Reducing Abdominal Aortic Aneurysm Progression by Blocking Neutrophil Extracellular Traps Depends on Thrombus Formation.

Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of abdominal aortic aneurysm (AAA), located in adventitia and intraluminal thrombus. We compared the therapeutic potential of targeting upstream or downstream effector molecules of NET formation in 2 murine AAA models based on angiotensin II or peri-adventitial elastase application. In both models, NETs were detected in formed aneurysms at treatment start. Although NET inhibitors failed in the elastase model, they prevented progression of angiotensin II-induced aneurysms with thrombus, which resembles established human disease (including thrombus development). Blockade of upstream NET mediators was more effective than interference with downstream NET molecules.

Quantitation of oxidized nuclear and mitochondrial DNA in plasma samples of patients with abdominal aortic aneurysm.

There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction.

3D Ultrasound Measurements Are Highly Sensitive to Monitor Formation and Progression of Abdominal Aortic Aneurysms in Mouse Models.

Background: Available mouse models for abdominal aortic aneurysms (AAAs) differ substantially in the applied triggers, associated pathomechanisms and rate of vessel expansion. While maximum aortic diameter (determined after aneurysm excision or by 2D ultrasound) is commonly applied to document aneurysm development, we evaluated the sensitivity and reproducibility of 3D ultrasound to monitor aneurysm growth in four distinct mouse models of AAA. Methods: The models included angiotensin-II infusion in ApoE deficient mice, topical elastase application on aortas in C57BL/6J mice (with or without oral administration of ß-aminoproprionitrile) and intraluminal elastase perfusion in C57BL/6J mice. AAA development was monitored using semi-automated 3D ultrasound for aortic volume calculation over 12 mm length and assessment of maximum aortic diameter. Results: While the models differed substantially in the time course of aneurysm development, 3D ultrasound measurements (volume and diameter) proved highly reproducible with concordance correlation coefficients > 0.93 and variations below 9% between two independent observers. Except for the elastase perfusion model where aorta expansion was lowest and best detected by diameter increase, all other models showed high sensitivity of absolute volume and diameter measurements in monitoring AAA formation and progression by 3D ultrasound. When compared to standard 2D ultrasound, the 3D derived parameters generally reached the highest effect size. Conclusion: This study has yielded novel information on the robustness and limitations of semi-automated 3D ultrasound analysis and provided the first direct comparison of aortic volume increase over time in four widely applied mouse models of AAA. While 3D ultrasound generally proved highly sensitive in detecting early AAA formation, the 3D based volume analysis was found inferior to maximum diameter assessment in the elastase perfusion model where the extent of inflicted local injury is determined by individual anatomical features.

Histone citrullination as a novel biomarker and target to inhibit progression of abdominal aortic aneurysms.

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of abdominal aortic aneurysms (AAAs). This study has addressed the notion that NET components might serve as AAA biomarkers or novel targets of AAA therapy. Thus, parameters of neutrophil activation and NET formation were measured in plasma. Their diagnostic marker value was explored in 41 AAA patients and 38 healthy controls. The NET parameter citrullinated histone H3 (citH3) was then validated in 63 AAA patients and 63 controls matched for cardiovascular disease. The prognostic marker potential was investigated in 54 observation periods of AAA growth over 6 months. NETs were further assessed in conditioned medium and sections of aortic tissue. CitH3 was found to be increased in blood (median 362 vs 304 ng/mL, P = 0.004) and aortic tissue (50 vs 1.5 ng/mg, P < 0.001) of AAA patients compared to healthy controls and accumulated in the intraluminal thrombus (629 ng/mg). The diagnostic potential of citH3 ranged at 0.705 area under the ROC curve (AUROC) and was validated with the independent sample set. Furthermore, plasma citH3 predicted AAA growth over the next 6 months (AUROC: 0.707, P = 0.015) and dropped significantly after surgical aneurysm repair. In an angiotensin II - based mouse model of experimental AAA, an inhibitor of histone citrullination was applied to block NET formation and AAA progression. Of note, further growth of an established aneurysm was prevented in mice treated with the NET inhibitor (P = 0.040). In conclusion, histone citrullination represents a promising AAA biomarker and potential therapeutic target to control disease progression.