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Aims: Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation., Methods and Results: ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-β2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into μMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-β2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining., Conclusion: Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-β2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Aims: Vascular remodelling and aortic aneurysm formation are induced mainly by inflammatory responses in the adventitia and media. However, relatively little is known about the mechanistic significance of endothelium in the pathogenesis of these vascular disorders. The transcription factor nuclear factor-kappa B (NF-κB) regulates the expressions of numerous genes, including those related to pro-inflammatory responses. Therefore, to investigate the roles of endothelial pro-inflammatory responses, we examined the impact of blocking endothelial NF-κB signalling on intimal hyperplasia and aneurysm formation., Methods and Results: To block endothelial NF-κB signalling, we used transgenic mice expressing dominant-negative IκBα selectively in endothelial cells (E-DNIκB mice). E-DNIκB mice were protected from the development of cuff injury-induced neointimal formation, in association with suppressed arterial expressions of cellular adhesion molecules, a macrophage marker, and inflammatory factors. In addition, the blockade of endothelial NF-κB signalling prevented abdominal aortic aneurysm formation in an experimental model, hypercholesterolaemic apolipoprotein E-deficient mice with angiotensin II infusion. In this aneurysm model as well, aortic expressions of an adhesion molecule, a macrophage marker, and inflammatory factors were suppressed with the inhibited expression and activity of matrix metalloproteinases in the aorta., Conclusion: Endothelial NF-κB activation up-regulates adhesion molecule expression, which may trigger macrophage infiltration and inflammation in the adventitia and media. Thus, the endothelium plays important roles in vascular remodelling and aneurysm formation through its intracellular NF-κB signalling.

Aims: Abdominal aortic aneurysm (AAA) is characterized by extensive aortic wall matrix degradation that contributes to the remodelling and eventual rupture of the arterial wall. Elastinolytic cathepsin S (Cat S) is highly expressed in human aneurysmal lesions, but whether it contributes to the pathogenesis of AAA remains unknown., Methods and Results: AAAs were induced in apolipoprotein E (ApoE) and Cat S compound mutant (Apoe(-/-)Ctss(-/-)) mice and in ApoE-deficient Cat S wild-type littermates (Apoe(-/-)Ctss(+/+)) by chronic angiotensin II infusion, and AAA lesions were analysed after 28 days. We found that Cat S expression increased significantly in mouse AAA lesions. The AAA incidence in Apoe(-/-)Ctss(-/-) mice was much lower than that in Apoe(-/-)Ctss(+/+) mice (10 vs. 80%). Cat S deficiency significantly reduced external and luminal abdominal aortic diameters, medial elastin fragmentation, and adventitia collagen content. Cat S deficiency reduced aortic lesion expression and the activity of matrix metalloproteinase (MMP)-2, MMP-9, and Cat K, but not the activity of other major cathepsins, such as Cat B and Cat L. Absence of Cat S significantly reduced AAA lesion media smooth muscle cell (SMC) apoptosis, lesion adventitia microvessel content, and inflammatory cell accumulation and proliferation. In vitro studies proved that Cat S helps promote SMC apoptosis, angiogenesis, monocyte and T-cell transmigration, and T-cell proliferation--all of which are essential to AAA pathogenesis., Conclusions: These data provide direct evidence that Cat S plays an important role in AAA formation and suggest that Cat S is a new therapeutic target for human AAA.

Aims: Mast cells participate importantly in abdominal aortic aneurysms (AAAs) by releasing inflammatory cytokines to promote vascular cell protease expression and arterial wall remodelling. Mast cells accumulate in AAA lesions during disease progression, but the exact chemokines by which mast cells migrate to the site of vascular inflammation remain unknown. This study tested the hypothesis that mast cells use chemokine (C-C motif) receptor 2 (CCR2) for their accumulation in experimental mouse AAA lesions., Methods and Results: We generated mast cell and apolipoprotein E double-deficient (Apoe(-/-)Kit(W-sh/W-sh)) mice and found that they were protected from angiotensin II (Ang II) chronic infusion-induced AAAs compared with Apoe(-/-) littermates. Using bone-marrow derived mast cells (BMMC) from Apoe(-/-) mice and CCR2 double-deficient (Apoe(-/-)Ccr2(-/-)) mice, we demonstrated that Apoe(-/-)Kit(W-sh/W-sh) mice receiving BMMC from Apoe(-/-)Ccr2(-/-) mice, but not those from Apoe(-/-) mice, remained protected from AAA formation. Adoptive transfer of BMMC from Apoe(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice also increased lesion content of macrophages, T cells, and MHC class II-positive cells; there was also increased apoptosis, angiogenesis, cell proliferation, elastin fragmentation, and medial smooth muscle cell loss. In contrast, adoptive transfer of BMMC from Apoe(-/-)Ccr2(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice did not affect these variables., Conclusions: The increased AAA formation and associated lesion characteristics in Apoe(-/-)Kit(W-sh/W-sh) mice after receiving BMMC from Apoe(-/-) mice, but not from Apoe(-/-)Ccr2(-/-) mice, suggests that mast cells use CCR2 as the chemokine receptor for their recruitment in Ang II-induced mouse AAA lesions.

Aims: The loss of the inhibitory receptor CD31 on peripheral T lymphocytes is associated with the incidence of atherosclerotic complications such as abdominal aortic aneurysms (AAA) in patients and plaque thrombosis in mice. However, we have recently discovered that a small fragment of extracellular CD31 remains expressed on the surface of the apparently 'CD31-negative' T-cells and that it is possible to restore the CD31-mediated T-cell inhibition in vivo by using a synthetic CD31-derived peptide. Here, we wanted to evaluate the therapeutic potential of the peptide in an experimental model of accelerated atherosclerosis and AAA formation., Methods and Results: The effect of the murine CD31-derived peptide (aa 551-574, 1.5 mg/kg/day, sc) was evaluated on the extent of atherosclerotic plaques and the incidence of AAA in 28-week-old apolipoprotein E knockout mice (male, n ≥ 8/group) submitted to chronic angiotensin II infusion. The therapeutic mechanisms of the peptide were assessed by evaluating its effect on immune cell functions in vivo and in vitro. The prevalence of angiotensin II-induced AAA correlated with the loss of extracellular CD31 on T-cells. CD31 peptide treatment reduced both aneurysm formation and plaque size (P < 0.05 vs. control). Protection was associated with reduced perivascular leucocyte infiltration and T-cell activation in vivo. Functional in vitro studies showed that the peptide is able to suppress both T-cell and macrophage activation., Conclusion: CD31 peptides could represent a new class of drugs intended to prevent the inflammatory cell processes, such as those underlying progression of atherosclerosis and development of AAA.

Aims: Increased angiogenesis, chronic inflammation, and extracellular matrix degradation are the major pathological features of abdominal aortic aneurysm (AAA). We sought to elucidate the role of vascular endothelial growth factor (VEGF)-A, a potent angiogenic and proinflammatory factor, in the development of AAA., Methods and Results: Human AAA samples showed increased VEGF-A expression, neovascularization, and macrophage infiltration compared with normal aortic walls. AAA was induced in mice by periaortic application of CaCl(2). AAA mice were treated with soluble VEGF-A receptor (sFlt)-1 or phosphate-buffered saline and sacrificed 6 weeks after the operation. Treatment with sFlt-1 resulted in reduced aneurysm size, restored wavy structure of the elastic lamellae, reduced Mac-2(+) monocytes/macrophages, CD3(+) T-lymphocytes, and CD31(+) vessels, and attenuated matrix metalloproteinase (MMP)-2 and 9 activity in periaortic tissue of AAA. Increased aortic mRNA expression of monocyte chemotactic protein-1, tumour necrosis factor-α, and intercellular adhesion molecule-1 in AAA was attenuated by sFlt-1 treatment., Conclusion: VEGF-A was overexpressed in the aortic wall of human and experimental AAA. Treatment with sFlt-1 inhibited AAA development in mice, in association with reduced neoangiogenesis, infiltration of inflammatory cells, MMP activity, and extracellular matrix degradation. These findings suggest a crucial role of VEGF-A in the development of AAA.

Leukotrienes (LTs) derived from 5-lipoxygenase (5-LO) activity are most widely known for their actions during acute inflammation and asthma. 5-LO/LT pathway involvement in cardiovascular disease (CVD) pathogenesis has come to the forefront based on provocative human genetic/population and animal studies leading to the hypothesis that this pathway promotes atherosclerosis, abdominal aortic aneurysm, and myocardial infarction/reperfusion injury via increased leucocyte chemotaxis, vascular inflammation and enhanced permeability, and subsequent tissue/matrix degeneration. A series of pre-clinical studies have tested this hypothesis by means of genetic or pharmacological inhibition of either the LT biosynthesis axis (5-LO, 5-LO-activating protein, LTA(4) hydrolase, LTC(4) synthase) or the cognate LT receptors. Here, we summarize, compare, and analyse these animal studies and relate their findings to human disease pathogenesis. We draw a complex picture of 5-LO/LT participation in cardiovascular disorders, which is further complicated by marked differences between species. Moreover, we discuss how the cytokine footprint of the respective pathological conditions determines the expression level and hence, the contribution of components of the pathway to the overall disease state. Current knowledge implies a role for 5-LO and LTs during the early/acute phase of CVD, but our understanding of a putative 5-LO/LT involvement in more advanced stages of CVD is limited, thereby preventing simple extrapolation of findings from animal studies to humans.

Aims: Neutrophils/platelet interactions are involved in abdominal aortic aneurysm (AAA). The intraluminal thrombus (ILT) is a human model of platelet/neutrophil interactions. The present study focused on mediators involved in neutrophil recruitment in AAA., Methods and Results: Conditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/alpha1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and alpha-defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2., Conclusion: Taken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT.

Traditional concepts of vascular inflammation are considered "inside-out" responses centered on the monocyte adhesion and lipid oxidation hypotheses. These mechanisms likely operate in concert, holding the central tenet that the inflammatory response is initiated at the luminal surface. However, growing evidence supports a new paradigm of an "outside-in" hypothesis, in which vascular inflammation is initiated in the adventitia and progresses inward toward the intima. Hallmarks of the outside-in hypothesis include population of the adventitia with exogenous cell types, including monocytes, macrophages, and lymphocytes, the phenotypic switch of adventitial fibroblasts into migratory myofibroblasts, and increased vasa vasorum neovascularization. The resident and migrating cells deposit collagen and matrix components, respond to and upregulate inflammatory chemokines and/or antigens, and regulate the local redox state of the adventitia. B cells and T cells generate local humoral immune responses against local antigen presentation by foam cells and antigen presenting cells. These events result in increased local expression of cytokines and growth factors, evoking an inflammatory response that propagates inward toward the intima. Ultimately, it appears that the basic mechanisms of cellular activation and migration in vascular inflammation are highly conserved across a variety of cardiovascular disease states and that major inflammatory events begin in the adventitia.

Objective: Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammation which contributes to the remodeling and eventual weakening of the vessel wall. Increased cyclooxygenase-2 (COX-2) expression is detected in human aneurysmal tissue and is suggested to contribute to the disease. The aim of the current study was to define the role of COX-2 expression in the development of AAAs, using a model of the disease., Methods: AAAs were induced in mice by chronic angiotensin II infusion, and were analyzed following 3, 7, 21 or 28 days of the infusion. AAA incidence and severity, together with the expression of inflammatory markers, were compared between abdominal aortas from COX-2-deficient mice and their wild-type littermate controls., Results: The AAA incidence in COX-2 wild-type mice was 54% (13/24), whereas AAAs were not detected in COX-2-deficient mice (0/23) following 28 days of angiotensin II infusion. The genetic deficiency of COX-2 also resulted in a 73% and 90% reduction in AAA incidence following 7 and 21 days of angiotensin II infusion, respectively. In COX-2 wild-type mice, COX-2 mRNA expression in the abdominal aorta was induced by angiotensin II beginning 3 days following initiation of the infusion, which continued throughout progression of the disease. Abundant COX-2 protein expression was detected in medial smooth muscle cells adjacent to the AAAs. The deficiency of COX-2 significantly attenuated mRNA expression in the abdominal aorta of the macrophage marker CD68, and the inflammatory cell recruitment chemokines, monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha., Conclusions: Our findings suggest that increased COX-2 expression in smooth muscle cells of the abdominal aorta contributes to AAA formation in mice by enhancing inflammatory cell infiltration.

Objectives: Produced by vascular walls, adrenomedullin (AM) exerts antifibrotic actions in the process of cardiovascular remodeling. The purpose of this study was to examine the pathophysiological role of AM in the development of human abdominal aortic aneurysm (AAA)., Methods and Results: Immunohistochemical analyses revealed that vascular smooth muscle cells in the media were positive for AM in the early stage of atherosclerotic aorta. Intense immunoreactivity was observed in mast cells of the outer media and adventitia of AAA, and the number of mast cells was greater (p < 0.01) in AAA than in atherosclerotic aorta without any aneurysmal change. To determine the role of AM in mast cells, we examined cultured human mast cell leukemia line-1 (HMC-1) and fibroblasts isolated from AAA patients. Cultured HMC-1 cells were found to express preproAM gene and release AM peptide into the cultured media. When assessed by collagenase-sensitive [3H]proline incorporation and procollagen type I C-peptide secretion, collagen synthesis in co-culture of HMC-1 and the fibroblasts was reduced by 10(-6) mol/L synthetic AM, while conversely, it increased following blockade of the action of endogenous AM with 10 microg/mL anti-AM monoclonal antibody., Conclusion: The present study suggests an anti-fibrotic role for AM released from mast cells, providing new insight into the biological actions of mast cell-derived AM in the development of AAA.

Objective: Resistin belongs to a family of cysteine-rich secreted polypeptides that are mainly produced by monocytes/macrophages in humans. Recently, high concentrations of resistin were shown to induce vascular endothelial dysfunction and vascular smooth muscle cell proliferation. We examined if resistin was secreted from macrophages locally in atheromas and if it affected vascular cell function in human., Methods and Results: Immunohistochemical staining of human vessels showed that aortic aneurysms exhibited resistin-positive staining areas along macrophage infiltration, while normal arteries and veins did not. Co-localization of resistin and CD68 (a marker for macrophages) was observed in immunofluorescent double staining of aneurysms. Resistin mRNA was expressed much higher in cultured monocytes/macrophages than in human vascular smooth muscle cells (VSMCs) and human umbilical venous endothelial cells (HUVECs). This suggested that the resistin in aneurysms originates from macrophages within the vessels. To determine the effects of resistin on atherosclerosis, HUVECs and human VSMCs were incubated with resistin (10-100 ng/mL for 4 approximately 24 h). In HUVECs, plasminogen activator inhibitor (PAI)-1 release was assayed by ELISA, while the PAI-1 and endothelin (ET)-1 mRNA levels were analyzed by Northern blotting. Both were increased significantly with resistin treatment by factors of 1.3-2.5 (p<0.05). Migratory activity of VSMCs measured by scratched wound assay also increased significantly (1.6 times, p<0.05). In summary, macrophages infiltrating atherosclerotic aneurysms secrete resistin, and resistin affects endothelial function and VSMC migration., Conclusions: Resistin secreted from macrophages may contribute to atherogenesis by virtue of its effects on vascular endothelial cells and smooth muscle cells in humans.