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Background: In abdominal aortic aneurysm (AAA), pathophysiology deterioration of the medial aortic layer plays a critical role. Key players in vessel wall degeneration are reactive oxygen species (ROS), smooth muscle cell apoptosis, and extracellular matrix degeneration by matrix metalloproteinase-9 (MMP-9). Lipocalin-2, also neutrophil gelatinase-associated lipocalin (NGAL), is suggested to be involved in these degenerative processes in other cardiovascular diseases. We aimed to further investigate the role of NGAL in AAA development and rupture., Methods: In this observational study, aneurysm tissue and blood of ruptured (n = 13) AAA patients were investigated versus nonruptured (n = 26) patients. Nondilated aortas (n = 5) from deceased patients and venous blood from healthy volunteers (n = 10) served as controls. NGAL concentrations in tissue and blood were measured by enzyme-linked immunosorbent assay and immunofluorescence microscopy. Nitrotyrosine (marker of ROS), MMP-9, and caspase-3 (marker of apoptosis) in aneurysm tissue were measured by immunofluorescence microscopy. AAA expansion rates were calculated retrospectively., Results: NGAL (in μg/mL) blood concentration in ruptured AAA was 46 (range 22-122) vs. 26 (range 6-55) in nonruptured AAA (P < 0.01) and 14 (range 12-22) in controls (P < 0.01). In the aneurysm wall of ruptured AAA, NGAL concentration was 4.7 (range 1.4-25) vs. 4.4 (range 0.2-14) in nonruptured AAA (not significant) and 1.8 (range 1.2-2.7) in nondilated aortas (P = 0.04). In the medial layer, NGAL correlated positively with nitrotyrosine (Rs = 0.80, P < 0.01), MMP-9 (Rs = 0.56, P = 0.02), and caspase-3 (Rs = 0.75, P = 0.01). NGAL did not correlate to AAA expansion rate in blood or tissue (P = 0.34 and P = 0.95, respectively)., Conclusions: This study demonstrates that NGAL blood concentration is higher in ruptured AAA patients than in nonruptured AAA. NGAL expression in the AAA wall is also higher than in nondilated aorta. Furthermore, its expression is associated with factors of vessel wall deterioration. Based on our study results, we could not determine NGAL as a biomarker for AAA growth or rupture. However, our findings do support a potential role of NGAL in the development of AAA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The natural course of abdominal aortic aneurysms (AAA) is growth and rupture if left untreated. Numerous markers have been investigated; however, none are broadly acknowledged. Our aim was to identify potential prognostic markers for AAA growth and rupture., Methods and Results: Potential circulating, biomechanical, and genetic markers were studied. A comprehensive search was conducted in PubMed, Embase, and Cochrane Library in February 2017, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Study selection, data extraction, and methodological quality assessment were conducted by 2 independent researchers. Plausibility of markers was based on the amount of publications regarding the marker (more than 3), pooled sample size (more than 100), bias risk and statistical significance of the studies. Eighty-two studies were included, which examined circulating (n=40), biomechanical (n=27), and genetic markers (n=7) and combinations of markers (n=8). Factors with an increased expansion risk included: AAA diameter (9 studies; n=1938; low bias risk), chlamydophila pneumonia (4 studies; n=311; medium bias risk), S-elastin peptides (3 studies; n=205; medium bias risk), fluorodeoxyglucose uptake (3 studies; n=104; medium bias risk), and intraluminal thrombus size (5 studies; n=758; medium bias risk). Factors with an increased rupture risk rupture included: peak wall stress (9 studies; n=579; medium bias risk) and AAA diameter (8 studies; n=354; medium bias risk). No meta-analysis was conducted because of clinical and methodological heterogeneity., Conclusions: We identified 5 potential markers with a prognostic value for AAA growth and 2 for rupture. While interpreting these data, one must realize that conclusions are based on small sample sizes and clinical and methodological heterogeneity. Prospective and methodological consonant studies are strongly urged to further study these potential markers., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

The pathophysiology of aortic aneurysms (AA) is far from being understood. One reason for this lack of understanding is basic research being constrained to fixated cells or isolated cell cultures, by which cell-to-cell and cell-to-matrix communications are missed. We present a new, in vitro method for extended preservation of aortic wall sections to study pathophysiological processes. Intraoperatively harvested, live aortic specimens were cut into 150 μm sections and cultured. Viability was quantified up to 92 days using immunofluorescence. Cell types were characterized using immunostaining. After 14 days, individual cells of enzymatically digested tissues were examined for cell type and viability. Analysis of AA sections (N = 8) showed a viability of 40% at 7 days and smooth muscle cells, leukocytes, and macrophages were observed. Protocol optimization (N = 4) showed higher stable viability at day 62 and proliferation of new cells at day 92. Digested tissues showed different cell types and a viability up to 75% at day 14. Aortic tissue viability can be preserved until at least 62 days after harvesting. Cultured tissues can be digested into viable single cells for additional techniques. Present protocol provides an appropriate ex vivo setting to discover and study pathways and mechanisms in cultured human aneurysmal aortic tissue.

Background: Marfan syndrome (MFS), a congenital connective tissue disorder leading to aortic aneurysm development, is caused by fibrillin-1 (FBN1) gene mutations. Transforming growth factor beta (TGF-β) might play a role in the pathogenesis. It is still a matter of discussion if and how TGF-β up-regulates the intracellular downstream pathway, although TGF-β receptor 3 (TGFBR3 or Betaglycan) is thought to be involved. We aimed to elucidate the role of TGFBR3 protein in TGF-β signaling in Marfan patients., Methods: Dermal fibroblasts of MFS patients with haploinsufficient (HI; n=9) or dominant negative (DN; n=4) FBN1 gene mutations, leading to insufficient or malfunctioning fibrillin-1, respectively, were used. Control cells (n=10) were from healthy volunteers. We quantified TGFBR3 protein expression by immunofluorescence microscopy and gene expression of FBN1, TGFB1, its receptors, and downstream transcriptional target genes by quantitative polymerase chain reaction., Results: Betaglycan protein expression in FBN1 mutants pooled was higher than in controls (P=.004) and in DN higher than in HI (P=.015). In DN, significantly higher mRNA expression of FBN1 (P=.014), SMAD7 (P=.019), HSP47 (P=.023), and SERPINE1 (P=.008), but a lower HSPA5 expression (P=.029), was observed than in HI. A pattern of higher expression was noted for TGFB1 (P=.059), FN1 (P=.089), and COL1A1 (P=.089) in DN as compared to HI. TGFBR3 protein expression in cells, both presence in the endoplasmic reticulum and amount of vesicles per cell, correlated positively with TGFB1 mRNA expression (Rs=0.60, P=.017; Rs=0.55, P=.029; respectively). TGFBR3 gene expression did not differ between groups., Conclusion: We demonstrated that activation of TGF-β signaling is higher in patients with a DN than an HI FBN1 gene mutation. Also, TGFBR3 protein expression is increased in the DN group and correlates positively with TGFB1 expression in groups pooled. We suggest that TGFBR3 protein expression is involved in up-regulated TGF-β signaling in MFS patients with a DN FBN1 gene mutation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Extensive reactive oxygen and nitrogen species (also reactive species) production is a mechanism involved in abdominal aortic aneurysm (AAA) development. White blood cells (WBCs) are a known source of reactive species. Their production may be decreased by statins, thereby reducing the AAA growth rate. Reactive species production in circulating WBCs of AAA patients and the effect of statins on their production was investigated., Methods: This observational study investigated reactive species production in vivo and ex vivo in circulating WBCs of AAA patients, using venous blood from patients prior to elective AAA repair (n = 34; 18 statin users) and from healthy volunteers (n = 10). Reactive species production was quantified in circulating WBCs using immunofluorescence microscopy: nitrotyrosine (footprint of peroxynitrite, a potent reactive nitrogen species) in snap frozen blood smears; mitochondrial superoxide and cytoplasmic hydrogen peroxide (both reactive oxygen species) by live cell imaging. Neutrophils, lymphocytes, and monocytes were examined individually., Results: In AAA patients using statins, the median nitrotyrosine level in neutrophils was 646 (range 422-2059), in lymphocytes 125 (range 74-343), and in monocytes 586 (range 291-663). Median levels in AAA patients not using statins were for neutrophils 928 (range 552-2095, p = .03), lymphocytes 156 (101-273, NS), and for monocytes 536 (range 535-1635, NS). The statin dose tended to correlate negatively with nitrotyrosine in neutrophils (R s  -0.32, p = .06). The median levels in controls were lower for neutrophils 466 (range 340-820, p < .01) and for monocytes 191 (range 102-386, p = .03), but similar for lymphocytes 99 (range 82-246) when compared to the AAA patients. There were no differences in mitochondrial superoxide and cytoplasmic hydrogen peroxide between statin and non-statin users within AAA patients., Conclusions: It was found that the peroxynitrite footprint in circulating neutrophils and monocytes of AAA patients is higher than in controls. AAA patients treated with statins had a lower peroxynitrite footprint in neutrophils than non-statin users., (Copyright © 2017 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Extracellular matrix degeneration, caused by matrix metalloproteinase-2, facilitates smooth muscle cell migration leading to medial layer decline and, ultimately, abdominal aortic aneurysm. It remains unclear what exactly causes aneurysms to rupture, which leads to death in most patients. The extracellular signal-related kinase may be linked to the latter process. We aimed to clarify the role of extracellular signal-related kinase in aortic aneurysm development and rupture in patients., Design: Aortic fragments were harvested during open repair of nonruptured (n = 20) and ruptured (n = 8) aneurysms. As control, nondilated aortas (n = 6) were obtained during autopsy. We determined levels of phosphorylated and total extracellular signal-related kinase by Western blot, matrix metalloproteinase-2 by immunohistochemistry and medial layer thickness by conventional microscopy., Results: Nonruptured aneurysms had 1·8 times higher activation of extracellular signal-related kinase (ratio: phosphorylated/total) than controls (P = 0·011). However, the ruptured aneurysms had only 0·9 times the activation of controls (ns). Both nonruptured and ruptured aneurysms showed significantly higher matrix metalloproteinase-2 than controls (3·8 and 4·0-times, respectively; P < 0·005). Of the medial layer thickness in controls, the median was 1·5 mm, in nonruptured 1·0 mm and in ruptured aneurysms 0·7 mm. Activation of extracellular signal-related kinase correlated positively to medial layer thickness (Rs  = 0·48; P = 0·014), but not to matrix metalloproteinase-2 (Rs  = -0·36; P = 0·10)., Conclusions: In this study, nonruptured aneurysms are associated with increased extracellular signal-related kinase activation while ruptured aneurysms are not. Extracellular signal-related kinase was not related to total matrix metalloproteinase-2 expression. We therefore speculate that increased extracellular signal-related kinase, in response to medial layer decline, could be protective against aneurysm rupture., (© 2016 Stichting European Society for Clinical Investigation Journal Foundation.)