperlipidemic diet and infused with angiotensin II. Aldosterone had no effect on the extent of atherosclerosis or AAAs. Concluding remarks. As can be seen from the selected studies from the recent literature summarized above (Table I, Fig. 1), the excitement in the field of AAA research is evident, so much so that the New York Academy of Sciences and Columbia University College of Physicians and Surgeons together with the National Heart, Lung, and Blood Institute of the National Institutes of Health are planning to sponsor an international conference, ‘‘The Abdominal Aortic Aneurysm: Genetics, Pathophysiology, and Molecular Biology.’’ The meeting will take place in New York City, April 3-5, 2006. The principal organizer of the convocation is M. David Tilson, a true pioneer of the modern research on AAA. The co-organizers are Gilbert R. Upchurch and Helena Kuivaniemi. Helena Kuivaniemi, MD, PhD Associate Editor e-mail: [email protected] Toru Ogata, MD, Detroit, MI, USA REFERENCES 1. Kuivaniemi H. Are there genes for aneurysms in the blueprint of the human genome? Ann Vasc Surg 2004;18:2-3. 2. Fatini C, Pratesi G, Sofi F, et al. ACE DD genotype: a predisposing factor for abdominal aortic aneurysm. Eur J Vasc Endovasc Surg 2005;29:227-232.

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3. Nishimoto M, Takai S, Fukumoto H, et al. Increased local angiotensin II formation in aneurysmal aorta. Life Sci 2002;71:2195-2205. 4. Danser AH, Schunkert H. Renin-angiotensin system gene polymorphisms: potential mechanisms for their association with cardiovascular diseases. Eur J Pharmacol 2000;410: 303-316. 5. Massart F, Marini F, Menegato A, et al. Allelic genes involved in artery compliance and susceptibility to sporadic abdominal aortic aneurysm. J Steroid Biochem Mol Biol 2004;92:413-418. 6. Register TC, Adams MR, Golden DL, et al. Conjugated equine estrogens alone, but not in combination with medroxyprogesterone acetate, inhibit aortic connective tissue remodeling after plasma lipid lowering in female monkeys. Arterioscler Thromb Vasc Biol 1998;18:1164-1171. 7. Ogata T, Shibamura H, Tromp G, et al. Genetic analysis of polymorphisms in biologically relevant candidate genes in patients with abdominal aortic aneurysms. J Vasc Surg 2005;41:1036-1042. 8. Jones GT, Phillips VL, Harris EL, et al. Functional matrix metalloproteinase-9 polymorphism (C-1562T) associated with abdominal aortic aneurysm. J Vasc Surg 2003;38: 1363-1367. 9. Shibamura H, Olson JM, van Vlijmen-Van Keulen C, et al. Genome scan for familial abdominal aortic aneurysm using sex and family history as covariates suggests genetic heterogeneity and identifies linkage to chromosome 19q13. Circulation 2004;109:2103-2108.

Annals of Vascular Surgery

10. Feezor RJ, Baker HV, Xiao W, et al. Genomic and proteomic determinants of outcome in patients undergoing thoracoabdominal aortic aneurysm repair. J Immunol 2004;172: 7103-7109. 11. Zhao L, Moos MP, Grabner R, et al. The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med 2004;10:966-973. 12. Shimizu K, Shichiri M, Libby P, et al. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest 2004;114:300308. 13. Kaschina E, Stoll M, Sommerfeld M, et al. Genetic kininogen deficiency contributes to aortic aneurysm formation but not to atherosclerosis. Physiol Genomics 2004;19:4149. 14. Sigala F, Papalambros E, Kotsinas A, et al. Relationship between iNOS expression and aortic cell proliferation and apoptosis in an elastase-induced model of aorta aneurysm and the effect of 1400 W administration. Surgery 2005;137: 447-456. 15. Sho E, Sho M, Nanjo H, et al. Comparison of cell-typespecific vs transmural aortic gene expression in experimental aneurysms. J Vasc Surg 2005;41:844-852. 16. Cassis LA, Helton MJ, Howatt DA, et al. Aldosterone does not mediate angiotensin II-induced atherosclerosis and abdominal aortic aneurysms. Br J Pharmacol 2005;144:443-448. DOI: 10.1007/s10016-005-9167-4 Published online: January 1, 2006