Glutamine Protects against Mouse Abdominal Aortic Aneurysm through Modulating VSMC Apoptosis and M1 Macrophage Activation.

Glutamine (Gln), known as the most abundant free amino acid, is widely spread in human body. In this study, we demonstrated the protective effects of glutamine against mouse abdominal aortic aneurysm (AAA) induced by both angiotensin II (AngII) and calcium phosphate (Ca3(PO4)2) in vivo, which was characterized with lower incidence of mouse AAA. Moreover, histomorphological staining visually presented more intact elastic fiber and less collagen deposition in abdominal aortas of mice treated by glutamine. Further, we found glutamine inhibited the excessive production of reactive oxide species (ROS), activity of matrix metalloproteinase (MMP), M1 macrophage activation, and apoptosis of vascular smooth muscle cells (VSMCs) in suprarenal abdominal aortas of mice, what's more, the high expressions of MMP-2 protein, MMP-9 protein, pro-apoptotic proteins, and IL-6 as well as TNF-α in protein and mRNA levels in cells treated by AngII were down-regulated by glutamine. Collectively, these results revealed that glutamine protected against mouse AAA through inhibiting apoptosis of VSMCs, M1 macrophage activation, oxidative stress, and extracellular matrix degradation.

Protective effects of safranal on kidney tissue in a rat model of distant ischemia-reperfusion injury with infrarenal aortic occlusion.

Background/aim: Ischemia-reperfusion (IR) injury to a part of the body can cause damage to distant organs such as the kidney and heart. This study investigated the protective effects of safranal against IR-induced renal injury. Materials and methods: Used in this study were 24 Wistar Albino male rats, which were divided into 3 equal and randomised groups. The sham group underwent laparotomy only. In the IR group, the infrarenal aorta was clamped for 1 h, and then reperfused for 2 h. In the IR-safranal group, safranal was administered 30 min before the procedure and IR injury was induced in the same way as in the IR group. After the procedure, blood and tissue samples were collected from the rats for biochemical and histopathological analyses. Antioxidant capacity and proinflammatory cytokine analyses were performed on the blood samples. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to determine the number of cells undergoing apoptosis in the kidney tissue. Results: The estimated glomerular filtration rate, an indicator of renal function, was lower in the IR group (p1 = 0.024 vs. p3 = 0.041, respectively) compared to the other groups, while creatinine levels were higher in the IR group compared to the other groups (p1 = 0.032 vs. p2 = 0.044, respectively). The blood urea nitrogen level was higher in the IR group than in the other groups (p1 = 0.001vs p2 = 0.035, respectively). The total antioxidant and total oxidant status, indicating tissue oxidative stress, did not differ between groups (p = 0.914 vs. p = 0.184, respectively). Among the proinflammatory cytokines, the interleukin-1ß (IL-1ß) and IL-6 levels were significantly higher in the IR group (p = 0.034 vs. p = 0.001, respectively), but the tumour necrosis factor-α (p = 0.19), and interferon-γ (p = 0.311) levels did not differ between groups. Histopathological examination showed significantly less damage to glomerular and tubular cells in the IR-safranal group (p < 0.001). The number of TUNEL-positive cells was higher in the IR group compared to the other groups (p < 0.001). Conclusion: Safranal may have protective effects against kidney damage caused by distant ischemia-reperfusion injury.

The Beneficial Impact of Zinc Supplementation on the Vascular Tissue of the Abdominal Aorta under Repeated Intoxication with Cadmium: A Study in an In Vivo Experimental Model.

In an in vivo rat model of human exposure to cadmium (Cd; 5 and 50 mg/L, 6 months), whether the supplementation with zinc (Zn; 30 and 60 mg/L, increasing its daily intake by 79% and 151%, respectively) protects against the unfavourable impact of this xenobiotic on the vascular tissue of the abdominal aorta was investigated. The treatment with Cd led to oxidative stress and increased the concentrations of pro-inflammatory interleukin 1ß (IL-1ß), total cholesterol (TC), triglycerides (TG), and endothelial nitric oxide synthase (eNOS) and decreased the concentration of anti-inflammatory interleukin 10 (IL-10) in the vascular tissue. Cd decreased the expression of intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and L-selectin on the endothelial cells. The administration of Zn prevented most of the Cd-induced alterations or at least weakened them (except for the expression of adhesive molecules). In conclusion, Zn supplementation may protect from the toxic impact of Cd on the blood vessels and thus exert a beneficial influence on the cardiovascular system. The increase in the intake of Zn by 79% may be sufficient to provide this protection and the effect is related to the antioxidative, anti-inflammatory, and antiatherogenic properties of this essential element.

Cilengitide Inhibits Neovascularization in a Rabbit Abdominal Aortic Plaque Model by Impairing the VEGF Signaling.

BACKGROUND: Cilengitide is a selective α v ß 3 and α v ß 5 integrin inhibitor. We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: For the in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism. RESULTS: CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, ß 3, and ß 5, but not α V. Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs. Conclusions. This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs.

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.

Allosteric activation of PP2A inhibits experimental abdominal aortic aneurysm.

Although extremely important, the molecular mechanisms that govern aortic aneurysm (AA) formation and progression are still poorly understood. This deficit represents a critical roadblock toward the development of effective pharmaceutical therapies for the treatment of AA. While dysregulation of protein phosphatase 2A (PP2A) is thought to play a role in cardiovascular disease, its role in aortic aneurysm is unknown. The objective of the present study is to test the hypothesis that PP2A regulates abdominal aortic aneurysm (AAA) progression in a murine model. In an angiotensin II-induced AAA murine model, the PP2A inhibitor, LB-100, markedly accelerated AAA progression as demonstrated by increased abdominal aortic dilation and mortality. AAA progression was associated with elevated inflammation and extracellular matrix fragmentation, concomitant with increases in both metalloproteinase activity and reactive oxygen species production. Conversely, administration of a novel class of small molecule activators of PP2A (SMAPs) resulted in an antithetical effect. SMAPs effectively reduced AAA incidence along with the corresponding pathologies that were increased with LB-100 treatment. Mechanistically, modulation of PP2A activities in vivo functioned in part via alteration of the ERK1/2 and NFκB signaling pathways, known regulators of AAA progression. These studies, for the first time, demonstrate a role of PP2A in AAA etiology and demonstrate that PP2A activation may represent a novel strategy for the treatment of abdominal aortic aneurysms.

Similar distribution of orally administered eicosapentaenoic acid and M2 macrophage marker in the hypoperfusion-induced abdominal aortic aneurysm wall.

Abdominal aortic aneurysm (AAA) is an aortic disease in which the aortic diameter is ≥3.0 cm; if left untreated, the aortic wall continues to weaken, resulting in progressive dilatation. Effective therapeutic drugs for AAA patients have not been discovered. Eicosapentaenoic acid (EPA) reportedly attenuates the development of AAA in experimental AAA animal models. However, the underlying mechanism of action is still not totally clear. To understand the mechanism, we visualized the distribution of EPA-containing phosphatidylcholine (PC) in the AAA wall by matrix-assisted laser desorption ionization-mass spectrometry imaging. EPA-containing PC was characteristically distributed in the AAA wall, and the positive area for the M2 macrophage marker was significantly higher in the region where EPA-containing PC was highly detected (region 2) than in the region where EPA-containing PC was poorly detected (region 1). The M1 macrophage marker levels were not different between regions 1 and 2. A comparative observation showed a similar distribution of the M2 macrophage marker and EPA-containing PC. These data suggest the preferential incorporation of EPA into M2 macrophages. Positive areas for matrix metalloproteinase 2 and malondialdehyde in region 2 were significantly lower than those in region 1. The reported suppressive effect of EPA on the development of AAA may be partly attributed to the increased anti-inflammatory property of M2 macrophages.

Kallikrein-1 Blockade Inhibits Aortic Expansion in a Mouse Model and Reduces Prostaglandin E2 Secretion From Human Aortic Aneurysm Explants.

Background Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. The kinin B2 receptor agonist, bradykinin, has been implicated in AAA pathogenesis through promoting inflammation. Bradykinin is generated from high- and low-molecular-weight kininogen by the serine protease kallikrein-1. The aims of this study were first to examine the effect of neutralizing kallikrein-1 on AAA development in a mouse model and second to test how blocking kallikrein-1 affected cyclooxygenase-2 and prostaglandin E2 in human AAA explants. Methods and Results Neutralization of kallikrein-1 in apolipoprotein E-deficient (ApoE-/-) mice via administration of a blocking antibody inhibited suprarenal aorta expansion in response to angiotensin (Ang) II infusion. Kallikrein-1 neutralization decreased suprarenal aorta concentrations of bradykinin and prostaglandin E2 and reduced cyclooxygenase-2 activity. Kallikrein-1 neutralization also decreased protein kinase B and extracellular signal-regulated kinase 1/2 phosphorylation and reduced levels of active matrix metalloproteinase 2 and matrix metalloproteinase 9. Kallikrein-1 blocking antibody reduced levels of cyclooxygenase-2 and secretion of prostaglandin E2 and active matrix metalloproteinase 2 and matrix metalloproteinase 9 from human AAA explants and vascular smooth muscle cells exposed to activated neutrophils. Conclusions These findings suggest that kallikrein-1 neutralization could be a treatment target for AAA.

Metformin Inhibits Abdominal Aortic Aneurysm Formation through the Activation of the AMPK/mTOR Signaling Pathway.

BACKGROUND AND OBJECTIVE: Epidemiological evidence suggests that the antidiabetic drug metformin (MET) can also inhibit abdominal aortic aneurysm (AAA) formation. However, the underlying protective mechanism remains unknown. It has been reported that phosphorylated AMP-activated protein kinase (AMPK) levels are significantly lower in AAA tissues than control aortic tissues. AMPK activation can inhibit the downstream signaling molecule called mechanistic target of rapamycin (mTOR), which has also been reported be upregulated in thoracic aneurysms. Thus, blocking mTOR signaling could attenuate AAA progression. MET is a known agonist of AMPK. Therefore, in this study, we investigated if MET could inhibit formation of AAA by activating the AMPK/mTOR signaling pathway. MATERIALS AND METHODS: The AAA animal model was induced by intraluminal porcine pancreatic elastase (PPE) perfusion in male Sprague Dawley rats. The rats were treated with MET or compound C (C.C), which is an AMPK inhibitor. AAA formation was monitored by serial ultrasound. Aortas were collected 4 weeks after surgery and subjected to immunohistochemistry, Western blot, and transmission electron microscopy analyses. RESULTS: MET treatment dramatically inhibited the formation of AAA 4 weeks after PPE perfusion. MET reduced the aortic diameter, downregulated both macrophage infiltration and matrix metalloproteinase expression, decreased neovascularization, and preserved the contractile phenotype of the aortic vascular smooth muscle cells. Furthermore, we detected an increase in autophagy after MET treatment. All of these effects were reversed by the AMPK inhibitor C.C. CONCLUSION: This study demonstrated that MET activates AMPK and suppresses AAA formation. Our study provides a novel mechanism for MET and suggests that MET could be potentially used as a therapeutic candidate for preventing AAA.

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 + sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions.

Ghrelin inhibited pressure overload-induced cardiac hypertrophy by promoting autophagy via CaMKK/AMPK signaling pathway.

Ghrelin, a novel gut hormone, has been shown to exert protective effects on cardiac dysfunction and remodeling. However, the underlying mechanisms of its protective effects remain unclear. Here, we investigated the effects of ghrelin on cardiac hypertrophy and explored the mechanisms involved. Ghrelin (30 µg.kg-1. day-1) was systemically administered to rats with cardiac hypertrophy induced by abdominal aortic constriction (AAC) by a mini-osmotic pump the next day after surgery continuously for 4 weeks. The AAC treated rats without ghrelin infusion showed decreased ghrelin content and expression of its receptors in the hearts. Exogenous ghrelin greatly attenuated cardiac hypertrophy as shown by heart weight to tibial length (HW/TL), hemodynamics, echocardiography, histological analyses, and expression of hypertrophic markers induced by AAC. This corresponded with decreased cardiac fibrosis and inflammation in the hearts of AAC rats treated with ghrelin. Moreover, ghrelin significantly increased the myocardial expression of autophagy markers, which was further confirmed in cultured cardiomyocytes. Concurrently, cardiomyocyte apoptosis in vivo and in vitro was ameliorated by ghrelin, which was reversed by inhibition of autophagy. The enhancement of autophagy and inhibition of apoptosis by ghrelin were eliminated on pretreatment with compound C, an AMP-activated protein kinase (AMPK) inhibitor. Furthermore, inhibition of Ca2+/Calmodulin-dependent protein kinase kinase (CaMKK), an upstream kinase of AMPK, made ghrelin fail to activate AMPK and simultaneously reversed ghrelin's promotion of autophagy. In conclusion, ghrelin could exert its cardioprotective effects on cardiac hypertrophy by promoting autophagy, possibly via CaMKK/AMPK signaling pathway.

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.

A Randomised Controlled Trial Assessing the Effects of Peri-operative Fenofibrate Administration on Abdominal Aortic Aneurysm Pathology: Outcomes From the FAME Trial.

OBJECTIVE: Experimental studies suggest that fenofibrate prevents abdominal aortic aneurysm (AAA) development by lowering aortic osteopontin (OPN) concentration and reducing the number of macrophages infiltrating the aortic wall. The current study examined the effects of a short course of fenofibrate on AAA pathology in people with large AAAs awaiting aortic repair. METHODS: This randomised double blind parallel trial included male and female participants aged ≥ 60 years who had an asymptomatic AAA measuring ≥ 50 mm and were scheduled to undergo open AAA repair. Participants were allocated to fenofibrate (145 mg/day) or matching placebo for at least two weeks before elective AAA repair. Blood samples were collected at recruitment and immediately prior to surgery. AAA biopsies were obtained during aortic surgery. The primary outcomes were (1) AAA OPN concentration; (2) serum OPN concentration; and (3) number of AAA macrophages. Exploratory outcomes included circulating and aortic concentrations of other proteins previously associated with AAA. Outcomes assessed at a single time point were compared using logistic regression. Longitudinal outcomes were compared using linear mixed effects models. RESULTS: Forty-three participants were randomised. After three withdrawals, 40 were followed until the time of surgery (21 allocated fenofibrate and 19 allocated placebo). As expected, serum triglycerides reduced significantly from recruitment to the time of surgery in participants allocated fenofibrate. No differences in any of the primary and exploratory outcomes were observed between groups. CONCLUSION: A short course of 145 mg of fenofibrate/day did not lower concentrations of OPN or aortic macrophage density in people with large AAAs.

Inhibition of Phosphatidylinositol 3-Kinase γ by IPI-549 Attenuates Abdominal Aortic Aneurysm Formation in Mice.

OBJECTIVE: The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway plays a pivotal role in abdominal aortic aneurysm (AAA). However, systemic inhibition of this pathway causes serious side effects, thus limiting the clinical use of pan-PI3K inhibitors. In this study, it was hypothesised that the γ subunit of PI3K plays an important role in the PI3K/AKT signalling pathway during AAA, and that specifically targeting PI3Kγ may prevent this process. METHODS: Aortic specimens were collected from AAA patients and organ donors. Furthermore, a classical AAA model in male C57BL/6 mice was created via an intra-aortic porcine pancreatic elastase (PPE) infusion and aortas were collected. A specific PI3Kγ inhibitor, IPI-549, was administered to mice orally. The protein expression level of PI3Kγ was examined by immunohistochemistry and western blotting. The aortic leukocytes were examined by immunohistochemistry and flow cytometry. RESULTS: PI3Kγ protein levels were elevated in the aortas of AAA patients and PPE infused mice. Three color immunofluorescence staining revealed the predominant area of PI3Kγ by T cells and macrophages in aneurysmal aortas. IPI-549 treatment significantly prevented AAA formation in mice. Aortic macrophages, T cells and neo-angiogenesis were significantly reduced in mice treated with IPI-549 compared with vehicle treated PPE infused mice. Flow cytometry analysis also revealed that CD45+ leukocytes and CD45+ F4/80+ macrophages in IPI-549 treated mouse aortas decreased dramatically. Additionally, IPI-549 treatment inhibited the phosphorylation of AKT in experimental aneurysmal lesions. CONCLUSION: Specific inhibition of PI3Kγ limits AAA formation. Targeting PI3Kγ prevents inflammatory cell infiltration through inhibition of AKT phosphorylation in AAA.

Targeting the NLRP3 Inflammasome With Inhibitor MCC950 Prevents Aortic Aneurysms and Dissections in Mice.

Background Aortic aneurysms and dissections are highly lethal diseases for which an effective treatment strategy is critically needed to prevent disease progression. The nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3)-caspase-1 inflammasome cascade was recently shown to play an important role in aortic destruction and disease development. In this study, we tested the effects of MCC950, a potent, selective NLRP3 inhibitor, on preventing aortic destruction and aortic aneurysm and dissection formation. Methods and Results In a model of sporadic aortic aneurysm and dissection induced by challenging wild-type mice with a high-fat, high-cholesterol diet and angiotensin II infusion, MCC950 treatment significantly inhibited challenge-induced aortic dilatation, dissection, and rupture in different thoracic and abdominal aortic segments in both male and female mice. Aortic disease reduction by MCC950 was associated with the prevention of NLRP3-caspase-1 upregulation, smooth muscle cell contractile protein degradation, aortic cell death, and extracellular matrix destruction. Further investigation revealed that preventing matrix metallopeptidase 9 (MMP-9) expression and activation in macrophages is an important mechanism underlying MCC950's protective effect. We found that caspase-1 directly activated MMP-9 by cleaving its N-terminal inhibitory domain. Moreover, the genetic knockdown of Nlrp3 or Casp-1 in mice or treatment of mice with MCC950 diminished the challenge-induced N-terminal cleavage of MMP-9, MMP-9 activation, and aortic destruction. Conclusions Our findings suggest that the NLRP3-caspase-1 inflammasome directly activates MMP-9. Targeting the inflammasome with MCC950 is a promising approach for preventing aortic destruction and aortic aneurysm and dissection development.

Factor XII blockade inhibits aortic dilatation in angiotensin II-infused apolipoprotein E-deficient mice.

Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Chronic inflammation and excessive matrix remodelling are considered important in AAA pathogenesis. Kinins are bioactive peptides important in regulating inflammation. Stimulation of the kinin B2 receptor has been previously reported to promote AAA development and rupture in a mouse model. The endogenous B2 receptor agonist, bradykinin, is generated from the kallikrein-kinin system following activation of plasma kallikrein by Factor XII (FXII). In the current study whole-body FXII deletion, or neutralisation of activated FXII (FXIIa), inhibited expansion of the suprarenal aorta (SRA) of apolipoprotein E-deficient mice in response to angiotensin II (AngII) infusion. FXII deficiency or FXIIa neutralisation led to decreased aortic tumor necrosis factor-α-converting enzyme (TACE/a disintegrin and metalloproteinase-17 (aka tumor necrosis factor-α-converting enzyme) (ADAM-17)) activity, plasma kallikrein concentration, and epithelial growth factor receptor (EGFR) phosphorylation compared with controls. FXII deficiency or neutralisation also reduced Akt1 and Erk1/2 phosphorylation and decreased expression and levels of active matrix metalloproteinase (Mmp)-2 and Mmp-9. The findings suggest that FXII, kallikrein, ADAM-17, and EGFR are important molecular mediators by which AngII induces aneurysm in apolipoprotein E-deficient mice. This could be a novel pathway to target in the design of drugs to limit AAA progression.

Factor Xa Inhibition by Rivaroxaban Modified Mitochondrial-Associated Proteins in Human Abdominal Aortic Aneurysms.

BACKGROUND: The presence of intraluminal thrombus and mitochondrial dysfunction in human abdominal aortic aneurysms (AAAs) have been associated with aneurysmal growth and rupture. The objective of the study was to study if endogenous factor Xa (FXa) may modulate mitochondrial functionality and expression of proteins associated with mitophagy in human AAAs. METHODS: AAA sites with intraluminal thrombus were obtained from 6 patients undergoing elective AAA surgery repair. Control samples were collected from 6 organ donors. The effect of FXa was analyzed by in vitro incubation of AAA with 50 nmol/L rivaroxaban, an oral FXa inhibitor. RESULTS: The enzymatic activities of citrate synthase, a biomarker of mitochondrial density, and cytochrome C oxidase, a biomarker of mitochondrial respiratory chain functionality, were significantly reduced in the AAA sites with respect to the healthy aorta (citrate synthase activity in µU/min/µg protein: control: 3.51 ± 0.22 vs. AAA: 0.37 ± 0.15.; P < 0.01; cytochrome C oxidase activity in µOD/min/µg protein: control: 8.05 ± 1.57 vs. AAA: 3.29 ± 1.05; P < 0.05). The addition of rivaroxaban to AAA reverted the activity of both citrate synthase and cytochrome C oxidase to similar values to control. Mitochondrial Drp-1 expression was higher in AAA sites than in either control aortas or rivaroxaban-incubated AAA sites. Cytosolic content of Drp-1 phosphorylated at Ser637, mitochondrial Parkin, and mitochondrial PINK1-Parkin interaction were significantly reduced in the AAA sites with respect to control aortas. For all these parameters, rivaroxaban-incubated AAA showed similar values compared with control aortas. CONCLUSIONS: In human AAA, rivaroxaban improved mitochondrial functionality that was associated with changes in proteins related to mitophagy. Its opens possible new effects of endogenous FXa on the mitochondria in the human AAA site.

Different Drugs Effect on Mesenchymal Stem Cells Isolated From Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a progressive dilation of the aortic wall, determined by the unbalanced activity of matrix metalloproteinase (MMPs). In vitro and in vivo studies support the pivotal role of MMP-9 to AAA pathogenesis. In our experience, we elucidated the expression of MMP-9 in an ex vivo model of human mesenchymal stem cells isolated from AAA specimen (AAA-MSCs). Thus, MMP-9 inhibition could be an attractive therapeutic strategy for inhibiting AAA degeneration and rupture. Our study was aimed at testing the effect of 3 different drugs (pioglitazone, doxycycline, simvastatin) on MMP-9 and peroxisome proliferator-activated receptor (PPAR)-γ expression in AAA-MSCs. METHODS: Aneurysmal aortic wall segments were taken from AAA patients after the open surgical treatment. MSCs were isolated from AAA (n = 20) tissues through enzymatic digestion. AAA-MSCs were exposed to different doses of pioglitazone (5-10-25 µM), doxycycline (10-25 µM), and simvastatin (10 µM) for 24 h. The effect of each drug was evaluated in terms of cell survival, by crystal violet stain. MMP-9 and PPAR-γ mRNA were analyzed using real-time PCR. RESULTS: AAA-MSCs were not affected by the exposure to the selected drugs, as shown by the analysis of cell viability. Interestingly, MMP-9 mRNA resulted significantly decreased after each treatment, recording a downregulation of 50% in presence of pioglitazone, 90% with doxycycline, and 40% with exposed to simvastatin, in comparison to untreated cells. We further analyzed the expression of PPAR-γ, target of pioglitazone, observing an upregulation in exposed AAA-MSCs to controls. CONCLUSIONS: Our data support the potential therapeutic effect of pioglitazone, doxycycline, and simvastatin on AAA by reducing the MMP-9 expression in a patient-specific model (AAA-MSCs). In addition, pioglitazone drives the increase of PPAR-G, another promising target for AAA therapy. Further studies are necessary to elucidate the mechanism driving this inhibitory pathway, which can reduces the mortality risk associated with AAA rupture.

Melatonin inhibits macrophage infiltration and promotes plaque stabilization by upregulating anti-inflammatory HGF/c-Met system in the atherosclerotic rabbit: USPIO-enhanced MRI assessment.

Macrophage plays critical roles in the pathogenesis of atherosclerosis (AS), and is an attractive target for detecting and treating vulnerable plaque. Our previous study showed that melatonin (MLT) ameliorated AS by suppressing the pro-inflammatory Toll-like receptor 4/nuclear factor kappa B system in high-fat-fed rabbit. However, it is unknown whether the anti-atherosclerotic properties of MLT are associated with the upregulation of anti-inflammatory hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) system. In present study, we examined whether MLT could inhibit macrophage infiltration and promote plaque stabilization by upregulating HGF/c-Met system with ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI) assessment in AS rabbit. Rabbits in this study were randomly divided into three groups and treated with a standard diet, high-fat diet, and high-fat diet plus 10 mg/kg/day MLT for 12 weeks, respectively. MLT treatment significantly reversed spotty signal void in 3D-TOF MRI, standard signal intensity reduction in T2WI MRI and aortic luminal area reduction in 2D-TOF MRI of the atherosclerotic abdominal aorta 72 h after USPIO injection. It also decreased serum interleukin-6 (IL-6), intima/media thickness ratio of the abdominal aorta, CD68 and iron-positive areas in the aortic intima, and increased serum IL-10, HGF and c-Met protein expression and the accumulation of vascular smooth muscle cell and collagen fiber in the aortic intima of AS rabbit. Our data demonstrated that MLT significantly decreased plaque macrophage infiltration and promoted plaque stability in AS rabbit assessed by USPIO-enhanced MRI. Remarkably, it was very first revealed that upregulation of anti-inflammatory HGF/c-Met system might contribute to the atheroprotective mechanisms of MLT.