From the Literature

Abdominal aortic aneurysm (AAA), characterized by a localized dilation of the abdominal aorta, is a life-threatening vascular pathology. Because of the current lack of effective treatment for AAA rupture, prevention is of prime importance for AAA management. Calorie restriction (CR) is a nonpharmacological intervention that delays the aging process and provides various health benefits. However, whether CR prevents AAA formation remains untested. In this study, we subjected Apoe-/- mice to 12 wk of CR and then examined the incidence of angiotensin II (AngII)-induced AAA formation. We found that CR markedly reduced the incidence of AAA formation and attenuated aortic elastin degradation in Apoe-/- mice. The expression and activity of Sirtuin 1 (SIRT1), a key metabolism/energy sensor, were up-regulated in vascular smooth muscle cells (VSMCs) upon CR. Importantly, the specific ablation of SIRT1 in smooth muscle cells abolished the preventive effect of CR on AAA formation in Apoe-/- mice. Mechanistically, VSMC-SIRT1-dependent deacetylation of histone H3 lysine 9 on the matrix metallopeptidase 2 (Mmp2) promoter was required for CR-mediated suppression of AngII-induced MMP2 expression. Together, our findings suggest that CR may be an effective intervention that protects against AAA formation.

A number of epidemiological studies have suggested an association of hyperhomocysteinemia (HHcy) and abdominal aortic aneurysm (AAA), but discrepancies exist. In addition, we lack direct evidence supporting a causal role. We determined the association and contribution of HHcy to AAA formation. We first performed a meta-analysis of studies involving 1489 subjects and found a strong association of HHcy and AAA (odds ratio, 7.39). Next, we used angiotensin II-infused male apolipoprotein E-deficient mice and tested whether HHcy contributes to AAA pathogenesis. Homocysteine (Hcy) supplement (1.8 g/L) in drinking water resulted in mild HHcy. Intriguingly, HHcy greatly increased the incidence of angiotensin II-induced AAA and aortic dissection in apolipoprotein E-deficient mice (vehicle versus Hcy: 50% versus 100%; P<0.05). Histology indicated HHcy markedly exaggerated aortic adventitial inflammation. Increased levels of proinflammatory interleukin-6 and monocyte chemoattractant protein-1 were preferentially colocalized within adventitial fibroblasts in HHcy plus angiotensin II mice, which suggested the importance of adventitial fibroblasts activation in Hcy-aggravated AAA. Hcy sequentially stimulated adventitial fibroblasts transformation into myofibroblasts, secretion of interleukin-6 and monocyte chemoattractant protein-1, and consequent recruitment of monocytes/macrophages to adventitial fibroblasts, which was abolished by the NADPH oxidase inhibitor diphenyliodonium. NADPH oxidase 4, but not other homologs of NADPH oxidase, was significantly upregulated by Hcy in adventitial fibroblasts, whereas NADPH oxidase 4 small interfering RNA silencing diminished Hcy-induced adventitial fibroblasts activation. Finally, folic acid supplement (0.071 μg/g per day) markedly reduced HHcy-aggravated angiotensin II-induced AAA formation in apolipoprotein E-deficient mice. HHcy may aggravate AAA formation at least partially via activating adventitial fibroblast NADPH oxidase 4.

Rupture of abdominal aortic aneurysm (AAA) is a devastating event that can be prevented by inhibiting the growth of small aneurysms. Therapeutic strategies targeting certain events that promote the development of AAA must be developed, in order to alter the course of AAA. Chronic inflammation of the aortic mural is a major characteristic of AAA and is related to AAA formation, development and rupture. Daphnetin (DAP) is a coumarin derivative with anti-inflammatory properties that is extracted from Daphne odora var. However, the effect of DAP on AAA development remains unclear. The present study investigated the effect of DAP on the formation and development of experimental AAAs and its potential underlying mechanisms. A mice AAA model was established by intra-aortic infusion of porcine pancreatic elastase (PPE), and mice were intraperitoneally injected with DAP immediately after PPE infusion. The maximum diameter of the abdominal aorta was measured by ultrasound system, and aortic mural changes were investigated by Elastica van Gieson (EVG) staining and immunohistochemical staining. The results demonstrated that DAP significantly suppressed PPE-induced AAA formation and attenuated the depletion of aortic medial elastin and smooth muscle cells in the media of the aorta. Furthermore, the density of mural macrophages, T cells and B cells were significantly attenuated in DAP-treated AAA mice. In addition, treatment with DAP resulted in a significant reduction in mural neovessels. These findings indicated that DAP may limit the formation and progression of experimental aneurysms by inhibiting mural inflammation and angiogenesis. These data confirmed the translational potential of DAP inclinical AAA inhibition strategies.

Invited commentary

Failing heart loses its metabolic flexibility, relying increasingly on glucose as its preferential substrate and decreasing fatty acid oxidation (FAO). Peroxisome proliferator-activated receptor α (PPAR-α) is a key regulator of this substrate shift. However, its role during heart failure is complex and remains unclear. Recent studies reported that heart failure develops in the heart of myosin heavy chain-PPAR-α transgenic mice in a manner similar to that of diabetic cardiomyopathy, whereas cardiac dysfunction is enhanced in PPAR-α knockout mice in response to chronic pressure overload. We created a pressure-overload heart failure model in mice through transverse aortic constriction (TAC) and activated PPAR-α during heart failure using an inducible transgenic model. After 8 wk of TAC, left ventricular (LV) function had decreased with the reduction of PPAR-α expression in wild-type mice. We examined the effect of PPAR-α induction during heart failure using the Tet-Off system. Eight weeks after the TAC operation, LV construction was preserved significantly by PPAR-α induction with an increase in PPAR-α-targeted genes related to fatty acid metabolism. The increase of expression of fibrosis-related genes was significantly attenuated by PPAR-α induction. Metabolic rates measured by isolated heart perfusions showed a reduction in FAO and glucose oxidation in TAC hearts, but the rate of FAO preserved significantly owing to the induction of PPAR-α. Myocardial high-energy phosphates were significantly preserved by PPAR-α induction. These results suggest that PPAR-α activation during pressure-overloaded heart failure improved myocardial function and energetics. Thus activating PPAR-α and modulation of FAO could be a promising therapeutic strategy for heart failure.NEW & NOTEWORTHY The present study demonstrates the role of PPAR-α activation in the early stage of heart failure using an inducible transgenic mouse model. Induction of PPAR-α preserved heart function, and myocardial energetics. Activating PPAR-α and modulation of fatty acid oxidation could be a promising therapeutic strategy for heart failure.

The maximum aortic diameter remains the diagnostic criteria and the indicator for prognosis prediction of abdominal aortic aneurysms (AAAs). An additional imaging modality is currently needed to help evaluate the prognosis of AAA as well as early detection of AAA formation. This study evaluated the most effective inflammatory markers for AAA using single-cell sequencing and, from these, developed probes to facilitate in vivo multimodal imaging of AAA inflammation. Single-cell RNA sequencing (scRNAseq) of the human aortic aneurysms, GSE155468 and GSE166676 data sets, identified CXCR4 as the most representative marker. Anti-CXCR4-PE antibody was conjugated to superparamagnetic iron oxide nanoparticles to synthesize Fe3O4-anti-CXCR4-PE probes. The biocompatibility and specificity of the probes were validated in vivo and in vitro. Magnetic particle imaging (MPI) and fluorescence imaging (FLI) were performed to assess inflammation in early and advanced AAA mouse models. CXCR4-specific receptor inhibitor, AMD3100, was used for confirming CXCR4 as an excellent target for AAA imaging and therapy. scRNAseq indicated that chemokine-related pathways were upregulated in aortic aneurysms, and CXCR4 was the chemokine receptor that marks all AAA-related immune cells and inflammatory vascular cells. Fe3O4-anti-CXCR4-PE effectively recognized immune cells and inflammatory vascular cells, as strong MPI and FLI signals corresponded to increased CXCR4, CD45, and CD68 levels that represented AAA severity and rupture risk. Importantly, Fe3O4-anti-CXCR4-PE can help identify early AAA formation when ultrasound is undiagnosable. Finally, AMD3100 treatment in AAA mouse model inhibited AAA expansion and rupture and reduced aortic wall inflammation by inhibiting accumulation of immune cells and haematopoietic stem cells. CXCR4 marks immune cells and inflammatory vascular cells in AAA and is associated with AAA prognosis in a mouse model of AAA. CXCR4-targeting multimodal MPI/FLI provides a novel approach for AAA prognosis prediction and early detection.

Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-β-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-β-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-β-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Although regulatory T cells (Tregs) have been shown to play a protective role in abdominal aortic aneurysm (AAA) formation, it remains unclear whether expansion of endogenous Foxp3(+) Tregs prevents AAA. In the current study, we determined the effects of endogenous Foxp3(+) Treg expansion or depletion in an experimental model of AAA. We continuously infused 12-week-old apolipoprotein E-deficient mice fed a high-cholesterol diet with angiotensin II (n=60) or normal saline (n=12) by implanting osmotic mini-pumps and evaluated AAA formation at 16 weeks. The angiotensin II-infused mice received interleukin-2/anti-interleukin-2 monoclonal antibody complex (interleukin-2 complex; n=31) or PBS (n=29). Eighty-one percent of angiotensin II-infused mice developed AAA, with 42% mortality possibly because of aneurysm rupture. Interleukin-2 complex treatment systemically increased the number of Foxp3(+) Tregs and significantly decreased the incidence (52%) and mortality (17%) of AAA. Immunohistochemical analysis showed reduced accumulation of macrophages and increased numbers of Foxp3(+) Tregs in aneurysmal tissues, suggesting that expansion of Tregs may suppress local inflammation in the vessel wall and provide protection against AAA formation. Furthermore, genetic depletion of Foxp3(+) Tregs led to a significant increase in the mortality of AAA, suggesting the protective role of Foxp3(+) Tregs against AAA. Our findings suggest that Foxp3(+) Tregs may play a protective role in AAA formation and that promotion of an endogenous regulatory immune response may be a potentially valuable therapeutic approach for preventing AAA.

Salvia miltiorrhiza-Derived Sal-miR-58 Induces Autophagy and Attenuates Inflammation in Vascular Smooth Muscle Cells

Antagonism of toll-like receptor 2 attenuates the formation and progression of abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a significant medical problem with a high mortality rate. Nevertheless, the underlying mechanism for the progression and regression of AAA is unknown. Experimental model of AAA was first created by porcine pancreatic elastase incubation around the infrarenal aorta of C57BL/6 mice. Then, AAA progression and regression were evaluated based on the diameter and volume of AAA. The aortas were harvested for hematoxylin-eosin staining (HE), orcein staining, sirius red staining, immunofluorescence analysis and perls' prussian blue staining at the indicated time point. Finally, β-aminopropionitrile monofumarate (BAPN) was used to explore the underlying mechanism of the regression of AAA. When we extended the observation period to 100 days, we not only observed an increase in the AAA diameter and volume in the early stage, but also a decrease in the late stage. Consistent with AAA diameter and volume, the aortic thickness showed the same tendency based on HE staining. The elastin and collagen content first degraded and then regenerated, which corresponds to the early deterioration and late regression of AAA. Then, endogenous up-regulation of lysyl oxidase (LOX) was detected, accompanying the regression of AAA, as detected by an immunofluorescent assay. BAPN and LOX inhibitor considerably inhibited the regression of AAA, paralleling the degradation of elastin lamella and collagen. Taken together, we tentatively conclude that endogenous re-generation of LOX played an influential role in the regression of AAA. Therefore, regulatory factors on the generation of LOX exhibit promising therapeutic potential against AAA.

Abdominal aortic aneurysm (AAA) is a degenerative disease with irreversible and progressive dilation of the artery. But there are few options for efficacious treatment except for traditional surgery. Probucol has been widely applied to treat hyperlipidaemia and atherosclerosis in clinic, but whether it can protect against AAA remains unknown. In this study, the protective effects of probucol against AAA and its related mechanisms were explored. The model of AAA was induced in mice by periaortic application of elastase (40 min) to the abdominal aorta. Probucol at different doses was administered by daily gavage, starting on the same day as AAA was induced, for 14days. In vitro, cultures of rat vascular smooth muscle cells (VSMCs) were stimulated with TNF-α. Haem oxygenase (HO)-1 siRNA and HO-1 plasmid were used to regulate the expression or activity of HO-1 in the VSMCs and to clarify the effects of HO-1. Probucol dose-dependently prevented the development of AAA, reflected by decreased incidence of AAA, diameter of aortic dilation, elastin degradation, and infiltration of inflammatory cells. Probucol also protected VSMCs from oxidative injury and enhanced elastin biosynthesis. This anti-inflammatory effects of probucol on VSMCs were significantly decreased when HO-1 was inhibited by siRNA. Probucol protected against AAA through inhibiting the degradation of elastin induced by inflammation and oxidation and by facilitating the biosynthesis of elastin. HO-1 played a crucial role in the anti-inflammatory effects of probucol in VSMCs.

Introduction: Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammation, which contributes to the pathological remodeling of extracellular matrix. Focal adhesion kinase (FAK) is involved in the interactions between cells and extracellular matrix, and has been suggested to regulate the remodeling of extracellular matrix. Hypothesis: We assessed the hypothesis that FAK plays a pivotal role in the development of AAA. Methods and Results: Aortic wall specimens were obtained from patients that underwent aortic surgery (AAA; n=25, non-AAA; n=8). Levels of activated FAK were significantly increased in AAA walls compared with non-AAA walls (2.8 ± 2.3 fold, p&lt;0.05). Activated FAK was localized to macrophages in the outer media and adventitia of AAA walls. We performed a comprehensive analysis of proteins secreted from mouse macrophages in vitro. The FAK inhibitor PF573228 significantly reduced the secretion of monocyte chemoattractant protein-1 and matrix metalloproteinase-9 in macrophages after stimulation with tumor necrosis factor-alpha. Further, we created AAA in C57BL/6 male mice by periaortic application of calcium chloride to investigate the role of FAK activation in vivo. From just after application of calcium chloride, mice were treated daily with PF573228 or vehicle for 6 weeks. Treatment of mice with PF573228 significantly prevented the increase in aortic diameter (PF573228; 0.84 ± 0.08 mm, vehicle; 1.19 ± 0.18 mm, p&lt;0.01). Treatment with PF573228 also reduced inflammatory cell infiltration and disruption of the elastic lamellae. In an additional experiment, PF573228 or vehicle was administered daily from weeks 3 to 6 after application of calcium chloride. Interestingly, delayed treatment with PF573228 significantly inhibited the progression of aneurysmal dilatation (PF573228; 0.88 ± 0.18 mm, vehicle; 1.25 ± 0.27 mm, p&lt;0.05). Conclusions: Our results indicate a critical role for FAK activation in the development and progression of AAA in vivo, indicating that FAK represents a novel therapeutic target for preventing and treating AAA.

Abdominal aortic aneurysm (AAA) is a major cardiovascular disease and involves enhancement of rennin‐angiotensin system and recruitment/activation of inflammatory factors such as matrix metalloproteinases (MMP's). Caveolae has been shown to play a role in a number of different cardiovascular diseases through different mechanisms including regulation of oxidative stress, inflammation and degradation of extracellular matrix components through MMP's. In addition, endothelial cell caveolae are known to localize the Ang‐II (AT1) receptor and regulate rennin‐angiotensin signaling. Based on these findings, we evaluated the role of caveolae in a murine model of AAA formation. Here, eight week old mice were co‐infused with AngII and BAPN, a lysyl oxidase inhibitor, to induce AAA. We found that mice lacking the main structural protein of caveolae, caveolin‐1, did not develop AAA compared to WT animals in spite of hypertensive blood pressures measured by telemetry in both groups. This finding suggests that intact Ang‐II signaling remains in place in caveolin‐1 knockout mice. To begin to address the underlying mechanism by which caveolae contributes to AAA, we measured the level of oxidative stress and MMP's in aneurysms. We found an increased expression of MMP‐2 and MMP‐9 in vessels of WT mice displaying aneurysms. This increase in expression was not observed in Cav‐1 knockout mice. Furthermore, KO mice showed less oxidative stress then their WT counterparts as assessed by anti‐nitrotyrosine staining. In conclusion, caveolae appears to play a critical role in the formation of AAA in mice via oxidative stress, and recruitment and/or activation of MMPs, specifically MMP‐2 and MMP‐9.

Background Phosphorylation of L-type calcium channels (LTCC) by cAMP-dependent protein kinase A (PKA) increases calcium current (ICa). However, it is unclear if PKA-dependent regulation of ICa is impaired in heart failure (HF) despite evidence for impaired β-adrenergic signaling. Recently, a novel PKA activation pathway by oxidation of regulatory subunit I (RI) has been identified. Objective We investigated the impact of redox-activated PKA for regulation of ICa, intracellular calcium (Ca) handling and contractile function in a pressure overload heart failure mouse model. Methods Knock-in mice (KI) that lack redox-dependent PKA activation (exchange of cysteine 17 of RI with serine) were compared to wild-type (WT) at baseline, 7 days and 6 weeks after transverse aortic constriction (TAC). Mouse echocardiography was performed to evaluate in-vivo cardiac function. ICa was measured by whole-cell patch clamp, PKA activity, cAMP levels, and protein levels of central Ca handling proteins were assessed at different time points in vitro. Results At baseline, no alterations of left ventricular (LV) function (echocardiography) were observed between WT and KI. TAC induced a significant RI oxidation in WT but not KI mice. Despite this difference, at 7 days after TAC, development of LV hypertrophy and impairment of systolic LV function in vivo were similar between WT and KI. Compared to baseline, 7 days after TAC a significant stimulation of peak ICa was observed in WT. In contrast to WT, the stimulation of peak ICa was absent in KI mice at 7 days after TAC. This impairment in peak ICa occurred despite a comparable increase in global PKA activity (ELISA), which was most likely due to increased cAMP levels in our TAC model (assessed by FLIM-FRET). Notably, cAMP levels were comparably increased in between groups (ELISA). Importantly, at 6 weeks after TAC, WT mice showed a mild additional deterioration of systolic LV function in vivo. In contrast, LV function was significantly more impaired in KI mice 6 weeks after TAC, which was accompanied by a significant increase in KI mice mortality. Comparing 6 weeks to 7 days after TAC, there was no stimulation of peak ICa in WT and even a significant decrease in peak ICa in KI mice. In accordance, PKA-dependent LTCC phosphorylation was absent in KI mice 6 weeks after TAC (western blotting). Conclusion Redox-activated PKA seems to exert a protective role by stimulation of ICa during pressure overload. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft