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Background and Purpose- Inflammation is a critical determinant of aneurysmal wall destabilization, growth, and rupture risk. Targeting inflammation may suppress aneurysm rupture. Vagus nerve stimulation (VNS) has been shown to suppress inflammation both systemically and in the central nervous system. Therefore, we tested the effect of a novel noninvasive transcutaneous VNS approach on aneurysm rupture and outcome in a mouse model of intracranial aneurysm formation with wall inflammation. Methods- Aneurysms were induced by a single stereotaxic injection of elastase into the cerebrospinal fluid at the skull base, combined with systemic deoxycorticosterone-salt hypertension, without or with high-salt diet, for mild or severe outcomes, respectively. Cervical VNS (two 2-minute stimulations 5 minutes apart) was delivered once a day starting from the day after elastase injection for the duration of follow-up. Transcutaneous stimulation of the femoral nerve (FNS) served as control. Multiple aneurysms developed in the circle of Willis and its major branches, resulting in spontaneous ruptures and subarachnoid hemorrhage, neurological deficits, and mortality. Results- In the milder model, VNS significantly reduced aneurysm rupture rate compared with FNS (29% versus 80%, respectively). Subarachnoid hemorrhage grades were also lower in the VNS group. In the more severe model, both VNS and FNS arms developed very high rupture rates (77% and 85%, respectively). However, VNS significantly improved the survival rate compared with FNS after rupture (median survival 13 versus 6 days, respectively), without diminishing the subarachnoid hemorrhage grades. Chronic daily VNS reduced MMP-9 (matrix metalloproteinase-9) expression compared with FNS, providing a potential mechanism of action. As an important control, chronic daily VNS did not alter systemic arterial blood pressure compared with FNS. Conclusions- VNS can reduce aneurysm rupture rates and improve the outcome from ruptured aneurysms.

Objective- Inflammation occurs during the progression of abdominal aortic aneurysm (AAA). IL (interleukin)-33 is a pleiotropic cytokine with multiple immunomodulatory effects, yet its role in AAA remains unknown. Approach and Results- Immunoblot, immunohistochemistry, and immunofluorescent staining revealed increased IL-33 expression in adventitia fibroblasts from mouse AAA lesions. Daily intraperitoneal administration of recombinant IL-33 or transgenic IL-33 expression ameliorated periaorta CaPO 4 injury- and aortic elastase exposure-induced AAA in mice, as demonstrated by blunted aortic expansion, reduced aortic wall elastica fragmentation, enhanced AAA lesion collagen deposition, attenuated T-cell and macrophage infiltration, reduced inflammatory cytokine production, skewed M2 macrophage polarization, and reduced lesion MMP (matrix metalloproteinase) expression and cell apoptosis. Flow cytometry analysis, immunostaining, and immunoblot analysis showed that exogenous IL-33 increased CD4 + Foxp3 + regulatory T cells in spleens, blood, and aortas in periaorta CaPO 4 -treated mice. Yet, ST2 deficiency muted these IL-33 activities. Regulatory T cells from IL-33-treated mice also showed significantly stronger activities in suppressing smooth muscle cell inflammatory cytokine and chemokine expression, macrophage MMP expression, and in increasing M2 macrophage polarization than those from vehicle-treated mice. In contrast, IL-33 failed to prevent AAA and lost its beneficial activities in CaPO 4 -treated mice after selective depletion of regulatory T cells. Conclusions- Together, this study established a role of IL-33 in protecting mice from AAA formation by enhancing ST2-dependent aortic and systemic regulatory T-cell expansion and their immunosuppressive activities.

Rationale: Histological examination of abdominal aortic aneurysm (AAA) tissues demonstrates extracellular matrix destruction and infiltration of inflammatory cells. Previous work with mouse models of AAA has shown that anti-inflammatory strategies can effectively attenuate aneurysm formation. Thrombospondin-1 is a matricellular protein involved in the maintenance of vascular structure and homeostasis through the regulation of biological functions, such as cell proliferation, apoptosis, and adhesion. Expression levels of thrombospondin-1 correlate with vascular disease conditions., Objective: To use thrombospondin-1-deficient (Thbs1(-/-)) mice to test the hypothesis that thrombospondin-1 contributes to pathogenesis of AAAs., Methods and Results: Mouse experimental AAA was induced through perivascular treatment with calcium phosphate, intraluminal perfusion with porcine elastase, or systemic administration of angiotensin II. Induction of AAA increased thrombospondin-1 expression in aortas of C57BL/6 or apoE-/- mice. Compared with Thbs1(+/+) mice, Thbs1(-/-) mice developed significantly smaller aortic expansion when subjected to AAA inductions, which was associated with diminished infiltration of macrophages. Thbs1(-/-) monocytic cells had reduced adhesion and migratory capacity in vitro compared with wild-type counterparts. Adoptive transfer of Thbs1(+/+) monocytic cells or bone marrow reconstitution rescued aneurysm development in Thbs1(-/-) mice., Conclusions: Thrombospondin-1 expression plays a significant role in regulation of migration and adhesion of mononuclear cells, contributing to vascular inflammation during AAA development., (© 2015 American Heart Association, Inc.)

Background and Purpose: Cerebral aneurysm (CA) affects 3% of the population and is associated with hemodynamic stress and inflammation. Myeloperoxidase, a major oxidative enzyme associated with inflammation, is increased in patients with CA, but whether myeloperoxidase contributes to CA is not known. We tested the hypotheses that myeloperoxidase is increased within human CA and is critical for formation and rupture of CA in mice., Methods: Blood was drawn from the lumen of CAs and femoral arteries of 25 patients who underwent endovascular coiling of CA, and plasma myeloperoxidase concentrations were measured with ELISA. Effects of endogenous myeloperoxidase on CA formation and rupture were studied in myeloperoxidase knockout mice and wild-type (WT) mice using an angiotensin II-elastase induction model of CA. In addition, effects of myeloperoxidase on inflammatory gene expression in endothelial cells were analyzed., Results: Plasma concentrations of myeloperoxidase were 2.7-fold higher within CA than in femoral arterial blood in patients with CA. myeloperoxidase-positive cells were increased in aneurysm tissue compared with superficial temporal artery of patients with CA. Incidence of aneurysms and subarachnoid hemorrhage was significantly lower in myeloperoxidase knockout than in WT mice. In cerebral arteries, proinflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2 (COX2), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C motif) ligand (XCL1), matrix metalloproteinase (MMP) 8, cluster of differentiation 68 (CD68), and matrix metalloproteinase 13, and leukocytes were increased, and α-smooth muscle actin was decreased, in WT but not in myeloperoxidase knockout mice after induction of CA. Myeloperoxidase per se increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells., Conclusions: These findings suggest that myeloperoxidase may contribute importantly to formation and rupture of CA., (© 2015 American Heart Association, Inc.)

Background: Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined., Methods and Results: Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening., Conclusions: The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA., (© 2015 American Heart Association, Inc.)

Background: Thoracic aortic aneurysms (TAAs) are common, but experimental TAA models are limited and the role of interleukin-1β (IL-1β) is undetermined., Methods and Results: IL-1β protein was measured in human TAAs and control aortas, and IL-1β protein was increased ≈20-fold in human TAAs. To develop an experimental model of TAAs, 8- to 10-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-1β expression. Next, TAAs were induced in mice deficient of IL-1β (IL-1β knockout) or IL-1 receptor (IL-1R knockout; n=10 each). Genetic deletion of IL-1β and IL-1R significantly decreased thoracic aortic dilation (IL-1β 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-1β knockout and IL-1R knockout aortas demonstrated preserved elastin and smooth muscle cells with fewer inflammatory cells. Correspondingly, IL-1β 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-1β decreased TAA formation and progression, indicating that IL-1β may be a potential target for TAA treatment., (© 2014 American Heart Association, Inc.)

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-α, MIP-1α, and IFN-γ) 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.

Background: Hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after lung injury. The goal of the present study was to characterize its role in pulmonary emphysema, which may lead to the development of new treatment strategies with HGF., Methods and Results: HGF mRNA and protein levels in lung tissue and plasma from elastase-induced emphysema rats transiently increased, then declined significantly to below the basal level in a time-dependent manner (P<0.01). Furthermore, changes in HGF were correlated with histologically progressive emphysematous changes and deterioration in pulmonary physiology. Use of the HVJ (hemagglutinating virus of Japan) envelope method resulted in successful transfection of cDNA encoding human HGF, as demonstrated by an efficient expression of HGF in alveolar endothelial and epithelial cells. Transfection of HGF resulted in a more extensive pulmonary vasculature and inhibition of alveolar wall cell apoptosis, and those effects led to improved exercise tolerance and gas exchange (P<0.05), which persisted for more than 1 month., Conclusions: Decreased HGF expression due to a failure in sustained endogenous production after injury was associated with emphysema-related histopathologic and physiological changes in the present rat model. In addition, induction of HGF expression by a gene-transfection method resulted in improved pulmonary function via inhibition of alveolar cell apoptosis, enhancement of alveolar regeneration, and promotion of angiogenesis.

Many mouse models of abdominal aortic aneurysms have been developed that use a diverse array of methods for producing the disease, including genetic manipulation and chemical induction. These models could provide insight into potential mechanisms in the development of this disease. Although experimental studies on abdominal aortic aneurysms (AAAs) have used a variety of mammalian and avian approaches, there is an increasing reliance on the use of mice. The models recapitulate some facets of the human disease including medial degeneration, inflammation, thrombus formation, and rupture. Most of the mouse models of AAA are evoked either by genetically defined approaches or by chemical means. The genetic approaches are spontaneous and engineered mutations. These include defects in extracellular matrix maturation, increased degradation of elastin and collagen, aberrant cholesterol homeostasis, and enhanced production of angiotensin peptides. The chemical approaches include the intraluminal infusion of elastase, periaortic incubations of calcium chloride, and subcutaneous infusion of AngII. A common feature of these models is the reduction of AAA incidence and severity by the prophylactic administration of matrix metalloproteinase (MMP) inhibitors or genetically engineered deficiencies of specific members of this proteolytic protein family. The validation of mouse models of AAAs will provide insight into the mechanisms of progression of the human disease.