Paul D. DiMusto, MD and Gilbert R. Upchurch, MD
The pathogenesis of abdominal aortic aneurysm (AAA) formation is complex and not fully understood. Certain epidemiologic risk factors are associated with AAA development, such as male sex, cigarette smoking, hypertension, advanced age, atherosclerosis, and a genetic predisposition. However, the relationship between these risk factors and the biological process of aneurysm formation is not clear.
It is known that AAAs are characterized by destruction of elastin and collagen in the media and adventitia, loss of medial smooth muscle cells with thinning of the vessel wall, and transmural infiltration of lymphocytes and macrophages. Atherosclerosis is a common underlying feature of aneurysms. However, atherosclerosis is not the primary driving factor in the development of AAAs. Atherosclerosis is a disease that is widespread throughout the vasculature, however aneurysms only form in specific locations and only in certain individuals. Additionally, atherosclerosis is primarily a disease of the intima, while aneurysm formation primarily affects the media and adventitia.
Currently there are thought to be four mechanisms relevant to AAA formation including: 1) proteolytic degradation of aortic wall connective tissue, 2) inflammation and immune responses, 3) biochemical wall stress, and 4) molecular genetics.
Proteolytic Degradation of Aortic Wall Connective Tissue
Aneurysm formation involves a complex process of destruction of the aortic media and supporting lamina through degradation of elastin and collagen. This leads to a decrease in tensile strength in the aortic wall which can then lead to aneurysm formation. In vivo models of AAA formation, including periadventitial application of calcium chloride, intraluminal aortic perfusion of elastase, and infusion of angiotensin II into ApoE -/- or LDL receptor -/- animals, have been used to elucidate the role of various proteases, including the serine proteases and cathepsins, during aneurysm formation. These models, as well as studies on human aortic tissue, suggest a critical role for the matrix metalloproteinase proteinases (MMPs), derived from macrophages and aortic smooth muscle cells, in aneurysm formation.
The MMPs play a central role in the control of inflammation, as well as break down the aortic wall. Several MMPs have been found to be important in aneurysm formation including MMP 1, 2, 3, 8, 9, 10, 12, and 13. MMP2 and MMP9 have both elastolytic and collagenolytic properties and appear to play a particularly critical role in AAA formation. Both are over expressed in both human and experimental AAAs. High levels of MMP2, a constitutive enzyme, are found in small aneurysmal aortas, suggesting a role for MMP2 in early aneurysm formation. MMP12 is highly expressed along the proximal leading edge of human AAAs and may also be important in aneurysm initiation. The inducible elastase MMP9 is elevated in aortic aneurysm tissue, as well as in serum from patients with AAAs. MMP9 is found at higher levels in larger aneurysms and is thought to play a role in aneurysm expansion and rupture. MMP9 has also been shown to decrease following endovascular exclusion of aortic aneurysms. Because this is an inducible MMP, transcription of the gene encoding for MMP9 is an important regulation point in the process of aneurysm formation.
Experimental aneurysm models also support a critical role for MMP9, as knockout mice do not form aneurysms. Importantly, when these knockout mice undergo wild type bone marrow transplantation, the aneurysm phenotype is restored, adding credence to a central role for MMP9. Additionally, pharmacologic inhibition of the MMPs using tissue inhibitors of metalloproteinases (TIMPs) and a2macrogloubin has been shown to suppress aneurysm formation in animal models. Finally, the tetracycline antibiotic doxycycline is also a nonselective MMP inhibitor. Standard doses of doxycycline have been shown to be effective in MMP inhibition; however, it is not the antibiotic moiety that confers this property as non-antibiotic tetracycline derivatives have also been shown to be effective MMP inhibitors and prevent AAA formation in animals.
In addition to the MMPs, the cystine proteases, including cathepsins K, L, and S, appear to play a role in AAA formation. Cathepsin K is the most potent elastolytic enzyme known. All three of these cathepsins have been found to be over expressed in human AAAs compared to normal aortic tissue. The protease activity of the cathepsins is normally balanced by inhibitors, the most abundant being cystatin C, which is expressed in virtually all organs. Serum and tissue levels of cystatin C, which is normally constitutively expressed, have been found to be significantly decreased in patients with AAAs. Immunohistochemical analysis of tissue from human AAAs confirms an increase in cathepsin S staining and a decrease in cystatin C staining when compared to normal aortic tissue. Cathepsin S deficient mice have shown a reduction in aortic dilatation in the elastase model of aneurysm formation when compared to normal mice. However, the biologic mechanisms underlying the shift in the natural balance towards proteolytic activity in the formation of AAAs are not yet fully known.
Degradation of the aortic wall also leads to the release of several inflammatory factors from the wall itself, including transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) that lead to further inflammation. Pharmacologic agents are also effective at reducing inflammation. Aspirin, as well as statins, have been shown to decrease inflammation in the aortic wall and confer some protection against AAA formation and progression in animal models. In addition, the angeotensin converting enzyme inhibitors have been shown to decrease the effectiveness of proteolytic enzymes, again providing some protection against AAA progression. However, the difficulty with these, like any daily pharmacologic regimen, includes poor patient compliance, which was a significant drawback in clinical trials examining the effectiveness of propranolol on slowing AAA growth in humans.
In summary, during AAA formation, the balance of vessel wall remodeling favors elastin and collagen degradation, leading to weakening of the aortic wall and aneurismal dilatation.
Inflammation and Immune Responses
A prominent histologic feature of AAAs is extensive transmural infiltration by macrophages and lymphocytes. It is hypothesized that these cells subsequently release a cascade of cytokines resulting in activation of many proteases. The trigger for influx and migration of leukocytes is unknown, but some “immune-inducer”, possibly including exposed elastin degradation products in the aortic wall, may serve as the primary chemotactic attractant for infiltrating macrophages.
The concept that AAA formation is an autoimmune response is supported by the extensive infiltration of B and T lymphocytes, dendritic cells, and monocytes into the aortic wall. These are primarily found in the adventitia. The presence of these cells suggests that a chronic immune response is present in the aneurysm; however, it is yet unclear whether this is a primary event in aneurysm formation or is due to chronic inflammation and tissue destruction.
The role of the cellular immune system in aneurysm formation and propagation requires further exploration, but there is some evidence for both T-helper type 1 and type 2 responses in human AAAs. There is a component of an active immune response in the aortic wall, as the infiltrating mononuclear cells express different activation antigens at variable times in aneurysm formation. CD69 is expressed early; CD25 and CD38 in the intermediate stages, and CD45RO and HLA class II late. Additionally, there has been found to be an association between particular HLA alleles and AAAs, as well as autoantibodies present in AAAs. Finally, monoclonal/oligoclonal αβ T-cell receptor+ and γδ T-cell receptor+ T-cells have been shown to be present in AAAs. It is thought that molecular mimicry may be responsible for the induction of T cell inflammatory responses in AAA lesions.
The adventitia appears to be the primary site of leukocyte infiltration and initial MMP activation. Macrophage and lymphocyte-generated cytokines are elevated in the aneurysmal aortic wall, including IL-1ß, TFN-α, IL-6, IL-8, MCP-1, IFN-γ, and GM-CSF. Many of these pro-inflammatory cytokines activate c-Jun N-terminal kinase (JNK) in the smooth muscle cells, leading to secretion of the MMPs and other pro-inflammatory cytokines. Pharmacologic inhibition of JNK in animal aortic smooth muscle cells in culture has been shown to suppress the secretion of both MMP2 and MMP9. Further studies using a mouse model of AAA showed that pharmacologic inhibition of JNK prevented aneurysm formation and inhibition of JNK is associated with regression of an already established AAA.
Another area that has recently been explored is gender differences in the expression of inflammatory cytokines and chemokines. This work was based on the clinical observation that men are four times as likely to develop an AAA as women, thus implying that female gender is protective in terms of AAA formation. These studies were conducted using the elastase perfusion model in rodents. Four days after perfusion, the female elastase perfused aortas had a five-fold decrease in the expression of several genes compared to males. These included several in the bone morphogenetic protein (BMP) family, including BMP1, BMP6, BMP7, and BMP15. The tumor necrosis factor superfamily ligands (TNFSF) particularly TNFSF4, 6, 9, and 14 also showed a five fold reduction in females compared to males. Genes in the transforming growth factor beta (TGFβ) family, specifically TGFβ1, TGFβ2, and TGFβ3, as well as the vascular endothelial growth factor (VEGF), specifically VEGF1 and VEGF2, were three to four times lower in females compared to males. The expression of several genes in the interleukin family (IL), including IL8, IL11, IL12, and IL13, and the CC chemokine receptor family (CCR) including CCR2, CCR6, CCR7, and CCR9, were undetectable in the females, but present in males. It is thought that these differences in gene expression are due to the anti-inflammatory effects of estrogen, which acts through multiple pathways. Further study in this area is needed to explore the exact mechanisms of hormonal protection.
Finally, nitric oxide (NO), a ubiquitous molecule known to alter vessel wall remodeling, also may play a role in AAA formation. However, there is some debate as to its exact role. Nitric oxide induces MMP9 expression and may be important in initiating vessel wall degradation leading to aneurysm formation. However, pharmacologic inhibition of nitric oxide synthase, the enzyme that converts L-arginine to NO, leads to decreased NO production and has been shown to attenuate aneurysm formation in animal models. Other studies have been conducted using the elastase perfusion model of AAA in inducible NOS -/- mice, one of the three isoforms of NOS which has been shown to have increased expression in elastase induced AAAs. These studies showed no significant difference in the size of AAA formed in the iNOS knock out mice when compared to controls. Immunohistochemical evaluation of normal aortic tissue and aneurysmal aortic tissue for nitrotyrosine, one of the endpoints of nitration, was also conducted. This revealed no detectable nitrotyrosine in the normal tissue, while there was extensive nitrotyrosine staining in the tissue from AAAs. The pattern of staining was widespread and appeared to be associated with mononuclear inflammatory cells, smooth-muscle like cells, and areas of degenerating extracellular matrix. Further studies are needed to clarify these issues.
Biochemical Wall Stress
The preferential infrarenal site for AAA formation suggests potential differences in aortic structure, biology, and stress along the length of the aorta. There is a natural reduction in the number of elastin layers in the aortic wall, with about half as many layers found in the infrarenal aorta compared to the proximal thoracic aorta. There is also a decrease in the collagen content from the proximal to the distal aorta. This is likely clinically relevant, since diminished elastin is associated with aortic dilation, while collagen degradation predisposes to aortic rupture. Increased shear and tension on the aortic wall also result in collagen remodeling.
Studies of rat aortas that were harvested and then incubated with IL-1β to stimulate MMP and TIMP production revealed an increase in MMP9 activity in the infrarenal aorta compared to the aortic arch and thoracic aorta. Additionally, aortic transplants were carried out, transplanting the thoracic or abdominal aorta of rats into the infrarenal aortic position of syngeneic rats. After four weeks, the aortas were harvested, stimulated with IL-1β, then analyzed. MMP9 activity was elevated in both the thoracic and abdominal aortic transplants when compared with controls. Both the thoracic and abdominal transplants had MMP9 activity similar to each other, as well as the native infrarenal aorta. This group of studies suggests that there is a difference in the biology of different segments of the aorta that may be related to differences in wall stress.
Finite element analysis has been used to determine the relationship between wall stress and the natural history of AAA disease. These studies revealed that a calcified plaque, a common feature of AAAs, causes increased local wall stress due to the focal stiffness of the calcium compared to the surrounding aortic wall. However, thrombus, also a common feature of AAAs, can actually reduce and redistribute stress along the aortic wall.
Once an AAA has developed, it is likely that increased wall stress is an important in accelerating dilation and increasing the risk of rupture. Additional studies have been done to create mathematical models of the wall stress in an AAA. Turbulent blood flow resulting from expansion of the flow stream from the normal to the dilated aorta does generate additional stress on the aortic wall. This can lead to vibration and further turbulence, leading to a self-perpetuating mechanism of aortic expansion. Also, the shear stress created when a patient exercises has higher peak values in the distal half of an AAA compared to the proximal half. There has also been found to be increased stress at the interface between the less distensible proximal part of the AAA and the more distensible distal portion of the AAA. This location corresponds to the most frequent area of rupture seen in human AAAs. ß-blockers serve to reduce wall stress and have been suggested to be protective of continued aneurysm dilation and rupture in animal models. Newer imaging techniques are being developed that measure aortic wall stress, which will provide a more accurate means of assessing the risk of rupture than the size based criteria currently used.
Family history is a known risk factor for development of an AAA, even if inherited connective tissue disorders, such as Ehlers-Danlos and Marfan syndrome, are excluded from analysis. It is estimated that approximately 15% of patients with an AAA will have a positive family history. Genetic linkage analysis of families with AAAs has identified two loci that correlate with a susceptibility to AAA formation when sex and family history are taken into account. These loci are on chromosomes 19q13 and 4q31. There are many candidate genes in these two regions that could be linked to AAA formation; however, no single genetic polymorphism or defect has been identified as positively being linked with AAA formation.
A targeted gene analysis looking at polymorphisms in the genes for TIMP1, HLA-DR-15, ferritin light chain (FTL), and collagen XI-α-1 was conducted, as these were thought to represent possible candidate genes associated with AAA formation. A polymorphism was identified in the TIMP1 gene, but this was found to not alter the amino acid coded for by the gene as the altered base pair was the third base in a codon. The FTL gene maps to chromosome 19q13 and remains a possible candidate gene related to AAA formation, as an amino acid changing polymorphism was identified. However further study using a larger sample of patients is required to verify these findings. A polymorphism has also been identified in the HLA-DR-15 gene that may be associated with AAA formation, but further confirmation is required. Finally, a polymorphism in the upstream untranslated region of the collagen XI-α-1 gene that may affect protein splicing and expression was identified, however, again, further studies are required to verify this finding and confirm any clinical significance.
Polymorphisms in the promoter region of several genes known to be important in AAA formation were studied in small aneurysms using patients from the surveillance arm of the U.K. Small Aneurysm Trial. These included MMPs 2, 3, 9, and 12, as well as TIMP1, and PAI1. There were no significant associations with any of the polymorphysims analyzed and AAA formation. There was some suggestion of accelerated aneurysm growth rate in patients who had the 5G5G polymorphism of PAI1, but this did not reach statistical significance.
Additionally, there are some specific phenotypes have been found to be associated with AAAs. For example, the Hp-2-1 haptoglobin phenotype and deficiencies in α1-antitrypsin are associated with aneurysm formation. In addition, there is a decreased frequency of AAAs in patients with Rh-negative blood group and an increased frequency in patients with MN or Kell-positive blood groups.
A combination of multiple factors, including localized hemodynamic biomechanical stress, medial fragmentation, and genetic predisposition, through a complex immunologic mechanism is likely to attract inflammatory cells into the aortic wall. Inflammatory cells then release chemokines and cytokines resulting activation of subcellular pathways, including JNK, and further influx of leukocytes. This leads to subsequent expression and activation of inducible and constitutive proteases, including the MMPs, cathepsins and serine proteases. These proteases result in medial degradation, SMC thinning and apoptosis with eventual aneurysmal dilation secondary to negative vessel wall remodeling. Increased wall stress then causes continued progressive proteolysis and aortic dilation, with eventual aortic rupture if untreated.
Areas of Future Research
Despite many recent advances in basic science, there remain many areas to explore in pathogenesis of the abdominal aortic aneurysm. These include the proteolytic enzymes and their inhibitors, the role of the immune response to inflammation and aortic wall degeneration, the role of smooth muscle cells, the role of endogenous repair mechanisms, and the role of bone marrow derived pluripotent cells. Continued research also needs to be performed in order to link the molecular etiology of the AAA with known risk factors for the disease, such as smoking, atherosclerosis, and hypertension. A third area of continued study is the molecular mechanisms of inheritance that produce a susceptibility to AAA formation within families. Additionally, specific genes that are involved in AAA formation remain to be elucidated. A fourth area of active research involves the use of provocative imaging to diagnose and follow patients with AAAs. Current aortic diameter based criteria for repair is likely a poor surrogate for rupture risk. Imaging using mathematical models that analyze the stress seen in the aortic wall of an aneurysm offer a much better insight into the individual patient’s risk of rupture. These models have been developed, but need further refinement before they are ready for widespread use. Finally, the use of nanoparticles to provide localized, direct therapy to the aortic wall is also currently under investigation. Nanoparticles could be used to deliver inhibitors of the MMPs or other enzymes that lead to the formation of AAA directly to the aortic wall, eliminating undesired systemic effects. This technology clearly requires further refinement before it is ready for widespread use.
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Updated November 2009