Nicolas H. Pope

Inhibition of Interleukin-1β Decreases Aneurysm Formation and Progression in a Novel Model of Thoracic Aortic Aneurysms

Background— Thoracic aortic aneurysms (TAAs) are common, but experimental TAA models are limited and the role of interleukin-1&bgr; (IL-1&bgr;) is undetermined. Methods and Results— IL-1&bgr; protein was measured in human TAAs and control aortas, and IL-1&bgr; protein was increased ≈20-fold in human TAAs. To develop an experimental model of TAAs, 8- to10-week-old male C57Bl/6 mice (wild type [WT]) underwent thoracotomy with application of periadventitial elastase (WT TAA) or saline (WT control; n=30 per group). Elastase treatment to thoracic aortas resulted in progressive dilation until day 14 with maximal dilation of 99.6±24.7% compared with 14.4±8.2% for WT saline control (P<0.0001). WT TAAs demonstrated elastin fragmentation, smooth muscle cell loss, macrophage infiltration, and increased IL-1&bgr; expression. Next, TAAs were induced in mice deficient of IL-1&bgr; (IL-1&bgr; knockout) or IL-1 receptor (IL-1R knockout; n=10 each). Genetic deletion of IL-1&bgr; and IL-1R significantly decreased thoracic aortic dilation (IL-1&bgr; knockout=54.2±16.8% and IL-1R knockout=62.6±17.2% versus WT TAA=104.7±23.8%; P<0.001for both). IL-1&bgr; knockout and IL-1R knockout aortas demonstrated preserved elastin and smooth muscle cells with fewer inflammatory cells. Correspondingly, IL-1&bgr; and IL-1R knockout aortas had decreased inflammatory cytokine and matrix metalloproteinase 9 expression. Separately, WT mice pretreated with either IL-1R antagonist anakinra (100 mg/kg per day) or vehicle alone (control) underwent elastase treatment. Pretreatment of WT mice with anakinra attenuated TAA formation (control: 99.2±15.5% versus anakinra: 68.3±19.2%; P<0.005). Finally, to investigate treatment of small TAAs, WT mice were treated with anakinra 3 days after TAA induction. Anakinra treatment in WT mice with small TAAs reduced aortic dilation on day 14 (control treatment: 89.1±18.6% versus anakinra treatment: 59.7±25.7%; P=0.01). Conclusions— Periadventitial application of elastase to murine thoracic aortas reproducibly produced aneurysms with molecular and histological features consistent with TAA disease. Genetic and pharmacological inhibition of IL-1&bgr; decreased TAA formation and progression, indicating that IL-1&bgr; may be a potential target for TAA treatment.

KLF4 Regulates Abdominal Aortic Aneurysm Morphology and Deletion Attenuates Aneurysm Formation

Background— KLF4 mediates inflammatory responses after vascular injury/disease; however, the role of KLF4 in abdominal aortic aneurysms (AAAs) remains unknown. The goals of the present study were to (1) determine the role of KLF4 in experimental AAA; and (2) determine the effect of KLF4 on smooth muscle (SM) cells in AAAs. Methods and Results— KLF4 expression progressively increased at days 3, 7, and 14 after aortic elastase perfusion in C57BL/6 mice. Separately, loss of a KLF4 allele conferred AAA protection using ERTCre+ KLF4 flx/wt mice in the elastase AAA model. In a third set of experiments, SM-specific loss of 1 and 2 KLF4 alleles resulted in progressively greater protection using novel transgenic mice (MYHCre+ flx/flx, flx/wt, and wt/wt) in the elastase AAA model compared with control. Elastin degradation, MAC2, and cytokine production (MCP1, tumor necrosis factor-&agr;, and interleukin-23) were significantly attenuated, whereas &agr;-actin staining was increased in KLF4 knockout mice versus controls. Results were verified in global KLF4 and SM-specific knockout mice using an angiotensin II model of aneurysm formation. KLF4 inhibition with siRNA attenuated downregulation of SM gene expression in vitro, whereas in vivo studies demonstrated that KLF4 binds to promoters of SM genes by chromatin immunoprecipitation analysis. Finally, human aortic aneurysms demonstrated significantly higher KLF4 expression that was localized to SM cells. Conclusions— KLF4 plays a critical role in aortic aneurysm formation via effects on SM cells. These results suggest that KLF4 regulates SM cell phenotypic switching and could be a potential therapeutic target for AAA disease.

D-series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization

The role of resolvins in abdominal aortic aneurysm (AAA) has not been established. We hypothesized that treatmentwithD‐series resolvins (RvD2 or RvD1)would attenuate murine AAA formation through alterations in macrophage polarization and cytokine expression. Male C57/B6 mice (n = 9 per group) 8 to 12 wk old received RvD2 (100 ng/kg/treatment), RvD1 (100 ng/kg/treatment), or vehicle only every third day beginning 3 d before abdominal aorticperfusionwith elastase asprevention. Aortaswere collected 14 d after elas taseperfusion. Cytokine analysis (n = 5 per group) or confocal microscopy (n = 4 per group) was performed. In a separate experiment, RvD2 was provided tomicewith small AAAs 3 d after elastase treatment (n = 8 per group). Additionally, apolipoprotein E knockoutmice treatedwith angiotensin II (1000 ng/kg) were treated with RvD2 or vehicle alone (n = 10 per group) in a nonsurgical model of AAA. To determine the effect of RvD2 on macrophage polarization, confocal staining for macrophages, M1 and M2 macrophage subtypes, α‐actin, and DAPI was performed. Mean aortic dilationwas 966 13% for vehicle‐treated mice ≤57 × 9.7% for RvD2‐treated mice, and 61 ± 11% for RvD1‐treated mice (P < 0.0001). Proinflammatory cytokines macrophage chemotactic protein 1, C‐X‐C motif ligand 1, and IL‐1β were significantly elevated in control animals compared to RvD2‐ and RvD1‐treated animals (P < 0.05), resulting in a reduction of matrix metalloproteinase 2 and 9 activity in resolvin‐treated mice in both elastase and angiotensin II models. Treatment of existing small AAAs with RvD2 demonstrated a 25%reduction in aneurysm size at d 14 compared to vehicle alone (P = 0.018). Confocal histology demonstrated a prevalence of M2 macrophages within the aortic medium in mice treated with RvD2. Resolvin D2 exhibits a potent protective effect against experimental AAA formation. Treatment with RvD2 significantly influences macrophage polarization and decreases several important proinflammatory cytokines. Resolvins and the alteration of macrophage polarization represent potential future targets for prevention of AAA.—Pope, N.H., Salmon, M.,Davis, J. P.,Chatterjee, A., Su, G., Conte, M. S.,Ailawadi, G., Upchurch, G. R., Jr. D‐series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization. FASEB J. 30, 4192–4201 (2016). www.fasebj.org

Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of lungs donated after cardiac death

OBJECTIVE Ex vivo lung perfusion has been successful in the assessment of marginal donor lungs, including donation after cardiac death (DCD) donor lungs. Ex vivo lung perfusion also represents a unique platform for targeted drug delivery. We sought to determine whether ischemia-reperfusion injury would be decreased after transplantation of DCD donor lungs subjected to prolonged cold preservation and treated with an adenosine A2A receptor agonist during ex vivo lung perfusion. METHODS Porcine DCD donor lungs were preserved at 4°C for 12 hours and underwent ex vivo lung perfusion for 4 hours. Left lungs were then transplanted and reperfused for 4 hours. Three groups (n = 4/group) were randomized according to treatment with the adenosine A2A receptor agonist ATL-1223 or the dimethyl sulfoxide vehicle: Infusion of dimethyl sulfoxide during ex vivo lung perfusion and reperfusion (DMSO), infusion of ATL-1223 during ex vivo lung perfusion and dimethyl sulfoxide during reperfusion (ATL-E), and infusion of ATL-1223 during ex vivo lung perfusion and reperfusion (ATL-E/R). Final Pao2/Fio2 ratios (arterial oxygen partial pressure/fraction of inspired oxygen) were determined from samples obtained from the left superior and inferior pulmonary veins. RESULTS Final Pao2/Fio2 ratios in the ATL-E/R group (430.1 ± 26.4 mm Hg) were similar to final Pao2/Fio2 ratios in the ATL-E group (413.6 ± 18.8 mm Hg), but both treated groups had significantly higher final Pao2/Fio2 ratios compared with the dimethyl sulfoxide group (84.8 ± 17.7 mm Hg). Low oxygenation gradients during ex vivo lung perfusion did not preclude superior oxygenation capacity during reperfusion. CONCLUSIONS After prolonged cold preservation, treatment of DCD donor lungs with an adenosine A2A receptor agonist during ex vivo lung perfusion enabled Pao2/Fio2 ratios greater than 400 mm Hg after transplantation in a preclinical porcine model. Pulmonary function during ex vivo lung perfusion was not predictive of outcomes after transplantation.

Mesenchymal Stem Cells Attenuate NADPH Oxidase-Dependent High Mobility Group Box 1 Production and Inhibit Abdominal Aortic Aneurysms

Objective—Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation, and matrix degradation. This study tests the hypothesis that macrophage-produced high mobility group box 1 (HMGB1) production is dependent on nicotinamide adenine dinucleotide phosphate oxidase (Nox2), which leads to increase in interleukin (IL)-17 production resulting in AAA formation and that treatment with human mesenchymal stem cells (MSCs) can attenuate this process thereby inhibiting AAA formation. Approach and Results—Human aortic tissue demonstrated a significant increase in HMGB1 expression in AAA patients when compared with controls. An elastase-perfusion model of AAA demonstrated a significant increase in HMGB1 production in C57BL/6 (wild-type [WT]) mice, which was attenuated by MSC treatment. Furthermore, anti-HMGB1 antibody treatment of WT mice attenuated AAA formation, IL-17 production, and immune cell infiltration when compared with elastase-perfused WT mice on day 14. Elastase-perfused Nox2−/y mice demonstrated a significant attenuation of HMGB1 and IL-17 production, cellular infiltration, matrix metalloproteinase activity, and AAA formation when compared with WT mice on day 14. In vitro studies showed that elastase-treated macrophages from WT mice, but not from Nox2−/y mice, produced HMGB1, which was attenuated by MSC treatment. The production of macrophage-dependent HMGB1 involved Nox2 activation and superoxide anion production, which was mitigated by MSC treatment. Conclusions—These results demonstrate that macrophage-produced HMGB1 leads to aortic inflammation and acts as a trigger for CD4+ T-cell–produced IL-17 during AAA formation. HMGB1 release is dependent on Nox2 activation, which can be inhibited by MSCs leading to attenuation of proinflammatory cytokines, especially IL-17, and protection against AAA formation.

Interleukin-6 Receptor Inhibition Prevents Descending Thoracic Aortic Aneurysm Formation

BACKGROUND Thoracic aortic aneurysms (TAA) and abdominal aortic aneurysms (AAA) represent related but distinct disease processes. Interleukin-6 (IL-6) is known to be significantly upregulated in human TAA and AAA. We hypothesize that loss of IL-6 is protective in experimental TAA and AAA. METHODS Murine TAAs or AAAs were created using a novel model in C57/B6 mice by treating the intact aorta with elastase. Cytokine profiles were analyzed with antibody arrays (n = 5 per group). Separately, to determine the role of IL-6, thoracic (n = 7) or abdominal (n = 7) aortas of wild type mice and IL-6 knockout (KO) mice were treated with elastase. Additionally, thoracic animals treated with either the IL-6 receptor antagonist tocilizumab (n = 8) or vehicle (n = 5). Finally, human TAA and AAA were analyzed with human cytokine array. RESULTS Elastase treatment of thoracic aortas yielded dilation of 86.8% ± 9.6%, and abdominal aortas produced dilation of 85.6% ± 16.2%. Murine IL-6, CXCL13, and matrix metalloproteinase-9 were significantly elevated in TAA compared with AAA (p = 0.004, 0.028, and 0.001, respectively). The IL-6KO mice demonstrated significantly smaller TAA size relative to wild type mice (wild type 100.1% versus IL-6KO 76.5%, p = 0.04). The IL-6KO mice did not show protection from AAA (p = 0.732). Pharmacologic inhibition of IL-6 resulted in significant reduction in TAA size (tocilizumab 71.5% ± 13.2% versus vehicle 103.6% ± 20.7%, p = 0.005). Human TAA showed significantly greater IL-6 (p < 0.0001) compared with AAA and normal thoracic and abdominal aorta. CONCLUSIONS Interleukin-6 is significantly greater in both murine and human TAA compared with AAA, suggesting fundamental differences in these disease processes. Interleukin-6 receptor antagonism attenuates experimental TAA formation, indicating that IL-6 may be a potential target for human thoracic aneurysmal disease.

Novel Role of IL (Interleukin)-1β in Neutrophil Extracellular Trap Formation and Abdominal Aortic Aneurysms

Objective— Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1&bgr; in the formation of murine experimental AAAs. Here, the hypothesis that IL-1&bgr;–induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested. Approach and Results— NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1&bgr;. In vitro, IL-1RA attenuated IL-1&bgr;–induced NETosis in human neutrophils. Mechanistically, IL-1&bgr; treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-&bgr;–induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage–day 3 of aneurysm induction. IL-1&bgr;–knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1&bgr;–knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation. Conclusions— Altogether, the results suggest a dominant role of IL-1&bgr;–induced NETosis in AAA formation.

Inferior Vena Cava Reconstruction with Tubularized Bovine Pericardium.

A 32-year-old man presented with a large, locally advanced sarcomatoid right renal cell carcinoma invading the duodenum and inferior vena cava (IVC). Because of persistent symptomatic gastrointestinal bleeding requiring repeated blood transfusion and the inability to use appropriate systemic chemotherapy, the patient was taken for palliative resection. En bloc pancreaticoduodenectomy, right nephrectomy, and IVC resection were performed with reconstruction of the IVC with tubularized bovine pericardium. Widespread availability, ease and speed of tubularized graft creation, lack of morbidity to the patient, and its inherent resistance to infection in contaminated fields make bovine pericardium an expedient reconstructive option in these challenging cases.

Iliac Artery Injuries

Injury to the iliac arteries remains a devastating injury with significant morbidity and mortality that continues to challenge trauma surgeons. Patients typically present in significant metabolic disarray as a result of massive hemorrhage. Damage control techniques should be employed for patients who are coagulopathic, hypothermic, or acidotic. Definitive arterial repair can be accomplished by various techniques, selection of which should depend upon the extent and location of injury. Penetrating injury typically requires open repair, whereas most blunt injuries are amenable to endovascular therapies. Early communication between the trauma surgeon, vascular surgeon, and interventional radiologist is necessary. Fasciotomies should be strongly considered in patients with injury to the common or external iliac arteries or in patients with crush injury in order to avoid development of compartment syndrome.

Iliac Vein Injuries

Iliac vein injuries are highly lethal and present a substantial challenge to manage. They account for ~5% of cardiovascular injuries following trauma and are difficult to diagnose and repair due to their location deep in the pelvis. A high index of suspicion must be maintained to make a rapid diagnosis and have a chance at repair. Injury most frequently follows penetrating trauma, but iliac vein damage can also result from blunt force. Injuries are frequently associated with concomitant injury to the bowel and bladder with mortality following repair ranging between 20% and 50%. Iliac vein injuries may either be repaired surgically with lateral venorrhaphy or ligation. Additionally, endovascular repair techniques are a promising adjunct to open repair. The following text will review the causes, diagnosis, and management of iliac vein injuries.