Hexarelin attenuates abdominal aortic aneurysm formation by inhibiting SMC phenotype switch and inflammasome activation.

Hexarelin, a synthetic growth hormone-releasing peptide, is shown to be protective in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of hexarelin in abdominal aortic aneurysm (AAA) remains undefined. The present study determined the effect of hexarelin administration (200 µg/kg twice per day) in a mouse model of elastase-induced abdominal aortic aneurysm. Echocardiography and in situ pictures showed hexarelin decreased infrarenal aorta diameter. Histology staining showed elastin degradation was improved in hexarelin-treated group. Hexarelin rescued smooth muscle cell contractile phenotype with increased α-SMA and decreased MMP2. Furthermore, hexarelin inhibited inflammatory cell infiltration, NLRP3 inflammasome activation and IL-18 production. Particularly, hexarelin suppressed NF-κB signaling pathway which is a key initiator of inflammatory response. These results demonstrated that hexarelin attenuated AAA development by inhibiting SMC phenotype switch and NF-κB signaling mediated inflammatory response.

Reversal of elastase-induced abdominal aortic aneurysm following the delivery of nanoparticle-based pentagalloyl glucose (PGG) is associated with reduced inflammatory and immune markers.

OBJECTIVE: An Abdominal aortic aneurysm (AAA), a deadly disease in elderly population, is featured by expansion of aortic diameter, degradation and weakening of vasculature. Its common and significant characteristics are disarray and inflammation in vasculature. We tested the hypothesis that the reversal of abdominal aortic aneurysm by pentagalloyl glucose-loaded nanoparticles (PGG-NPs) therapy that targets degraded elastin suppresses inflammatory and immune markers to ameliorate the pathophysiology of the disease in advance stage aneurysm in a porcine pancreatic elastase (PPE)-induced mouse model of AAA. METHODS AND RESULTS: After induction of aneurysm in pathogen-free C57BL/6 male mice by applying PPE peri-adventitially to the abdominal aorta, once a week for two doses of intravenous injections of pentagalloyl glucose-loaded nanoparticles (PGG-NPs) conjugated with elastin targeted antibody were used to reverse the aneurysms. We showed that PGG-NPs therapy could suppress infiltration of macrophages, CD8 and CD4 subsets of T cells, matrix metalloproteinases (MMPs), inflammatory cytokines interferon (IFN-γ) and interleukin (IL)-6 at the local and systemic level. Moreover, such PGG-NPs therapy increases the induction of anti-inflammatory cytokines IL-13, IL-27 and IL-10 at the local and systemic level. The therapy also led to remodeling of elastic lamina at the aneurysm site. CONCLUSION: Nanoparticles-loaded pentagalloyl glucose therapy can be an effective treatment option against advanced stage aneurysms to reverse the disease by ameliorating inflammation and restoring arterial homeostasis.

Paeonol Ameliorates Abdominal Aortic Aneurysm Progression by the NF-κB Pathway.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease characterized by localized progressive dilatation. Currently, paeonol has been shown to possess anti-inflammatory and protective cardiovascular properties. Our study aimed to investigate the potential influences of paeonol on AAA progression. METHODS: Experimental AAAs were created in C57BL/6J mice by intra-aortic infusion of porcine pancreatic elastase, and then intragastrically administered paeonol (20 mg/kg/day) for 14 days. The effects of paeonol on experimental AAA were measured by ultrasound imaging, histopathology, and western blot analyses. RESULTS: Paeonol treatment limited the enlargement of the aneurysmal diameter and alleviated the depletion of elastic fibers and vascular smooth muscle cells (VSMCs). Furthermore, the infiltration of CD68+ macrophages and CD8+ lymphocytes was obviously attenuated after paeonol administration, along with mural neoangiogenesis. Western blot results showed that paeonol inhibited the expression of matrix metalloproteinase (MMP) and the NF-κB pathway activation. CONCLUSIONS: Paeonol might prevent experimental AAA progression by inhibiting the NF-κB pathway, which suggests that it is a potential drug for AAA.

Adventitial recruitment of Lyve-1- macrophages drives aortic aneurysm in an angiotensin-2-based murine model.

OBJECTIVE: Aortic macrophage accumulation is characteristic of the pathogenesis of abdominal aortic aneurysm (AAA) but the mechanisms of macrophage accumulation and their phenotype are poorly understood. Lymphatic vessel endothelial receptor-1 (Lyve-1+) resident aortic macrophages independently self-renew and are functionally distinct from monocyte-derived macrophages recruited during inflammation. We hypothesized that Lyve-1+ and Lyve-1- macrophages differentially contribute to aortic aneurysm. Approach and results: Angiotensin-2 and ß-aminopropionitrile (AT2/BAPN) were administered to induce AAA in C57BL/6J mice. Using immunohistochemistry (IHC), we demonstrated primarily adventitial accumulation of aortic macrophages, and in association with areas of elastin fragmentation and aortic dissection. Compared with controls, AAA was associated with a relative percent depletion of Lyve-1+ resident aortic macrophages and accumulation of Lyve-1- macrophages. Using CD45.1/CD45.2 parabiosis, we demonstrated aortic macrophage recruitment in AAA. Depletion of aortic macrophages in CCR2-/- mice was associated with reduced aortic dilatation indicating the functional role of recruitment from the bone marrow. Depletion of aortic macrophages using anti-macrophage colony-stimulating factor 1 receptor (MCSF1R)-neutralizing antibody (Ab) reduced the incidence of AAA. Conditional depletion of Lyve-1+ aortic macrophages was achieved by generating Lyve-1wt/cre Csf1rfl/fl mice. Selective depletion of Lyve-1+ aortic macrophages had no protective effects following AT2/BAPN administration and resulted in increased aortic dilatation in the suprarenal aorta. CONCLUSIONS: Aortic macrophage accumulation in AAA derives from adventitial recruitment of Lyve-1- macrophages, with relative percent depletion of Lyve-1+ macrophages. Selective targeting of macrophage subtypes represents a potential novel therapeutic avenue for the medical treatment of AAA.

Prevention of CaCl2-induced aortic inflammation and subsequent aneurysm formation by the CCL3-CCR5 axis.

Inflammatory mediators such as cytokines and chemokines are crucially involved in the development of abdominal aortic aneurysm (AAA). Here we report that CaCl2 application into abdominal aorta induces AAA with intra-aortic infiltration of macrophages as well as enhanced expression of chemokine (C-C motif) ligand 3 (CCL3) and MMP-9. Moreover, infiltrating macrophages express C-C chemokine receptor 5 (CCR5, a specific receptor for CCL3) and MMP-9. Both Ccl3-/- mice and Ccr5-/- but not Ccr1-/- mice exhibit exaggerated CaCl2-inducced AAA with augmented macrophage infiltration and MMP-9 expression. Similar observations are also obtained on an angiotensin II-induced AAA model. Immunoneutralization of CCL3 mimics the phenotypes observed in CaCl2-treated Ccl3-/- mice. On the contrary, CCL3 treatment attenuates CaCl2-induced AAA in both wild-type and Ccl3-/- mice. Consistently, we find that the CCL3-CCR5 axis suppresses PMA-induced enhancement of MMP-9 expression in macrophages. Thus, CCL3 can be effective to prevent the development of CaCl2-induced AAA by suppressing MMP-9 expression.

Ex vivo magnetic particle imaging of vascular inflammation in abdominal aortic aneurysm in a murine model.

Abdominal aortic aneurysms (AAAs) are currently one of the leading causes of death in developed countries. Inflammation is crucial in the disease progression, having a substantial impact on various determinants in AAAs development. Magnetic particle imaging (MPI) is an innovative imaging modality, enabling the highly sensitive detection of magnetic nanoparticles (MNPs), suitable as surrogate marker for molecular targeting of vascular inflammation. For this study, Apolipoprotein E-deficient-mice underwent surgical implantation of osmotic minipumps with constant Angiotensin II infusion. After 3 and 4 weeks respectively, in-vivo-magnetic resonance imaging (MRI), ex-vivo-MPI and ex-vivo-magnetic particle spectroscopy (MPS) were performed. The results were validated by histological analysis, immunohistology and laser ablation-inductively coupled plasma-mass spectrometry. MR-angiography enabled the visualization of aneurysmal development and dilatation in the experimental group. A close correlation (R = 0.87) with histological area assessment was measured. Ex-vivo-MPS revealed abundant iron deposits in AAA samples and ex-vivo histopathology measurements were in good agreement (R = 0.76). Ex-vivo-MPI and MPS results correlated greatly (R = 0.99). CD68-immunohistology stain and Perls'-Prussian-Blue-stain confirmed the colocalization of macrophages and MNPs. This study demonstrates the feasibility of ex-vivo-MPI for detecting inflammation in AAA. The quantitative ability for mapping MNPs establishes MPI as a promising tool for monitoring inflammatory progression in AAA in an experimental setting.

Interleukin-1 Beta-Mediated Sex Differences in Kawasaki Disease Vasculitis Development and Response to Treatment.

OBJECTIVE: Kawasaki disease (KD) is the leading cause of acute vasculitis and acquired heart disease in children in developed countries. Notably, KD is more prevalent in males than females. We previously established a key role for IL (interleukin)-1 signaling in KD pathogenesis, but whether this pathway underlies the sex-based difference in susceptibility is unknown. Approach and Results: The role of IL-1 signaling was investigated in the Lactobacillus casei cell wall extract-induced experimental mouse model of KD vasculitis. Five-week-old male and female mice were injected intraperitoneally with PBS, Lactobacillus caseicell wall extract, or a combination of Lactobacillus caseicell wall extract and the IL-1 receptor antagonist Anakinra. Aortitis, coronary arteritis inflammation score and abdominal aorta dilatation, and aneurysm development were assessed. mRNA-seq (messenger RNA sequencing) analysis was performed on abdominal aorta tissue. Publicly available human transcriptomics data from patients with KD was analyzed to identify sex differences and disease-associated genes. Male mice displayed enhanced aortitis and coronary arteritis as well as increased incidence and severity of abdominal aorta dilatation and aneurysm, recapitulating the increased incidence in males that is observed in human KD. Gene expression data from patients with KD and abdominal aorta tissue of Lactobacillus caseicell wall extract-injected mice showed enhanced Il1b expression and IL-1 signaling genes in males. Although the more severe IL-1ß-mediated disease phenotype observed in male mice was ameliorated by Anakinra treatment, the milder disease phenotype in female mice failed to respond. CONCLUSIONS: IL-1ß may play a central role in mediating sex-based differences in KD, with important implications for the use of anti-IL-1ß therapies to treat male and female patients with KD.

Ex vivo expansion of regulatory T cells from abdominal aortic aneurysm patients inhibits aneurysm in humanized murine model.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease. Studies of human aneurysm tissue demonstrate dense inflammatory cell infiltrates with CD4+ T cells predominating. Regulatory T cells (Tregs) play an important role in inhibiting pro-inflammatory T cell proliferation, therefore, limiting collateral tissue destruction. The aim of this study was to investigate whether ex vivo augmentation of human Tregs attenuates aneurysm formation in humanized murine model of AAA. METHODS: Circulating Treg population in AAA patients and age- and gender-matched controls were determined by real-time polymerase chain reaction and flow cytometry. To create humanized murine model of AAA, irradiated Rag1-deficient (Rag1-/-) mice, without mature T lymphocytes, at 7 weeks of age were given 5 × 106 of human CD4+ T cells intraperitoneally. Then the mice underwent CaCl2 aneurysm induction. Aortic diameters were measured before and at 6 weeks after aneurysm induction. Aortic tissue was collected for histology and protein extraction. Verhoeff-Van Gieson stain was used for staining elastic fiber. CD4+ T cells in the aortic tissue were detected by immunohistochemical staining. RESULTS: In human peripheral blood mononuclear cells, the proportion of Tregs are decreased in AAA patients compared with matched control patients with significant vascular disease. We first validated the role of Tregs in the CaCl2 model of AAA. To determine the role of human T cells in AAA formation, Rag1-/- mice, resistant to CaCl2-aneurysm induction, were transplanted with human CD4+ T cells. Human CD4+ T cells were able to drive aneurysm formation in Rag1-/- mice. We show that ex vivo augmentation of human Tregs by interleukin-2 resulted in decreased aneurysm progression. CONCLUSIONS: These data suggest that the ex vivo expansion of human Tregs may be a potential therapeutic strategy for inhibiting progression of AAA.

B cell-derived anti-beta 2 glycoprotein I antibody contributes to hyperhomocysteinaemia-aggravated abdominal aortic aneurysm.

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-ß2GPI) 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-ß2GPI 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-ß2GPI 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-ß2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

T Cells Are Dominant Population in Human Abdominal Aortic Aneurysms and Their Infiltration in the Perivascular Tissue Correlates With Disease Severity.

Abdominal Aortic Aneurysm (AAA) is a major cause of cardiovascular mortality. Adverse changes in vascular phenotype act in concert with chronic inflammation to promote AAA progression. Perivascular adipose tissue (PVAT) helps maintain vascular homeostasis but when inflamed and dysfunctional, can also promote vascular pathology. Previous studies suggested that PVAT may be an important site of vascular inflammation in AAA; however, a detailed assessment of leukocyte populations in human AAA, their anatomic location in the vessel wall and correlation to AAA size remain undefined. Accordingly, we performed in depth immunophenotyping of cells infiltrating the pathologically altered perivascular tissue (PVT) and vessel wall in AAA samples at the site of maximal dilatation (n = 51 patients). Flow cytometry revealed that T cells, rather than macrophages, are the major leukocyte subset in AAA and that their greatest accumulations occur in PVT. Both CD4+ and CD8+ T cell populations are highly activated in both compartments, with CD4+ T cells displaying the highest activation status within the AAA wall. Finally, we observed a positive relationship between T cell infiltration in PVT and AAA wall. Interestingly, only PVT T cell infiltration was strongly related to tertiles of AAA size. In summary, this study highlights an important role for PVT as a reservoir of T lymphocytes and potentially as a key site in modulating the underlying inflammation in AAA.

The effects of ultrasound-targeted microbubble destruction (UTMD) carrying IL-8 monoclonal antibody on the inflammatory responses and stability of atherosclerotic plaques.

This study investigated the value of using ultrasound-targeted microbubble destruction (UTMD) to deliver an IL-8 monoclonal antibody to inhibit the inflammatory response and increase plaque stability in a rabbit model of atherosclerosis (AS). An abdominal aortic atherosclerotic plaque model was established in sixty 4-week-old male New Zealand rabbits. The rabbits were fed a high-fat diet for 12 weeks. On the 12th week, the abdominal aorta was subjected to both balloon-induced mechanical injury and bovine serum albumin-induced immunological injury. After these injuries were established, the rabbits were fed a high-fat diet for 8 additional weeks. On the 20th week, the rabbits were divided into three groups: the pretreatment (PT) group, the control group, and the IL-8 group. The ultrasonic parameters and histological data associated with the plaques from the PT group were acquired on the 20th week after targeted contrast-enhanced ultrasonography (CEUS) was performed. The rabbits in the IL-8 and control groups received targeted CEUS and UTMD every 2 weeks. A targeted contrast agent carrying IL-8 monoclonal antibody was used for the IL-8 group, whereas normal saline was used for the control group. The rabbits in these two groups underwent the same procedure four times beginning during the 20th week. On the 26th week after UTMD, ultrasonic parameters and histological data were collected. The peak intensity (PI), microvessel density (MVD), and macrophage count of the PT group were significantly higher than those of both the control and IL-8 groups (P < 0.05). Additionally, these three parameters were higher in the control group than in the IL-8 group (P < 0.05). The two-dimensional (2D) ultrasonic parameters, including the maximum thickness of the plaque and the intima-media thickness (IMT), did not differ significantly among the three groups (P > 0.05). PI, MVD, and macrophage count were positively correlated with each other (r=0.564, r=0.6034, and r=0.536, respectively; P<0.05). UTMD-delivered IL-8 monoclonal antibodies alleviate inflammation within atherosclerotic plaques. UTMD is a novel and effective method for plaque stabilization.

Mfn2 inhibits chronic rejection of the rat abdominal aorta by regulating TGF-ß1 levels.

OBJECTIVES: The various forms of chronic rejection share a common histological appearance termed allograft arteriosclerosis. In the early stages thereof, apoptosis of vascular smooth muscle cells (VSMC) is obviously reduced, associated with vascular intimal thickening. High-level expression of the HSG/Mfn2 gene promotes apoptosis of rat VSMC. However, the role and mechanism of Mfn2 in inhibition of chronic allograft rejection have not been described. METHODS: In the present study, we transfected transplanted abdominal aortas of donor Lewis rats with an Mfn2-encoding or control lentivirus. And then We transplanted the donor aortas to the corresponding aortal positions in recipient rats. Transplanted aortas were collected on days 30, 60, and 90 and Masson stained to measure intimal thicknesses. Immunohistochemistry would be used to confirm TGF-ß1, Mfn2 and TGF-ß-R2 expression in different groups. RESULTS: Our results confirm that high-level expression of Mfn2 lowers the expression of TGF-ß1, reduces the intimal thickness of transplanted rat abdominal aorta, and retards the process of chronic rejection. CONCLUSION: Mfn2 influences TGF-ß/smad pathway and may function as potential chronic rejection inhibitor.

CTLA-4 Protects against Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice.

Vascular inflammation via T-cell-mediated immune responses has been shown to be critically involved in the pathogenesis of abdominal aortic aneurysm (AAA). T-cell coinhibitory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is known to act as a potent negative regulator of immune responses. However, the role of this molecule in the development of AAA remains completely unknown. We determined the effects of CTLA-4 overexpression on experimental AAA. We continuously infused CTLA-4 transgenic (CTLA-4-Tg)/apolipoprotein E-deficient (Apoe-/-) mice or control Apoe-/- mice fed a high-cholesterol diet with angiotensin II by implanting osmotic mini-pumps and evaluated the development of AAA. Ninety percent of angiotensin II-infused mice developed AAA, with 50% mortality because of aneurysm rupture. Overexpression of CTLA-4 significantly reduced the incidence (66%), mortality (26%), and diameter of AAA. These protective effects were associated with a decreased number of effector CD4+ T cells and the downregulated expression of costimulatory molecules CD80 and CD86, ligands for CTLA-4, on CD11c+ dendritic cells in lymphoid tissues. CTLA-4-Tg/Apoe-/- mice had reduced accumulation of macrophages and CD4+ T cells, leading to attenuated aortic inflammation, preserved vessel integrity, and decreased susceptibility to AAA and aortic rupture. Our findings suggest T-cell coinhibitory molecule CTLA-4 as a novel therapeutic target for AAA.

Direct CD137 costimulation of CD8 T cells promotes retention and innate-like function within nascent atherogenic foci.

Effector CD8 T cells infiltrate atherosclerotic lesions and are correlated with cardiovascular events, but the mechanisms regulating their recruitment and retention are not well understood. CD137 (4-1BB) is a costimulatory receptor induced on immune cells and expressed at sites of human atherosclerotic plaque. Genetic variants associated with decreased CD137 expression correlate with carotid-intimal thickness and its deficiency in animal models attenuates atherosclerosis. These effects have been attributed in part to endothelial responses to low and disturbed flow (LDF), but CD137 also generates robust effector CD8 T cells as a costimulatory signal. Thus, we asked whether CD8 T cell-specific CD137 stimulation contributes to their infiltration, retention, and IFNγ production in early atherogenesis. We tested this through adoptive transfer of CD8 T cells into recipient C57BL/6J mice that were then antigen primed and CD137 costimulated. We analyzed atherogenic LDF vessels in normolipidemic and PCSK9-mediated hyperlipidemic models and utilized a digestion protocol that allowed for lesional T-cell characterization via flow cytometry and in vitro stimulation. We found that CD137 activation, specifically of effector CD8 T cells, triggers their intimal infiltration into LDF vessels and promotes a persistent innate-like proinflammatory program. Residence of CD137+ effector CD8 T cells further promoted infiltration of endogenous CD8 T cells with IFNγ-producing potential, whereas CD137-deficient CD8 T cells exhibited impaired vessel infiltration, minimal IFNγ production, and reduced infiltration of endogenous CD8 T cells. Our studies thus provide novel insight into how CD137 costimulation of effector T cells, independent of plaque-antigen recognition, instigates their retention and promotes innate-like responses from immune infiltrates within atherogenic foci. NEW & NOTEWORTHY Our studies identify CD137 costimulation as a stimulus for effector CD8 T-cell infiltration and persistence within atherogenic foci, regardless of atherosclerotic-antigen recognition. These costimulated effector cells, which are generated in pathological states such as viral infection and autoimmunity, have innate-like proinflammatory programs in circulation and within the atherosclerotic microenvironment, providing mechanistic context for clinical correlations of cardiovascular morbidity with increased CD8 T-cell infiltration and markers of activation in the absence of established antigen specificity.

Establishment of Novel Murine Model showing Vascular Inflammation-derived Cognitive Dysfunction.

Inflammation is a critical feature of aging and its related diseases, including cardiovascular diseases. Recent epidemiological studies demonstrated that abdominal aortic aneurysm (AAA), an aging-related vascular pathological condition, is associated with cognitive decline. However, the underlying mechanism, especially the role of vascular inflammation, is largely unknown because of lack of an available animal model. In this study, we examined whether vascular inflammation affects synaptic and cognitive dysfunction, using an AAA mouse model. In young (3 months) and middle-aged (12 months) C57BL/6J mice, AAA was induced by angiotensin II infusion with calcium chloride application. After 4 weeks of induction, aortic diameter was significantly increased and excessive Mac3-positive inflammatory cells infiltrated the destroyed aorta in middle-aged mice. AAA-induced middle-aged mice further exhibited cognitive impairment. Neuronal loss was observed in the CA3 region of the hippocampus. IBA1/MHCII-double-positive microglia activation was also seen in the hippocampus, suggesting that vascular inflammation drives neuroinflammation and subsequent cognitive dysfunction. Furthermore, we found that senescence-accelerated mice prone 8 exhibited robust AAA formation and a marked decrease of cognitive and synaptic function in the hippocampus mediated by inflammation. In conclusion, this novel murine model convincingly suggested the occurrence of vascular inflammation-derived cognitive dysfunction.

Transforming growth factor ß neutralization finely tunes macrophage phenotype in elastase-induced abdominal aortic aneurysm and is associated with an increase of arginase 1 expression in the aorta.

OBJECTIVE: Macrophages play a critical role in the initiation and progression of abdominal aortic aneurysm (AAA) and are classically distinguished into M1 "proinflammatory" and M2 "anti-inflammatory" macrophages. Topical application of elastase associated with transforming growth factor ß (TGF-ß) systemic neutralization reproduces the main pathologic features of human AAA, offering a new model to investigate their role. The aim of this study was to investigate whether macrophages contribute to the expression of canonical M1/M2 markers in the aorta in the AAA model induced by elastase and systemic blockade of TGF-ß and whether blocking of TGF-ß activity affects macrophage phenotype and the expression of the M2 marker arginase 1 (ARG1). METHODS: C57Bl/6J male mice (6-8 weeks old) were randomly assigned to three experimental groups: mice that had local application of heat-inactivated elastase or elastase and mice that had elastase application and received injection of anti-TGF-ß (elastase + anti-TGF-ß group). Monocyte-macrophage depletion was achieved in the elastase + anti-TGF-ß group using liposome clodronate. Macrophage phenotype was characterized by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. Human infrarenal AAA tissues (n = 10) were obtained to analyze ARG1 expression. RESULTS: Analysis of gene expression in the infrarenal aortic wall revealed that after 14 days, no significant difference for the expression of CCL2, NOS2, and Ym1/2 was observed in the elastase group compared with the elastase + anti-TGF-ß group, whereas the expression of ARG1, interleukin (IL) 1ß, and IL-6 was significantly increased. Macrophage depletion in the elastase + anti-TGF-ß group led to a significant decrease of IL-1ß, IL-6, ARG1, and Ym1/2 gene expression. Immunofluorescent staining confirmed that TGF-ß neutralization significantly enhanced ARG1 protein expression in the aneurysmal tissue. Flow cytometry analysis revealed an increase of macrophages expressing ARG1 in the aorta of mice treated with elastase + anti-TGF-ß compared with the elastase group, and their proportion increased with aneurysmal dilation. In humans, ARG1 protein expression was increased in aneurysmal tissues compared with controls, and positive cells were mainly found in the adventitia. CONCLUSIONS: TGF-ß neutralization finely tunes macrophage phenotype in elastase-induced AAA and leads to an increase in ARG1 gene and protein expression in the aortic wall. Even if further studies are required to elucidate its role in AAA development, ARG1 could represent a new prognostic or therapeutic target in aneurysmal disease.

Necrotic cell debris induces a NF-κB-driven inflammasome response in vascular smooth muscle cells derived from abdominal aortic aneurysms (AAA-SMC).

Abdominal aortic aneurysm (AAA) is a multi-factorial progressive vascular disease with life-threatening complications. Increasing evidence suggests that smooth muscle cell (SMC) dysfunction and cell death contribute to dilatation and rupture of the aorta by inducing an inflammatory response. The exact mechanism of this response however, is incompletely understood. We here investigated in vitro the capacity of autologous necrotic cell debris (CD) to induce inflammasome components and inflammatory mediators in aortic SMC (AAA-SMC) isolated from patients with AAA undergoing surgical repair. AAA-SMCs were additionally primed with Interferon- γ (IFN-γ) before treatment with CD in order to mimic the proinflammatory status caused by higher IFN-γ concentrations that have been demonstrated in the wall of AAAs. Real-time RT-PCR revealed that CD significantly increased NLRP3 and IL1B mRNA expression in different SMC cultures within 6 h of exposure. Priming of the AAA-SMC with IFN-γ significantly increased expression of NLRP3, AIM2, IFI16 and CASP1 mRNAs, whereas IL1B mRNA was reduced. Additional exposure of IFN-γ-primed AAA-SMC to CD for 6-24 h, further augmented expression of AIM2, NLRP3, and Caspase-1 protein levels. Analysis of the SMC supernatants by ELISA revealed CD-induced release of the senescence-associated cytokines IL-6 and MCP-1 in native and IFN-γ-primed SMC, whereas no secretion of Interleukin-(IL) 1α and IL-1ß secretion were observed. Our results implicate a role of necrotic cell debris derived from dead neighboring cells in SMC dysfunction and in inflammatory response of AAA tissue.

Incense smoke exposure augments systemic oxidative stress, inflammation and endothelial dysfunction in male albino rats.

Incense smoke is reported to increase cardiovascular disease (CVD) risk in exposed individuals. However, the mechanism underlying the toxic effect of incense smoke on cardiovascular system is unclear. To test this, we chronically exposed male albino rats to two different types of Arabian incense smoke and studied their effects on oxidative stress, inflammation, and endothelial function. Rats exposed to either of incense smoke showed a significant increase in malondialdehyde (MDA) and a significant decline in superoxide dismutase (SOD) and reduced glutathione (GSH). Endothelial functional marker, nitric oxide (NO) was significantly decreased while endothelin-1 was significantly increased in rats exposed to both the incense types. Incense smoke exposure also led to a significant increase in chemokines and inflammatory mediators including monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage-colony stimulating factor (GM-CSF), regulated on activation normal T cell expressed and secreted (RANTES), interleukin-4 (IL-4), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α). Besides, incense smoke-exposed rats demonstrated a significant increase in the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecules-1 (VCAM-1), and E-selectin. Importantly, cessation of incense smoke exposure for 30 days led to a significant reversal in the levels of all the studied markers. Collectively, this study describes oxidative stress, endothelial dysfunction, and inflammation as possible underlying mechanisms in the toxic effects of incense smoke on increased CVD risk in exposed individuals. Findings also underscore that avoiding incense smoke exposure may have beneficial health effects.

Chronic Periaortitis: an Update.

PURPOSE OF REVIEW: We aim to review traditional concepts and recent developments on the nosology, pathophysiology, clinical phenotypes and treatment of chronic periaortitis (CP). RECENT FINDINGS: CP is a rare disorder hallmarked by a periaortic fibro-inflammatory tissue. It can present as an isolated disease, but it can also be associated with other autoimmune and fibro-inflammatory lesions (e.g., fibrosing mediastinitis, sclerosing pancreato-cholangitis) that are part of the spectrum of IgG4-related disease. In a subgroup of patients, it also involves the thoracic aorta (so-called "diffuse periaortitis"), which supports the notion of an inflammatory disorder of large arteries. The pathogenesis of CP is multifactorial: recent studies have elucidated the predisposing role of immunogenetic variants and exposures to environmental agents such as smoking and asbestos. CP is a rare immune-mediated disease that affects the abdominal aorta and the iliac arteries and, in some cases, the thoracic aorta. It may overlap with manifestations of IgG4-related disease, and its treatment comprises glucocorticoids, conventional and biological immunosuppressive agents.

Dietary DNA Attenuates the Degradation of Elastin Fibers in the Aortic Wall in Nicotine-Administrated Mice.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by chronic inflammation in the infrarenal aorta. Epidemiologic data have clearly linked tobacco smoking to aneurysm formation and a faster rate of expansion. It suggested that nicotine, one of the main ingredients of tobacco, has been suggested to be associated with AAA development and rupture. In the condition where no established drugs are available; therefore, an effective approach to prevent the vascular damage from nicotine consumption may be the use of dietary functional food factors. However, little is known about the relationship between dietary components and AAA. In this study, we estimated the effect of dietary deoxyribonucleic acid (DNA) on the vascular wall. After habituation for 5 d, the mice were divided into four groups: control diet and distilled water group (C), DNA-Na diet and distilled water group (DNA), control diet and 0.5 mg/mL nicotine solution group (C-Nic), DNA-Na diet, and 0.5 mg/mL nicotine solution group (DNA-Nic). The dietary DNA attenuated the degradation of elastin fibers induced by nicotine administration. The areas stained positive for MMP-2 in the DNA-Nic group were significantly suppressed compared to C-Nic mice. These data suggest that the dietary DNA may prevent the weakening of the aortic wall via inhibition of the MMP-2-dependent pathway. In conclusion, we have revealed the protective effect of dietary DNA on the vascular pathology of nicotine-administrated mice. A nucleic acid-rich diet might be useful for people who consume nicotine via smoking, chewing tobacco, or nicotine patches.