The receptor for advanced glycation end products and risk of peripheral arterial disease, amputation or death in type 2 diabetes: a population-based cohort study.

BACKGROUND: Patients with type 2 diabetes have a high risk for early and extensive development of peripheral arterial disease (PAD) and this excess risk is not explained by increased burden of traditional atherosclerotic risk factors. Activation of the receptor for advanced glycation end products (RAGE) could be one additional mechanism for accelerated PAD and increased risk for amputation and death. We investigated the association between RAGE plasma components and the risk for PAD, amputation and death in patients with type 2 diabetes. We also estimated the rate of amputation-free survival and survival without PAD. METHODS: We investigated if plasma levels of carboxymethyl-lysine, S100A12 and endosecretory RAGE (esRAGE) were associated with two endpoints: survival without development of PAD and survival without amputation in a 12 years prospective population-based cohort of 146 patients with type 2 diabetes, free from PAD at inclusion. Influence of baseline plasma levels of RAGE ligands (individually and combined by a RAGE-score) were evaluated for both endpoints in the Cox-regression analysis. RESULTS: 106 patients survived without amputation and 93 survived without signs of PAD during follow up. Higher levels of S100A12 and RAGE-score were associated with increased risk for amputation or death, hazard ratios (HR) 1.29; 95% confidence interval (CI) [1.04, 1.59] and 1.79; 95% CI [1.07, 2.99] and with increased risk for PAD or death, HR 1.22; 95% CI [1.00, 1.49] and 1.56; [1.00, 2.44] after adjustment for age and sex. The effect was decreased after adjustment for Framingham cardiovascular disease score: risk for amputation or death, HR 1.17; 95% CI [0.94, 1.46] and 1.54; [0.95, 2.49], and risk for PAD or death, HR 1.12; 95% CI [0.91, 1.38] and 1.38; [0.91, 2.11] for S100A12 and RAGE-score respectively. The incidence for amputation or death was 2.8 per 100 person-years; 95% CI [2.0, 3.7] and the incidence rate for PAD or death was 3.6 per 100 person-years; 95% CI [2.7, 4.8]. CONCLUSION: Higher plasma levels of S100A12 and the combined effect (RAGE-score) of esRAGE, carboxymethyl-lysine and S100A12 seem to be associated with shorter PAD- and amputation-free survival in patients with type 2 diabetes. This may indicate a role for S100A12 in PAD by activation of the RAGE system.

Multidimensional growth measurements of abdominal aortic aneurysms.

BACKGROUND: Monitoring the expansion of abdominal aortic aneurysms (AAAs) is critical to avoid aneurysm rupture in surveillance programs, for instance. However, measuring the change of the maximum diameter over time can only provide limited information about AAA expansion. Specifically, regions of fast diameter growth may be missed, axial growth cannot be quantified, and shape changes of potential interest for decisions related to endovascular aneurysm repair cannot be captured. METHODS: This study used multiple centerline-based diameter measurements between the renal arteries and the aortic bifurcation to quantify AAA growth in 51 patients from computed tomography angiography (CTA) data. Criteria for inclusion were at least 1 year of patient follow-up and the availability of at least two sufficiently high-resolution CTA scans that allowed an accurate three-dimensional reconstruction. Consequently, 124 CTA scans were systematically analyzed by using A4clinics diagnostic software (VASCOPS GmbH, Graz, Austria), and aneurysm growth was monitored at 100 cross-sections perpendicular to the centerline. RESULTS: Monitoring diameter development over the entire aneurysm revealed the sites of the fastest diameter growth, quantified the axial growth, and showed the evolution of the neck morphology over time. Monitoring the development of an aneurysm's maximum diameter or its volume over time can assess the mean diameter growth (r = 0.69, r = 0.77) but not the maximum diameter growth (r = 0.43, r = 0.34). The diameter growth measured at the site of maximum expansion was ~16%/y, almost four times larger than the mean diameter expansion of 4.4%/y. The sites at which the maximum diameter growth was recorded did not coincide with the position of the maximum baseline diameter (ρ = 0 .12; P = .31). The overall aneurysm sac length increased from 84 to 89 mm during the follow-up (P < .001), which relates to the median longitudinal growth of 3.5%/y. The neck length shortened, on average, by 6.2% per year and was accompanied by a slight increase in neck angulation. CONCLUSIONS: Neither maximum diameter nor volume measurements over time are able to measure the fastest diameter growth of the aneurysm sac. Consequently, expansion-related wall weakening might be inappropriately reflected by this type of surveillance data. In contrast, localized spots of fast diameter growth can be detected through multiple centerline-based diameter measurements over the entire aneurysm sac. This information might further reinforce the quality of aneurysm surveillance programs.

Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm.

Leukotrienes (LTs) are arachidonic acid-derived lipid mediators involved in the pathogenesis and progression of diverse inflammatory disorders. The cysteinyl-leukotrienes LTC(4), LTD(4), and LTE(4) are important mediators of asthma, and LTB(4) has recently been implicated in atherosclerosis. Here we report that mRNA levels for the three key enzymes/proteins in the biosynthesis of cysteinyl-leukotrienes, 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and LTC(4) synthase (LTC(4)S), are significantly increased in the wall of human abdominal aortic aneurysms (AAAs). In contrast, mRNA levels of LTA(4) hydrolase, the enzyme responsible for the biosynthesis of LTB(4), are not increased. Immunohistochemical staining of AAA wall revealed focal expression of 5-LO, FLAP, and LTC(4)S proteins in the media and adventitia, localized in areas rich in inflammatory cells, including macrophages, neutrophils, and mast cells. Human AAA wall tissue converts arachidonic acid and the unstable epoxide LTA(4) into significant amounts of cysteinyl-leukotrienes and to a lesser extent LTB(4). Furthermore, challenge of AAA wall tissue with exogenous LTD(4) increases the release of matrix metalloproteinase (MMP) 2 and 9, and selective inhibition of the CysLT1 receptor by montelukast blocks this effect. The increased expression of LTC(4)S, together with the predominant formation of cysteinyl-leukotrienes and effects on MMPs production, suggests a mechanism by which LTs may promote matrix degradation in the AAA wall and identify the components of the cysteinyl-leukotriene pathway as potential targets for prevention and treatment of AAA.

Mast cells: important players in the orchestrated pathogenesis of abdominal aortic aneurysms.

Mast cells (MCs) regulate inflammation and immunity. Their granular content includes heparin, histamine, and several enzymes (tryptase, chymase, carboxypeptidase, and cathepsin G). In addition, activated MCs synthesize and release eicosanoids and a large number of cytokines and chemokines. Recent findings suggest a role of MCs in abdominal aortic aneurysms (AAAs) in humans, where they are found in the media and adventitia. Experimentally induced AAA in MC-deficient animals and animals treated with MC inhibitors demonstrate that MCs are involved in the pathogenesis of AAA via several different mechanisms. MC-dependent activation of metalloproteinases and the renin-angiotensin system, contribution to smooth muscle cell apoptosis, and release of proteolytic enzymes are some key examples. Human studies indicate that MCs are the main source of cathepsin G in AAAs and contribute to activation of the renin-angiotensin system via chymase and cathepsin G. Activated MCs also contribute to neovascularization, inflammation, and atherosclerosis, all hallmarks of AAA. Thus, we may envision that MC stabilizing agents, as well as leukotriene receptor antagonists and histamine receptor blockers already in clinical use for treatment of other diseases, could also be tested for their efficacy in preventing development and growth of AAA.

Female and elderly abdominal aortic aneurysm patients more commonly have concurrent thoracic aortic aneurysm.

BACKGROUND: A recent report unexpectedly revealed that one-fourth of abdominal aortic aneurysm (AAA) patients also have an aneurysm in the thoracic aorta (TAA). It remains to be investigated which AAA patients have a higher risk of also developing TAAs. The aim of this study was to identify possible differences in the risk factor profile in AAA patients with or without a TAA. METHODS: All AAA patients attending an outpatient clinic and investigated using an abdominal and thoracic computed tomography scan were included (n = 354). Image analysis and hospital chart review were conducted. The association between comorbidity and TAA was estimated by logistic regression and odds ratios (ORs) with 95% confidence intervals (CIs). Gender-specific and neutral criteria were used. Validation of excluded patients was performed. RESULTS: Ninety-four (27%) of 354 AAA patients had a concurrent descending TAA (AAA/TAA). AAA/TAA patients were older than AAA patients (76 vs. 73 years). More women were identified in the AAA/TAA group (39% vs. 16%, P < 0.001). In the univariate logistic regression model, female gender (OR: 3.3, 95% CI: 1.9-5.6), hypertension (OR: 1.8, 95% CI: 1.1-3.0), and age (70-79 years-OR: 2.4, 95% CI: 1.3-4.6; 80-89 years-OR: 3.0, 95% CI: 1.5-6.0) were associated with concurrent TAA. In the multivariate model, only female gender and age were associated with TAA. CONCLUSIONS: AAA patients, in general, should be offered examination of the thoracic aorta, and special attention needs to be paid to female AAA patients and AAA patients at high age, if the AAA patient is considered operable. Surveillance of AAA patients must improve to enhance identification of the large group of patients who have developed, or will develop, TAAs. Future strategies will, of course, address pathophysiological aspects of aneurysmal development in the thoracic and infrarenal aorta.

High frequency of thoracic aneurysms in patients with abdominal aortic aneurysms.

OBJECTIVE: To investigate the prevalence of thoracic aortic aneurysms (TAA) in patients with abdominal aortic aneurysms (AAA). BACKGROUND: Different disease profiles between men and women treated for AAA have been reported. Reports regarding causes of death for treated AAA patients have shown an increased risk of death because of other aneurysms for women, possibly TAA. The prevalence of TAA in AAA patients is not well known. METHODS: Data for AAA patients attending the outpatient clinic at a university hospital were analyzed (N = 1055). Patients who had undergone abdominal and thoracic computed tomography were included (n = 354). The diameter of the aorta was measured in the ascending, descending, and infrerenal aorta. Comorbid conditions were analyzed. RESULTS: Mean age was 74 years, 23% were women. The presence of comorbid conditions did not differ between men and women except for a higher proportion of female smokers (P = 0.003). When sex-specific criteria were used, 100 patients (28%) had a TAA, 38 (48%) of the women compared with 62 (23%) of the men (P < 0.0001). Odds ratio for women compared with those of men to have a concurrent TAA was 3.09 (95% confidence interval, 1.84-5.22). CONCLUSIONS: More than one fourth of patients with AAA attending a regular outpatient clinic have a concomitant TAA, and women are particularly affected. During the last decades, the therapeutic options for TAA patients have changed considerably. Physicians need to increase the efforts to investigate also the thoracic aorta in AAA patients.

Perforin-independent extracellular granzyme B activity contributes to abdominal aortic aneurysm.

Granzyme B (GZMB) is a serine protease that is abundantly expressed in advanced human atherosclerotic lesions and may contribute to plaque instability. Perforin is a pore-forming protein that facilitates GZMB internalization and the induction of apoptosis. Recently a perforin-independent, extracellular role for GZMB has been proposed. In the current study, the role of GZMB in abdominal aortic aneurysm (AAA) was assessed. Apolipoprotein E (APOE)(-/-) x GZMB(-/-) and APOE(-/-) x perforin(-/-) double knockout (GDKO, PDKO) mice were generated to test whether GZMB exerted a causative role in aneurysm formation. To induce aneurysm, mice were given angiotensin II (1000 ng/kg/min) for 28 days. GZMB was found to be abundant in both murine and human AAA specimens. GZMB deficiency was associated with a decrease in AAA and increased survival compared with APOE-KO and PDKO mice. Although AAA rupture was observed frequently in APOE-KO (46.7%; n = 15) and PDKO (43.3%; n = 16) mice, rupture was rarely observed in GDKO (7.1%; n = 14) mice. APOE-KO mice exhibited reduced fibrillin-1 staining compared with GDKO mice, whereas in vitro protease assays demonstrated that fibrillin-1 is a substrate of GZMB. As perforin deficiency did not affect the outcome, our results suggest that GZMB contributes to AAA pathogenesis via a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity.

Reproductive history in women with abdominal aortic aneurysms.

BACKGROUND: The prevalence of abdominal aortic aneurysms (AAAs) differs considerably between the sexes, illustrated by the male/female ratio 4-6:1. Women are also reported to have a higher risk of rupture, and a poorer outcome compared with men. The primary aim of this study was to investigate if women with AAA have a different reproductive history compared with other women. The secondary aim was to study if women with a larger AAA differ in their reproductive history from women with a smaller AAA. METHOD: This case-control study was performed in October 2009 and included 140 consecutively monitored women with AAA and 140 with peripheral arterial disease (PAD) at the Department of Vascular Surgery at Karolinska University Hospital, Stockholm. AAA was defined as AAA diameter >3 cm, and women with AAA were subdivided into groups with AAA diameter ≥5 cm and diameter <5 cm. A validated questionnaire was used to obtain information about participants' reproductive history and general health. The response rate was 70% (n = 196). RESULTS: Women with AAA were smokers to a greater extent than women with PAD (previous, 52% vs 46%; current, 46% vs 34%, P = .001). Diabetes mellitus was more prevalent in women with PAD (28%) than in women with AAA (15%, P = .034). Angina pectoris occurred more often in women with AAA (26%) than in women with PAD (11%, P = .026). No significant difference was found between PAD and AAA women regarding statin use, treatment for hypertension, prior myocardial infarction, and body mass index (BMI). The 54 women with AAA ≥5 cm and the 44 women with AAA <5 cm were similar in age (76 vs 76 years, P = .908) and BMI (25.7 vs 24.0 kg/m(2), P = .66). No difference was noted in the occurrence of other risk factors between women with AAA ≥5 cm and women with AAA <5 cm. Mean age at menopause was lower in women with AAA ≥5 cm than in women with AAA <5 cm and in women with PAD (47.7 vs 49.9 vs 49.7 years, P = .011). Apart from menopausal age, the groups had a similar reproductive history, including hormone replacement therapy, parity, use of contraceptives, prior gynecological surgery, and breast cancer. CONCLUSION: These findings suggest that women with larger AAA reach menopausal age earlier, and this could influence an earlier onset of aneurysmatic disease or an increase in aneurysm growth. The true role of endogenous estrogen in aneurysm development and expansion is yet to be investigated.

Analysis of aortic wall stress and rupture risk in patients with abdominal aortic aneurysm with a gender perspective.

OBJECTIVE: The most commonly used predictor of rupture of an abdominal aortic aneurysm (AAA) is the diameter; however, this does not estimate the true risk for each patient. Why women with AAAs have an increased growth rate, weaker aortic wall, and increased risk for rupture is yet unclear. It is likely that geometrical and biomechanical properties contribute to found gender differences. Several studies have shown that peak wall stress (PWS) and peak wall rupture risk (PWRR), predicted by a finite element (FE) analysis of AAAs derived from computed tomography (CT), is a better predictor of rupture than maximum diameter. The purpose of this study was to investigate if women with AAAs have an increased PWS and PWRR using an FE model compared to men. METHOD: Fifteen men and 15 women (AAAs 4-6 cm) were included. AAA geometry was derived from CT scans, and PWS and PWRR were estimated using the FE method. Comparisons were made by t test and Mann-Whitney test. RESULTS: Mean age (women 73 years old vs men 71 years old) and mean AAA diameter was similar (49.7 mm vs 50.1 mm) for women and men. PWS did not differ for women 184 and men 198 kPa. PWRR was 0.54 (0.28-0.85) for women and 0.43 (0.24-0.66) for men, P = .06. CONCLUSION: This is the first analysis of stress and strength of the aneurysm wall with a gender perspective. The reported higher rupture risk for women has previously not been tested with geometrical and biomechanical properties. PWS did not differ, but the PWRR was slightly higher in women. However, the difference did not reach statistical significance, probably due to the small sample size. In summary, the results in the present study suggest that differences in biomechanical properties could be a contributing explanation for the higher rupture risk reported for female patients with AAAs.

Imaging the intraluminal thrombus of abdominal aortic aneurysms: techniques, findings, and clinical implications.

Intraluminal thrombus (ILT) is known to influence the natural history of abdominal aortic aneurysms, and its effect on the arterial wall may predict the risks of rupture. The main features of ILT believed to be associated with aneurysm growth and increased rupture risk are size; presence of fissures, dissections, or calcifications in the ILT; and inhomogeneity in its internal structure. Modern imaging allows for detailed depiction of the ILT. This review describes the techniques, findings, clinical implications, advantages, and disadvantages of imaging the ILT by ultrasound, contrast-enhanced computed tomography, and magnetic resonance imaging.

Genetic and environmental contributions to abdominal aortic aneurysm development in a twin population.

BACKGROUND: The contribution of hereditary and environmental factors to the development of abdominal aortic aneurysms (AAAs) is still partly unknown. The aim of this study was to analyze the role of these factors in a large population-based sample of twins. METHODS: 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 nonshared environmental factors for development of AAA. RESULTS: There were 172,890 twins registered at the time of the study including 265 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. The odds ratio (OR) was 71 (95% confidence interval [CI] 27-183) for MZ twins and 7.6 (95% CI 3.0-19) for DZ twins. 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 nonshared environmental effects for 30% (95% CI 0.17-0.46) of the phenotypic variance among twins. CONCLUSION: These data provide robust epidemiologic evidence that heritability contributes to aneurysm formation. Concordances and correlations were higher in MZ compared with DZ twins, indicating genetic effects. There was a 24% probability that an MZ twin of a person with AAA will have the disease. The twin of an MZ twin with AAA had a risk of AAA that was 71 times that of the MZ twin of a person without AAA. A heritability of 70% of the total trait variance was estimated. The remaining variance was explained by nonshared environmental factors with no support for a role of shared environmental influences.

A population-based case-control study of the familial risk of abdominal aortic aneurysm.

BACKGROUND: Several studies have reported a familial clustering of abdominal aortic aneurysm (AAA) supporting that AAA is an inheritable disease, but few population-based studies can be found. Possible gender differences regarding hereditary patterns have been reported. OBJECTIVE: The aim of this study was to investigate the risk of developing an AAA for first-degree relatives of patients with AAA in Sweden and compare them with matched controls and their relatives. METHODS AND MATERIALS: 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. RESULTS: 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. 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.

Mast cells associate with neovessels in the media and adventitia of abdominal aortic aneurysms.

OBJECTIVE: Mast cells (MCs) are inflammatory cells present in atherosclerotic lesions and neovascularized tissues. Recently, MCs were shown to modulate abdominal aortic aneurysm (AAA) formation in a mouse model. Progression of aneurysmatic disease process may also depend on intraluminal thrombus and neovascularization of the aneurysm wall. Here we investigated the relationship between MCs and inflammation, neovascularization, and the presence of intraluminal thrombus in human AAA. METHODS AND RESULTS: Specimens from AAAs and normal control aortas were analyzed with basic histology, immunohistochemical staining, and quantitative real-time polymerase chain reaction (PCR). Double immunostainings with endothelial cell markers CD31/CD34 and MC tryptase showed that, in contrast to histologically normal aorta, MCs in AAA were abundant in the media, but absent from the intima. Medial MCs and (CD31/CD34)(+) neovessels increased significantly in AAA compared with normal aorta (P < .0001 for both), and the highest densities of neovessels and MCs were observed in the media of thrombus-covered AAA samples. Also, the proportional thickness of aortic wall penetrated by the neovessels was significantly higher in the AAA samples (P < .0001), and the neovascularized area correlated with the density of medial MCs (P < .0001). In histologic analysis, the medial MCs were mainly located adjacent to the stem cell factor (SCF)(+) medial neovessels. Real-time PCR analysis also showed that mRNA levels of genes associated with neovascularization (vascular endothelial growth factor [VEGF], FLT1, VE-cadherin, CD31), and MCs (tryptase, chymase, cathepsin G) were higher in AAA samples than in controls. Demonstration of adhered platelets by CD42b staining and lack of endothelial cell (CD31/CD34) staining in the luminal surface of AAA specimens suggest endothelial erosion of the aneurysm walls. CONCLUSIONS: The results support participation of MCs in the pathogenesis of AAA, particularly regarding neovascularization of aortic wall.

More patients are treated for nonruptured abdominal aortic aneurysms, but the proportion of women remains unchanged.

BACKGROUND: Large variations in the intervention rates for ruptured and nonruptured abdominal aortic aneurysm (AAA) over time have been reported, both decreasing and increasing numbers. Women have been reported to constitute an increasing proportion of patients treated for several manifestations of cardiovascular disease; whether a similar trend is true for AAA is not well understood. This study investigated recent temporal trends in a complete national register regarding the number and type of procedure performed for AAA, and outcome, with special emphasis on gender differences. METHODS: Data for all individuals treated for nonruptured or ruptured AAA in Sweden (1990 to 2005) were obtained from the Swedish National Board of Health and Welfare (NBHW). A total of 14369 individuals were identified; 2327 (16%) were women. Date and type of intervention, date and cause of death, age, and sex were included in the statistical model. RESULTS: There was a relative annual increase in interventions for nonruptured AAA; 4% for women (P < .0001) and 2% for men (P < .0001). No significant trends were observed for interventions for rupture during the observation period. No significant increase in the proportion of women was recorded for nonrupture (17%) or rupture (15%). Women had higher crude 30-day mortality rate than men after treatment for both nonruptured (5.7% vs 4.9%) and ruptured (41.9% vs 36.8%) AAA. In a logistic regression model, survival improved over time after intervention for nonrupture (P < .0001) and rupture (P < .0001). Increasing age (P < .0001 for both nonrupture and rupture) but not sex (P = .49 for non rupture and P = .42 for rupture) had a negative influence on mortality. CONCLUSION: Interventions for nonruptured but not for ruptured AAA increased over time, with an expected rapid increase of endovascular repair in the nonruptured group. The unchanged fraction of women over time possibly reflects the true distribution of AAA between the sexes. The outcome after treatment for both nonruptured and ruptured AAA improved, as anticipated, over time. No increase in mortality among women was recorded after adjustment for age.

Bleeding into the intraluminal thrombus in abdominal aortic aneurysms is associated with rupture.

OBJECTIVE: The aim of this study was to determine signs of bleeding in the intraluminal thrombus and the site of rupture using multislice computed tomography (CT) imaging in patients with abdominal aortic aneurysms (AAA). METHODS: We analyzed CT images of 42 patients with ruptured infrarenal AAA in two hospitals in Stockholm, Sweden during a 3-year period. A "crescent sign" or localized areas with higher attenuation in the thrombus were interpreted as signs of bleeding in the thrombus. A localized area of hyperattenuation did not have the typical crescent shape and was distinguished from calcifications in the thrombus. We measured the attenuation in Hounsfield units in the intraluminal thrombus using CT software to quantify the presence of blood in the thrombus. As controls, we analyzed 36 patients with intact AAA and a comparable aneurysm diameter and age. RESULTS: The crescent sign was more frequent in the ruptured group (38% vs 14%, P = .02), but there was no significant difference in the presence of localized areas of hyperattenuation in the two groups. The attenuation in the thrombus was significantly higher in patients with rupture than in those with intact aneurysms (P = .02). The site of rupture could be localized in 29/42 patients. Ruptures occurred both through the thrombus-covered and the thrombus free wall. In 45% of the patients, the rupture site was localized in the left lateral wall, in 24% in the anterior wall, in 24% in the right lateral wall, but only in 7% in the posterior wall. CONCLUSION: The site of rupture could be identified in a majority of cases of AAA with routine multislice CT. This study demonstrates an association between the presence of blood in the thrombus as suggested by higher attenuation levels and a crescent sign and AAA rupture. If these findings also predict AAA rupture, remains to be established.

Reduced perlecan expression and accumulation in human carotid atherosclerotic lesions.

Heparan sulfate in the extracellular matrix of the artery wall has been proposed to possess anti-atherogenic properties by interfering with lipoprotein retention, suppression of inflammation, and inhibition of smooth muscle cell growth. Previously, the amount of heparan sulfate in atherosclerotic lesions from humans and animals has been shown to be reduced but the identity or identities of the heparan sulfate molecules being down regulated in this disease are not known. In this study, atherosclerotic lesions were retrieved from 44 patients undergoing surgery for symptomatic carotid stenosis. Normal iliac arteries from organ donors were used as controls. Analysis of the specimens by gene microarray showed a selective reduction in perlecan gene expression, whereas, expression of the other heparan sulfate proteoglycans in the artery wall, agrin and collagen XVIII, remained unchanged. Expression of the large chondroitin sulfate proteoglycan, versican, also remained unchanged. Real-time PCR confirmed the decrease in perlecan gene expression and the unchanged expression of versican. The findings were supported by immunohistochemical analysis demonstrating a reduced accumulation of both perlecan core protein and heparan sulfate in carotid lesions. The study demonstrates a reduction of perlecan mRNA-expression and protein deposition in human atherosclerosis, which in part explains the low levels of heparan sulfate in this disease.

Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms.

It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA enlargement and rupture. The growth of the AAA is dependent on proteolytic degradation of elastin. Here, we analysed whether the neutrophil gelatinase-associated lipocalin (NGAL) is expressed within the thrombus and the aneurysm wall. NGAL can bind to metalloproteinase-9 (MMP-9) and inhibit its degradation, thereby preserving enzymatic activity. Biopsies were obtained from thrombus-free and thrombus-covered aneurysm wall and the intraluminal thrombus from patients undergoing elective surgery for AAA. Immunohistochemistry and real-time PCR were used to study NGAL and MMP-9 expression. Immunoprecipitation, gel zymography, Western blot and ELISA were used to detect and quantify NGAL/MMP-9 complexes. NGAL was detected in the thrombus, the interface between the thrombus and the underlying wall and in the wall itself. Double staining showed that neutrophils are the major source of NGAL expression. Immunoprecipitation of MMP-9 with antibody against NGAL showed that complexes of NGAL and active MMP-9 were present in thrombus, the interface fluid and the aneurysm wall. Western blot analyses using non-reducing conditions and gel zymography demonstrated that high-molecular-weight complexes of NGAL/MMP-9 were present within the different regions. The concentration of the NGAL/MMP-9 complex was highest in the luminal part of the thrombus. In conclusion, NGAL in complex with activated MMP-9 is present in AAA wall and thrombus. Neutrophil-derived NGAL could enhance the proteolytic activity associated with AAA, but the importance of this mechanism for aneurysm growth remains to be shown.

The intraluminal thrombus as a source of proteolytic activity.

Most abdominal aortic aneurysms (AAAs) with a diameter indicating need for surgical repair contain intraluminal thrombus (ILT). The development of AAA is linked to degradation of elastin and collagen. These changes are more pronounced in the aneurysm wall covered by the ILT, which also shows more signs of inflammation and is thinner compared to the aneurysm wall exposed to flowing blood. The rate of increase in diameter of AAA correlates with increased thrombus growth and rupture. CT examinations of patients with rupture have demonstrated contrast appearing in the thrombus suggesting bleeding into it. Studies using gene array of human aneurysm specimens have shown that most matrix metalloproteinases (MMP) were upregulated in the thrombus-free wall. Analyses by zymography, however, demonstrate gelatinase activity in the interface between the thrombus and the underlying wall and in the media of the wall not covered by a thrombus. The thrombus contains large amounts of neutrophils. Neutrophil gelatinase associated lipocalin (NGAL) is involved in the regulation of MMP-9 activity and prevents its inactivation, thus augmenting the proteolytic effect. It has been identified in all layers of the ILT. The presence of NGAL/MMP-9 complexes throughout the thrombus and in the thrombus-covered wall may contribute to the increased proteolytic degradation seen in this wall segment. In conclusion, the presence, growth, and thickness of the ILT have been shown to be associated with growth and risk of rupture. The wall underlying the thrombus is thinner and shows more signs of proteolytic degradation. Increased proteolytic activity by MMP-9 may be mediated by binding to NGAL.

Difference in matrix-degrading protease expression and activity between thrombus-free and thrombus-covered wall of abdominal aortic aneurysm.

OBJECTIVE: It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA rupture. Here, we examined the possibility that the intraluminal thrombus influences expression and activity of matrix-degrading proteases in the AAA wall. METHODS AND RESULTS: Twenty patients undergoing elective repair of AAAs were included. From each patient, specimens from both thrombus-covered and thrombus-free wall were taken for analysis. Gene arrays and quantitative real-time polymerase chain reaction showed that matrix metalloproteinase (MMP)-1, -7, -9, and -12 expressions were upregulated in the thrombus-free wall compared with the thrombus-covered wall. Immunohistochemistry confirmed the differential expression of MMP-9 but also localized MMP-9 to the interface between the thrombus and the underlying vessel wall. MMP-9 expression was colocalized with the presence of macrophages. Similar expression patterns were observed for urokinase plasminogen activator (uPA), uPA receptor, and plasminogen activator inhibitor-1. Gelatinase activity was detected in the same regions as MMP-9 protein expression, ie, within the thrombus-free wall and in the interface between the thrombus and the underlying wall. CONCLUSIONS: The present work demonstrates that protease expression and activity differs within the aneurysm wall. The source and activity of the proteases responsible for the degradation of the thrombus-covered wall need to be further determined.