Necrotic Core Size Research Articles

Cyclophilin D induces necrotic core formation by mediating mitochondria-associated macrophage death in advanced atherosclerotic lesions

Background and aimsIn advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation. MethodsTo clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe−/−Ppif−/−) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development. ResultsDeletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe−/− mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. ConclusionsThese results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.

Urolithin A promotes atherosclerotic plaque stability by limiting inflammation and hypercholesteremia in Apolipoprotein E-deficient mice.

Urolithin A (UroA), a dietary phytochemical, is produced by gut bacteria from fruits rich in natural polyphenols ellagitannins (ETs). The efficiency of ETs metabolism to UroA in humans depends on gut microbiota. UroA has shown a variety of pharmacological activities. In this study we investigated the effects of UroA on atherosclerotic lesion development and stability. Apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat and high-cholesterol diet for 3 months to establish atherosclerosis model. Meanwhile the mice were administered UroA (50 mg·kg-1·d-1, i.g.). We showed that UroA administration significantly decreased diet-induced atherosclerotic lesions in brachiocephalic arteries, macrophage content in plaques, expression of endothelial adhesion molecules, intraplaque hemorrhage and size of necrotic core, while increased the expression of smooth muscle actin and the thickness of fibrous cap, implying features of plaque stabilization. The underlying mechanisms were elucidated using TNF-α-stimulated human endothelial cells. Pretreatment with UroA (10, 25, 50 μM) dose-dependently inhibited TNF-α-induced endothelial cell activation and monocyte adhesion. However, the anti-inflammatory effects of UroA in TNF-α-stimulated human umbilicalvein endothelial cells (HUVECs) were independent of NF-κB p65 pathway. We conducted RNA-sequencing profiling analysis to identify the differential expression of genes (DEGs) associated with vascular function, inflammatory responses, cell adhesion and thrombosis in UroA-pretreated HUVECs. Human disease enrichment analysis revealed that the DEGs were significantly correlated with cardiovascular diseases. We demonstrated that UroA pretreatment mitigated endothelial inflammation by promoting NO production and decreasing YAP/TAZ protein expression and TEAD transcriptional activity in TNF-α-stimulated HUVECs. On the other hand, we found that UroA administration modulated the transcription and cleavage of lipogenic transcription factors SREBP1/2 in the liver to ameliorate cholesterol metabolism in ApoE-/- mice. This study provides an experimental basis for new dietary therapeutic option to prevent atherosclerosis.

Circulating LIGHT/TNFSF14 levels are negatively associated with cardiovascular mortality, while intra-plaque levels are associated with symptomatic cerebrovascular disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): The Swedish Heart and Lung Foundation, Åke Wiberg's Foundation Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it is associated with a high-risk plaque phenotype and cardiovascular events is not known. Purpose This study investigates the association between intra-plaque and circulating LIGHT levels and cardiovascular disease. Method Plaque levels of LIGHT/TNFSF14 were measured in plasma and homogenised carotid endarterectomy plaques by Proximity Extension Assay in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=557 and 201, respectively). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through immunohistochemistry. Multivariate Cox proportional hazards analysis was performed on normalized (logarithmically transformed) values. Results Plaque, but not circulating, levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, intra-plaque levels of LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, fibromodulin, ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7) and matrix metalloproteinases 1, 2, 9 and 10. While plaque LIGHT levels were not predictive of cardiovascular death, circulating LIGHT levels were negatively associated with risk of cardiovascular death (after 6 ½ years) after correcting for the possible confounders age, sex, cerebrovascular symptoms, diabetes, high-sensitivity C-reactive protein, inflammatory disease, hypertension and smoking (hazard ratio=0.032, confidence interval=0.420-0.951, p=0.028). Conclusion Though intra-plaque LIGHT levels are associated with a rupture-prone plaque phenotype and pre-operative symptoms, circulating levels are associated with a lower risk of future cardiovascular mortality. This suggest that upregulation of LIGHT in atherosclerotic plaques may occur as part of a reparative rupture response and, systemically, reflect efficient wound-healing capacity.

Abstract 1033: Loss Of Serine Hydroxymethyltransferase 2 In Macrophages Blunts Glutathione And Enhances Ceramide Synthesis To Accelerate Atherosclerosis

Increasing evidence suggests an important role for amino acid metabolism in atherosclerotic cardiovascular disease (ASCVD), highlighting potential biomarkers, causative factors, and therapeutic targets. Among all amino acids, lower circulating glycine is consistently reported to be associated with enhanced ASCVD severity. The amino acid serine is the main precursor for glycine, and a lower glycine:serine ratio is associated with more advanced and unstable atherosclerotic plaques. The mitochondrial serine hydroxymethyltransferase 2 (SHMT2) catalyzes the bulk of glycine formation from serine, and is critical in development since global knockout mice are nonviable. We found that SHMT2 is highly expressed in macrophages in both human and mouse atherosclerotic plaques. LPS-treated macrophages displayed reduced SHMT2 expression concomitant with a lower intracellular glycine:serine ratio. To study the role of SHMT2 in macrophages, we developed novel myeloid-specific Shmt2 knockout mice ( Shmt2 MKO ). Metabolomics and stable isotope tracing studies revealed that macrophages lacking SHMT2 have a lower glycine:serine ratio leading to attenuated de novo synthesis of glutathione and enhanced formation of the ceramide precursor, sphinganine. Accordingly, the loss of SHMT2 in macrophages enhanced superoxide production and ceramide accumulation. To determine the role of macrophage SHMT2 in atherosclerosis, we crossed the Shmt2 MKO mice with apolipoprotein E-deficient ( Apoe -/- ) mice. After 12 weeks on Western diet, Shmt2 MKO / Apoe -/- mice showed no differences in plasma lipid or amino acid profiles as compared to Shmt2 flox / Apoe -/- mice. Nevertheless, plaque area and necrotic core size were significantly enhanced in Shmt2 MKO / Apoe -/- mice. Taken together, macrophage SHMT2 plays a critical role in atherosclerosis by regulating redox and ceramide metabolism.

Heparanase promotes the onset and progression of atherosclerosis in apolipoprotein E gene knockout mice

Background and aimsAtherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo. MethodsWe generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE−/−) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated. ResultsHpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE−/− mice were significantly decreased compared to control ApoE−/−mice at both disease stages. ConclusionsThis study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease.

Nicotinamide mononucleotide protects against high-fat-diet-induced atherosclerosis in mice and dampens aortic inflammation and oxidative stress

Atherosclerosis, a chronic inflammatory disease, is the major cause of arteriosclerotic cardiovascular diseases. Nicotinamide mononucleotide (NMN), an effective NAD+ booster, has been shown to reduce inflammation and protect vascular cell function, which are strongly associated with the progression of atherosclerosis. In this study, we explored the anti-atherosclerotic effects of NMN and its potential mechanism in ApoE−/− mice. We found that NMN significantly reduced the size of atherosclerotic plaque (36 %) and necrotic core (48 %) in aortic sinus. Additionally, NMN decreased lipid area (43 %) and increased collagen content (51 %) in atherosclerotic lesions. In addition, NMN reduced the level of malondialdehyde (MDA) in serum while elevating the enzymatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX). Moreover, NMN reduced the expression of pro-inflammatory cytokines (Tnfα, Il-1β, Il-6 and Mcp-1) while increased the expression of anti-inflammatory factors (Arg-1, Mrc-1, Retlna and Irf-4) in aortic tissues. These results indicate that NMN exerts anti-atherosclerotic effects and inhibits oxidative stress and inflammatory response in HFD-fed ApoE−/− mice.

Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids.

Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue. dROb spheroids in control and mineralization media at different seeding densities (1 × 104, 5 × 104, and 1 × 105 cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin® by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects. Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (p < 0.001), and upregulated alkaline phosphatase (p < 0.01). With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.

Investigating the Association of Carotid Atherosclerotic Plaque MRI Features and Silent Stroke After Carotid Endarterectomy.

The predictive value of carotid plaque characteristics for silent stroke (SS) after carotid endarterectomy (CEA) is unclear. To investigate the associations between carotid plaque characteristics and postoperative SS in patients undergoing CEA. Prospective. One hundred fifty-three patients (mean age: 65.4 ± 7.9 years; 126 males) with unilateral moderate-to-severe carotid stenosis (evaluated by CT angiography) referred for CEA. 3 T, brain-MRI:T2-PROPELLER, T1-/T2-FLAIR, diffusion weighted imaging (DWI) and T2*, carotid-MRI:black-blood T1-/T2W, 3D TOF, Simultaneous Non-contrast Angiography intraplaque hemorrhage. Patients underwent carotid-MRI within 1-week before CEA, and brain-MRI within 48-hours pre-/post-CEA. The presence and size (volume, maximum-area-percentage) of carotid lipid-rich necrotic core (LRNC), intraplaque hemorrhage (Type-I/Type-II IPH) and calcification were evaluated on carotid-MR images. Postoperative SS was assessed from pre-/post-CEA brain DWI. Patients were divided into moderate-carotid-stenosis (50%-69%) and severe-carotid-stenosis (70%-99%) groups and the associations between carotid plaque characteristics and SS were analyzed. Independent t test, Mann-Whitney U-test, chi-square test and logistic regressions (OR: odds ratio, CI: confidence interval). P value <0.05 was considered statistically significant. SS was found in 8 (16.3%) of the 49 patients with moderate-carotid-stenosis and 21 (20.2%) of the 104 patients with severe-carotid-stenosis. In patients with severe-carotid-stenosis, those with SS had significantly higher IPH (66.7% vs. 39.8%) and Type-I IPH (66.7% vs. 38.6%) than those without. The presence of IPH (OR 3.030, 95% CI 1.106-8.305) and Type-I IPH (OR 3.187, 95% CI 1.162-8.745) was significantly associated with SS. After adjustment, the associations of SS with presence of IPH (OR 3.294, 95% CI 1.122-9.669) and Type-I IPH (OR 3.633, 95% CI 1.216-10.859) remained significant. Moreover, the volume of Type-II IPH (OR 1.014, 95% CI 1.001-1.028), and maximum-area-percentage of Type-II IPH (OR 1.070, 95% CI 1.002-1.142) and LRNC (OR 1.030, 95% CI 1.000-1.061) were significantly associated with SS after adjustment. No significant (P range: 0.203-0.980) associations were found between carotid plaque characteristics and SS in patients with moderate-carotid-stenosis. In patients with unilateral severe-carotid-stenosis, carotid vulnerable plaque MR features, particularly presence and size of IPH, might be effective predictors for SS after CEA. 2 TECHNICAL EFFICACY: Stage 2.

Mechanical wall stress and wall shear stress are associated with atherosclerosis development in non-calcified coronary segments

Background and aimsAtherosclerotic plaque onset and progression are known to be affected by local biomechanical factors. Whereas the role of wall shear stress (WSS) has been studied, the impact of another biomechanical factor, namely mechanical wall stress (MWS), remains poorly understood. In this study, we investigated the association of MWS, independently and combined with WSS, towards atherosclerosis in coronary arteries. MethodsThirty-four human coronary arteries were analyzed using near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) and optical coherence tomography (OCT) at baseline and after 12 months. Baseline WSS and MWS were calculated using computational models, and wall thickness (ΔWT) and lipid-rich necrotic core size (ΔLRNC) change were measured in non-calcified coronary segments. The arteries were further divided into 1.5 mm/45° sectors and categorized as plaque-free or plaque sectors. For each category, associations between the biomechanical factors (WSS & MWS) and changes in the coronary wall (ΔWT & ΔLRNC) were studied using linear mixed models. ResultsIn plaque-free sectors, higher MWS (p < 0.001) was associated with greater vessel wall growth. Plaque sectors demonstrated wall thickness reduction over time, likely due to the medical therapy, where higher levels of WSS and WMS, individually and combined, (p < 0.05) were associated with a greater reduction. Sectors with low MWS combined with high WSS demonstrated the highest LRNC increase (p < 0.01). ConclusionsIn this study, we investigated the association of the (largely-overlooked) biomechanical factor, MWS with coronary atherosclerosis, individually and combined with WSS. Our results demonstrated that both MWS and WSS significantly correlate with atherosclerotic plaque initiation and development.

LIGHT/TNFSF14 levels in carotid atherosclerotic plaques are associated with symptomatic cerebrovascular disease

Abstract Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it has a driving role in promoting a high-risk plaque phenotype is not known. Purpose This study investigates the association between intra-plaque LIGHT and plaque vulnerability. Methods Plaque levels of LIGHT/TNFSF14 were measured in homogenised carotid endarterectomy plaques by Proximity Extension Assay (PEA) in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=201). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through histology/immunohistochemistry. Results Plaque levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, the collagen cross-linking proteoglycans fibromodulin and lumican, the matricellular protein COMP (cartilage oligomeric matrix protein), and the remodelling regulator ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7). Levels also correlated with matrix metalloproteinases (MMPs) 1, 2, 9 and 10, and tissue inhibitor of metalloproteinase (TIMP)-1. Conclusion Expression of LIGHT in atherosclerotic plaques does not only correlate with markers of plaque destabilisation¸ but is also significantly elevated in plaques from symptomatic, compared to asymptomatic, patients. These results connect LIGHT content to a rupture-prone plaque phenotype and suggest the LIGHT signalling pathway as a potential therapeutic target to promote atherosclerotic plaque stability.

Rational Design of a Double-Locked Photoacoustic Probe for Precise In Vivo Imaging of Cathepsin B in Atherosclerotic Plaques.

Atherosclerotic plaque rupture is a significant cause of acute cardiovascular events such as heart attack and stroke, triggered by the decomposition of fiber caps induced by cysteine cathepsin. However, the accurate measurement of cathepsin B (CTB) activity in plaques is challenging due to the low specificity and insufficient penetration depth of available atherosclerosis-associated cathepsin fluorescent probes, hampering reliable assessment of plaque vulnerability. To address these limitations, we added both lipophilic alkyl chain and hydrophilic CTB substrate to the hemicyanine scaffold to develop a lipid-unlocked CTB responsive probe (L-CRP) that uses lipids and CTB as two keys to unlock photoacoustic (PA) signals for measuring CTB activity in lipophilic environments. Such properties allow L-CRP for the reliable imaging of specific CTB activities in foam cells and atherosclerotic plaques while keeping in silence toward CTB in lipid-deficient environments, such as M1-type macrophages and LPS-induced inflammatory lesions. Moreover, the activatable PA signals of L-CRP exhibit a deeper tissue penetration ability (>1.0 cm) than current CTB probes based on near-infrared fluorescent imaging (∼0.3 cm), suitable for atherosclerosis imaging in living mice. In atherosclerotic mice, L-CRP dynamically reports intraplaque CTB levels, which is well-correlated with the plaque vulnerability characteristics such as fiber cap thickness, macrophage recruitment, and necrotic core size, thus enabling risk stratification of atherosclerotic mice complicated with pneumonia. Moreover, L-CRP successfully identifies atherosclerotic plaques in excised human artery tissues, promising for auxiliary diagnosis of plaque vulnerability in clinical application.

Drug transport modeling in solid tumors: A computational exploration of spatial heterogeneity of biophysical properties

Inadequate uptake of therapeutic agents by tumor cells is still a major barrier in clinical cancer therapy. Mathematical modeling is a powerful tool to describe and investigate the transport phenomena involved. However, current models for interstitial flow and drug delivery in solid tumors have not yet embedded the existing heterogeneity of tumor biomechanical properties. The purpose of this study is to introduce a novel and more realistic methodology for computational models of solid tumor perfusion and drug delivery accounting for these regional heterogeneities as well as lymphatic drainage effects.Several tumor geometries were studied using an advanced computational fluid dynamics (CFD) modeling approach of intratumor interstitial fluid flow and drug transport. Hereby, the following novelties were implemented: (i) the heterogeneity of tumor-specific hydraulic conductivity and capillary permeability; (ii) the effect of lymphatic drainage on interstitial fluid flow and drug penetration.Tumor size and shape both have a crucial role on the interstitial fluid flow regime as well as drug transport illustrating a direct correlation with interstitial fluid pressure (IFP) and an inverse correlation with drug penetration, except for large tumors having a diameter larger than 50 mm. The results also suggest that the interstitial fluid flow and drug penetration in small tumors depend on tumor shape. A parameter study on the necrotic core size illustrated that the core effect (i.e. fluid flow and drug penetration alteration) was only profound in small tumors. Interestingly, the impact of a necrotic core on drug penetration differs depending on the tumor shape from having no effect in ideally spherical tumors to a clear effect in elliptical tumors with a necrotic core. A realistic presence of lymphatic vessels only slightly affected tumor perfusion, having no substantial effect on drug delivery.In conclusion, our findings illustrated that our novel parametric CFD modeling strategy in combination with accurate profiling of heterogeneous tumor biophysical properties can provide a powerful tool for better insights into tumor perfusion and drug transport, enabling effective therapy planning.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

High levels of lipoprotein(a) in transgenic mice exacerbate atherosclerosis and promote vulnerable plaque features in a sex-specific manner

Background and aimsDespite increased clinical interest in lipoprotein(a) (Lp(a)), many questions remain about the molecular mechanisms by which it contributes to atherosclerotic cardiovascular disease. Existing murine transgenic (Tg) Lp(a) models are limited by low plasma levels of Lp(a) and have not consistently shown a pro-atherosclerotic effect of Lp(a). MethodsWe generated Tg mice expressing both human apolipoprotein(a) (apo(a)) and human apoB-100, with pathogenic levels of plasma Lp(a) (range 87–250 mg/dL). Female and male Lp(a) Tg mice (Tg(LPA+/0;APOB+/0)) and human apoB-100-only controls (Tg(APOB+/0)) (n = 10–13/group) were fed a high-fat, high-cholesterol diet for 12 weeks, with Ldlr knocked down using an antisense oligonucleotide. FPLC was used to characterize plasma lipoprotein profiles. Plaque area and necrotic core size were quantified and immunohistochemical assessment of lesions using a variety of cellular and protein markers was performed. ResultsMale and female Tg(LPA+/0;APOB+/0) and Tg(APOB+/0) mice exhibited proatherogenic lipoprotein profiles with increased cholesterol-rich VLDL and LDL-sized particles and no difference in plasma total cholesterol between genotypes. Complex lesions developed in the aortic sinus of all mice. Plaque area (+22%), necrotic core size (+25%), and calcified area (+65%) were all significantly increased in female Tg(LPA+/0;APOB+/0) mice compared to female Tg(APOB+/0) mice. Immunohistochemistry of lesions demonstrated that apo(a) deposited in a similar pattern as apoB-100 in Tg(LPA+/0;APOB+/0) mice. Furthermore, female Tg(LPA+/0;APOB+/0) mice exhibited less organized collagen deposition as well as 42% higher staining for oxidized phospholipids (OxPL) compared to female Tg(APOB+/0) mice. Tg(LPA+/0;APOB+/0) mice had dramatically higher levels of plasma OxPL-apo(a) and OxPL-apoB compared to Tg(APOB+/0) mice, and female Tg(LPA+/0;APOB+/0) mice had higher plasma levels of the proinflammatory cytokine MCP-1 (+3.1-fold) compared to female Tg(APOB+/0) mice. ConclusionsThese data suggest a pro-inflammatory phenotype exhibited by female Tg mice expressing Lp(a) that appears to contribute to the development of more severe lesions with greater vulnerable features.

Heterozygosity for ADP-ribosylation factor 6 suppresses the burden and severity of atherosclerosis.

Atherosclerosis is the root cause of major cardiovascular diseases (CVD) such as myocardial infarction and stroke. ADP-ribosylation factor 6 (Arf6) is a ubiquitously expressed GTPase known to be involved in inflammation, vascular permeability and is sensitive to changes in shear stress. Here, using atheroprone, ApoE-/- mice, with a single allele deletion of Arf6 (HET) or wildtype Arf6 (WT), we demonstrate that reduction in Arf6 attenuates atherosclerotic plaque burden and severity. We found that plaque burden in the descending aorta was lower in HET compared to WT mice (p˂0.001) after the consumption of an atherogenic Paigen diet for 5 weeks. Likewise, luminal occlusion, necrotic core size, plaque grade, elastic lamina breaks, and matrix deposition were lower in the aortic root atheromas of HET compared to WT mice (all p≤0.05). We also induced advanced human-like complex atherosclerotic plaque in the left carotid artery using partial carotid ligation surgery and found that atheroma area, plaque grade, intimal necrosis, intraplaque hemorrhage, thrombosis, and calcification were lower in HET compared to WT mice (all p≤0.04). Our findings suggest that the atheroprotection afforded by Arf6 heterozygosity may result from reduced immune cell migration (all p≤0.005) as well as endothelial and vascular smooth muscle cell proliferation (both p≤0.001) but independent of changes in circulating lipids (all p≥0.40). These findings demonstrate a critical role for Arf6 in the development and severity of atherosclerosis and suggest that Arf6 inhibition can be explored as a novel therapeutic strategy for the treatment of atherosclerotic CVD.

Abstract 689: Loss of Smooth Muscle Cell Nogo-B Protects from Atherosclerosis Plaque Development

Background: Atherosclerosis, a major cause of cardiovascular diseases, is a lipid-driven inflammatory disease that occurs as fatty deposits, immune cells, calcium, and waste products accumulate on the walls of arteries over decades and often result in myocardial infarction or stroke. A member of the reticulon family of proteins, Nogo-B localizes in endothelial cells, vascular smooth muscle cells (VSMCs), and macrophages. Global and macrophage-specific Nogo-B deletion exacerbated atherogenesis by increasing lesion area and necrotic core size while decreasing cap thickness and collagen deposition. Our previous studies also suggest Nogo-B attenuates growth factor induced VSMC proliferation and neointimal formation after vascular injury. This study aims to elucidate the role of VSMC-specific Nogo-B in plaque development and stability in mice. Methods and Results: A murine atherosclerosis model was generated by specifically deleting Nogo-B in VSMCs of ApoE -/- mice with a Cre-recombinase system (Nogo-B iSMCKO ) and feeding a Western diet (1.25% cholesterol) for 14 weeks. VSMC-Nogo-B loss demonstrates a decreased necrotic core, minimized lesion size, and increased collagen content in aortic root lesions, all of which are indices of a more stable plaque phenotype. Nogo-B deletion in cultured VSMCs stimulated with β-CD cholesterol (20ug/mL) was performed to investigate phenotypic modulation and trans-differentiation in vitro . After cholesterol stimulation, expression of smooth muscle contractile genes was maintained in cells deficient in Nogo-B while decreasing in WT cells. Furthermore, VSMCs deficient in Nogo-B had decreased cholesterol uptake in cholesterol-loading experiments. Mechanistically, our results show Nogo-B modulates VSMC plasticity through transcription factor Krüppel-like factor 4. Conclusions: VSMC-specific Nogo-B deficiency attenuates atherogenesis and promotes stable plaque phenotypes. VSMC-specific Nogo-B deficiency may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.

Hematopoietic NLRP3 and AIM2 Inflammasomes Promote Diabetes-Accelerated Atherosclerosis, but Increased Necrosis Is Independent of Pyroptosis.

The contribution of hematopoietic cell inflammasome activation to atherosclerosis associated with type 1 diabetes is unknown. The goal of this study was to address whether hematopoietic NOD, LRR, and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2) inflammasomes, or the pyroptosis executioner gasdermin D (GSDMD) contributes to atherosclerosis in mouse models of type 1 diabetes. Diabetic mice exhibited increased inflammasome activation, with hematopoietic deletions of NLRP3, AIM2, or GSDMD causing smaller atherosclerotic lesions in diabetic mice, but the increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis. Further studies on whether inflammasome activation contributes to cardiovascular complications in people with type 1 diabetes are warranted.

GPR55 in B cells limits atherosclerosis development and regulates plasma cell maturation

Dissecting the pathways regulating the adaptive immune response in atherosclerosis is of particular therapeutic interest. Here we report that the lipid G-protein-coupled receptor GPR55 is highly expressed by splenic plasma cells (PCs), upregulated in mouse spleens during atherogenesis and human unstable or ruptured compared to stable plaques. Gpr55-deficient mice developed larger atherosclerotic plaques with increased necrotic core size compared to their corresponding controls. Lack of GPR55 hyperactivated B cells, disturbed PC maturation and resulted in IgG overproduction. B-cell-specific Gpr55 depletion or adoptive transfer of Gpr55-deficient B cells was sufficient to promote plaque development and elevated IgG titers. In vitro, the endogenous GPR55 ligand lysophsophatidylinositol (LPI) enhanced PC proliferation, whereas GPR55 antagonism blocked PC maturation and increased their mitochondrial content. Collectively, these discoveries provide previously undefined evidence for GPR55 in B cells as a key modulator of the adaptive immune response in atherosclerosis.

Brussels Chicory Stabilizes Unstable Atherosclerotic Plaques and Reshapes the Gut Microbiota in Apoe−/− Mice

Adherence to a Mediterranean dietary pattern can protect against atherosclerosis in part by reducing intestinal permeability and gut microbial LPS production. Brussels chicory, a typical Mediterranean vegetable, has been shown to inhibit the formation of early-stage atherosclerosis in mice. We evaluated whether Brussels chicory affects advanced atherosclerosis progression, intestinal permeability, and gut microbial LPS production. Thirty-week-old male apoE-deficient mice with unstable atherosclerotic plaques in the brachiocephalicarterywere fed the AIN-93G diet alone (control) or supplemented with 0.5% freeze-dried Brussels chicory for 20 wk. Plaque volume and features of plaque stability, plaque macrophage polarization, fecal and serum LPS concentrations, serum lipid profiles and inflammation-related cytokines, and gut microbial profiles were measured. Compared with the control treatment, Brussels chicory consumption did not significantly change plaque volume and serum lipid profiles. However, it increased plaque stability (P < 0.05), as evidenced by reduced necrotic core size (42.3%), and increased fibrous cap thickness (55.0%) and collagen content (68.4%). Moreover, Brussels chicory consumption reduced intestinal permeability (56.3%), fecal and serum LPS concentrations (52.2% and 39.4%), serum IL1β and TNFα (52.0% and 33.8%), promoted plaquemacrophage polarization towards the M2-likephenotype, and altered gut microbial composition, the latter indicated by increased relative abundance of certain members of the Ruminococcaceae family, such as Ruminiclostridium_9, Ruminiclostridium_5, and Intestinimonas (P < 0.05). Spearman correlation analyses further showed that these bacterial genera were significantly correlated with intestinal permeability, fecal and serum LPS, serum proinflammatory cytokines, and several features of plaque stability. Brussels chicory might help stabilize atherosclerotic plaques in mice by reducing intestinal permeability and gut microbial LPS production. This study provides a promising approach to slow the progression of atherosclerosis.

Myeloid CD40 deficiency reduces atherosclerosis by impairing macrophages' transition into a pro-inflammatory state.

CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice. Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe-/- background were generated (CD40wt and CD40mac-/-, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac-/- compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac-/- atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b- macrophages in the atherosclerotic aorta of CD40mac-/- compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac-/- mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1). We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.