William F. Johnston

Genetic and Pharmacologic Disruption of Interleukin-1β Signaling Inhibits Experimental Aortic Aneurysm Formation

Objective—Abdominal aortic aneurysms (AAAs) are common, but their exact pathogenesis remains unknown and no specific medical therapies are available. We sought to evaluate interleukin-1&bgr; (IL-1&bgr;) and interleukin-1 receptor (IL-1R) in an experimental AAA model to identify novel therapeutic targets for AAA treatment. Methods and Results—IL-1&bgr; mRNA and protein levels were significantly elevated in abdominal aortas of 8- to 12-week-old male C57Bl/6 mice after elastase aortic perfusion (wild-type [WT]) compared with saline perfusion. Mice with genetic deletion of IL-1&bgr; (IL-1&bgr; knockout [KO]) or IL-1R (IL-1R KO) that underwent elastase perfusion demonstrated significant protection against AAA formation, with maximal aortic dilations of 38.0±5.5% for IL-1&bgr; KO and 52.5±4.6% for IL-1R KO, compared with 89.4±4.0% for WT mice (P<0.005). Correspondingly, IL-1&bgr; KO and IL-1R KO aortas had reduced macrophage and neutrophil staining with greater elastin preservation compared with WT. In WT mice pretreated with escalating doses of the IL-1R antagonist anakinra, there was a dose-dependent decrease in maximal aortic dilation (R=–0.676; P<0.0005). Increasing anakinra doses correlated with decreasing macrophage staining and elastin fragmentation. Lastly, WT mice treated with anakinra 3 or 7 days after AAA initiation with elastase demonstrated significant protection against AAA progression and had decreased aortic dilation compared with control mice. Conclusion—IL-1&bgr; is critical for AAA initiation and progression, and IL-1&bgr; neutralization through genetic deletion or receptor antagonism attenuates experimental AAA formation. Disrupting IL-1&bgr; signaling offers a novel pathway for AAA treatment.

Experimental Abdominal Aortic Aneurysm Formation Is Mediated by IL-17 and Attenuated by Mesenchymal Stem Cell Treatment

Background— Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation and matrix degradation. This study tests the hypothesis that CD4+ T-cell–produced IL-17 modulates inflammation and smooth muscle cell activation, leading to the pathogenesis of AAA and that human mesenchymal stem cell (MSC) treatment can attenuate IL-17 production and AAA formation. Methods and Results— Human aortic tissue demonstrated a significant increase in IL-17 and IL-23 expression in AAA patients compared with control subjects as analyzed by RT-PCR and ELISA. AAA formation was assessed in C57BL/6 (wild-type; WT), IL-23−/− or IL-17−/− mice using an elastase-perfusion model. Heat-inactivated elastase was used as control. On days 3, 7, and 14 after perfusion, abdominal aorta diameter was measured by video micrometry, and aortic tissue was analyzed for cytokines, cell counts, and IL-17–producing CD4+ T cells. Aortic diameter and cytokine production (MCP-1, RANTES, KC, TNF-&agr;, MIP-1&agr;, and IFN-&ggr;) was significantly attenuated in elastase-perfused IL-17−/− and IL-23−/− mice compared with WT mice on day 14. Cellular infiltration (especially IL-17–producing CD4+ T cells) was significantly attenuated in elastase-perfused IL-17−/− mice compared with WT mice on day 14. Primary aortic smooth muscle cells were significantly activated by elastase or IL-17 treatment. Furthermore, MSC treatment significantly attenuated AAA formation and IL-17 production in elastase-perfused WT mice. Conclusions— These results demonstrate that CD4+ T-cell–produced IL-17 plays a critical role in promoting inflammation during AAA formation and that immunomodulation of IL-17 by MSCs can offer protection against AAA formation.

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.

Sphingosine-1-phosphate receptor 1 agonism attenuates lung ischemia-reperfusion injury

Outcomes for lung transplantation are the worst of any solid organ, and ischemia-reperfusion injury (IRI) limits both short- and long-term outcomes. Presently no therapeutic agents are available to prevent IRI. Sphingosine 1-phosphate (S1P) modulates immune function through binding to a set of G protein-coupled receptors (S1PR1-5). Although S1P has been shown to attenuate lung IRI, the S1P receptors responsible for protection have not been defined. The present study tests the hypothesis that protection from lung IRI is primarily mediated through S1PR1 activation. Mice were treated with either vehicle, FTY720 (a nonselective S1P receptor agonist), or VPC01091 (a selective S1PR1 agonist and S1PR3 antagonist) before left lung IR. Function, vascular permeability, cytokine expression, neutrophil infiltration, and myeloperoxidase levels were measured in lungs. After IR, both FTY720 and VPC01091 significantly improved lung function (reduced pulmonary artery pressure and increased pulmonary compliance) vs. vehicle control. In addition, FTY720 and VPC01091 significantly reduced vascular permeability, expression of proinflammatory cytokines (IL-6, IL-17, IL-12/IL-23 p40, CC chemokine ligand-2, and TNF-α), myeloperoxidase levels, and neutrophil infiltration compared with control. No significant differences were observed between VPC01091 and FTY720 treatment groups. VPC01091 did not significantly affect elevated invariant natural killer T cell infiltration after IR, and administration of an S1PR1 antagonist reversed VPC01091-mediated protection after IR. In conclusion, VPC01091 and FTY720 provide comparable protection from lung injury and dysfunction after IR. These findings suggest that S1P-mediated protection from IRI is mediated by S1PR1 activation, independent of S1PR3, and that selective S1PR1 agonists may provide a novel therapeutic strategy to prevent lung IRI.

Staged hybrid approach using proximal thoracic endovascular aneurysm repair and distal open repair for the treatment of extensive thoracoabdominal aortic aneurysms

OBJECTIVE Repair of patients with extent I and II thoracoabdominal aortic aneurysms (TAAAs) is associated with significant morbidity and mortality, whereas repair of more distal extent III and IV TAAAs has a lower risk of paraplegia and death. Therefore, we describe an approach using thoracic endovascular aneurysm repair (TEVAR) as the index operation to convert extent I and II TAAAs to extent III and IV TAAAs amenable to subsequent open aortic repair to minimize patient risk. METHODS Between July 2007 and March 2012, 10 staged hybrid operations were performed to treat one extent I and nine extent II TAAAs. Aortic aneurysm pathology included five chronic type B dissections, three acute type B dissections, and two penetrating aortic ulcers. Initially, the proximal descending thoracic aorta was repaired with TEVAR for coverage of the most proximal fenestration or penetrating ulcer, with seven elective and three emergent repairs. Interval open distal aortic replacement was performed in a short-term planned setting or for progressive dilation of the distal aortic segment. In the open repair, the proximal end of the graft was sewn directly to the distal end of the TEVAR and outer wall of the aorta. RESULTS Average patient age was 48 years, and 60% were men. Risk factors included hypertension (80%), current tobacco use (50%), and Marfan syndrome (30%). Complications after TEVAR included type IA (n=1) and type II (n=3) endoleaks, pleural effusions (n=3), and acute kidney injury (n=1). Three patients required endovascular reinterventions. In patients with dissection, persistent filling of the false lumen was common and associated with distal thoracic aortic dilation. Complications of open repair included acute kidney injury in two patients, but no cardiac, pulmonary, or neurologic morbidity. Median time between TEVAR and open repair was 14 weeks. Most importantly, no deaths or neurologic deficits occurred after either procedure during a median follow-up of 35 weeks. CONCLUSIONS A staged hybrid approach to extensive TAAAs combining proximal TEVAR, followed by interval open distal TAAA repair, is safe and appears to be an effective alternative to traditional open repair. This approach may decrease the significant morbidity associated with single-stage open extent I and II TAAA repairs and may be applicable to multiple TAAA etiologies.

Aromatase is required for female abdominal aortic aneurysm protection

OBJECTIVE The protective effects of female gender on the development of abdominal aortic aneurysms (AAAs) have been attributed to anti-inflammatory effects of estrogen. Estrogen synthesis is dependent on the enzyme aromatase, which is located both centrally in the ovaries and peripherally in adipose tissue, bone, and vascular smooth muscle cells. It is hypothesized that deletion of aromatase in both ovarian and peripheral tissues would diminish the protective effect of female gender and would be associated with increased aortic diameter in female mice. METHODS Male and female 8- to 10-week-old mice with aromatase (wild type: WT) and without aromatase (ArKO) underwent elastase aortic perfusion with aortic harvest 14 days following. For the contribution of central and peripheral estrogen conversion to be evaluated, female WT mice were compared with female WT and ArKO mice that had undergone ovariectomy (ovx) at 6 weeks followed by elastase perfusion at 8 to 10 weeks. At aortic harvest, maximal aortic dilation was measured and samples were collected for immunohistochemistry and protein analysis. Serum was collected for serum estradiol concentrations. Groups were compared with analysis of variance. Human and mouse AAA cross sections were analyzed with confocal immunohistochemistry for aromatase, smooth muscle markers, and macrophage markers. RESULTS Female WT mice had significant reduction in aortic dilation compared with male WT mice (F WT, 51.5% ± 15.1% vs M WT, 78.7% ± 14.9%; P < .005). The protective effects of female gender were completely eliminated with deletion of aromatase (F ArKO, 82.6% ± 13.8%; P < .05 vs F WT). Ovariectomy increased aortic dilation in WT mice (F WT ovx, 70.6% ± 11.7%; P < .05 vs F WT). Aromatase deletion with ovariectomy further increased aortic dilation compared with WT ovx mice (F ArKO ovx, 87.3% ± 14.7%, P < .001 vs F WT and P < .05 vs F WT ovx). Accordingly, female ArKO ovx mice had significantly higher levels of the proinflammatory cytokines monocyte chemoattractant protein 1 and interleukin-1β and were associated with increased macrophage staining and decreased elastin staining. Regarding serum hormone levels, decreasing estradiol levels correlated with increasing aortic diameter (R = -0.565; P < .01). By confocal immunohistochemistry, both human and mouse AAA smooth muscle cells (smooth muscle α-actin positive) and macrophages (CD68 positive or Mac-2 positive) expressed aromatase. CONCLUSIONS The protective effect of female gender on AAAs is due to estrogen synthesis and requires the presence of both ovarian and extragonadal/peripheral aromatase. Peripheral estrogen synthesis accounts for roughly half of the protective effect of female gender. If peripheral aromatase could be targeted, high levels of local estrogen could be produced and may avoid the side effects of systemic estrogen.

Staged hybrid repair of extensive thoracoabdominal aortic aneurysms secondary to chronic aortic dissection

OBJECTIVE Many patients with aortic dissection develop Crawford extent I or II thoracoabdominal aortic aneurysms (TAAA). Because open repair is associated with a high morbidity and mortality, hybrid approaches to TAAA repair are emerging. In this study, we evaluated the midterm outcomes and aortic remodeling of a hybrid technique that combines proximal thoracic endovascular aneurysm repair (TEVAR), followed by staged distal open thoracoabdominal repair for patients with Crawford extent I or II TAAAs secondary to chronic aortic dissection. METHODS We identified 19 patients with Crawford extent I (n = 1) or extent II (n = 18) TAAAs secondary to chronic aortic dissection who underwent a staged hybrid repair from 2007 to 2014 at our institution. Nine patients had previous open ascending aortic surgery for type I aortic dissection. Stage 1 TEVAR was performed via percutaneous (n = 8), femoral cutdown (n = 8), or iliac exposure (n = 3). The left subclavian artery was covered in nine patients and revascularized in eight patients using carotid-subclavian bypass (n = 7) or laser fenestration (n = 1). Stage 2 open repair was performed a median of 18 weeks later with partial cardiopulmonary bypass via left femoral arterial and venous cannulation for visceral and lower body perfusion. The open thoracoabdominal graft was anastomosed proximally in an end to end fashion with the endograft. We then assessed surgical morbidity and mortality, midterm survival, and freedom from reintervention. Aortic remodeling was measured and change in maximum aortic and false lumen diameter at last follow-up (median, 3 years) from baseline was assessed. RESULTS There were no deaths, strokes, or chronic renal failure in this cohort. After stage 1 TEVAR, three patients required repeat intervention for endoleak (type Ia, n = 1; type Ib, n = 1; type II, n = 1) before open repair. After stage 2 open repair, there was a single delayed permanent paralysis 2 weeks after discharge. At a median 3-year follow-up (range, 6 months-6.2 years), there were no deaths, neurologic events, endoleaks, or TAAA reinterventions. Complete false lumen thrombosis occurred in 100% of the patients, with maximum false lumen diameter decreasing from 34.3 ± 15.3 mm to 13.2 ± 12.0 mm (P < .01) and total aortic diameter decreasing from 60.2 ± 9.0 mm to 49.4 ± 9.6 mm (P < .01). CONCLUSIONS Staged hybrid TAAA repair, using a combination of proximal TEVAR with open distal repair, can be performed using established endovascular skills and technology coupled with traditional open aortic surgical techniques, with low surgical morbidity and mortality. In the midterm, staged hybrid TAAA repair was associated favorable survival, aortic remodeling, and freedom from reintervention.

Surgical Management of Minimally Invasive Aortic Valve Operations

Although there is still a role for conventional sternotomy for aortic valve replacement, minimally invasive techniques are increasing in popularity and may benefit the patient with shorter postoperative course, less morbidity, and decreased overall cost. Additionally, transcatheter procedures have recently shown promising results in high-risk patients. This article provides an overview of the development of minimally invasive aortic valve operations, including a brief history of minimally invasive approaches, surgical considerations during minimally invasive aortic valve replacement, and the technical approach to performing a hemisternotomy with aortic valve replacement. In addition, the authors review transcatheter techniques, including aortic valve replacement via a sheath placed in the apex of the left ventricle or through a sheath placed in the femoral vessels. Finally, the exciting results of the PARTNER trial and the effect of these results on the future of aortic valve surgery are discussed.

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.