SPAG5 deficiency activates autophagy to reduce atherosclerotic plaque formation in ApoE-/- mice.

BACKGROUND: Autophagy, as a regulator of cell survival, plays an important role in atherosclerosis (AS). Sperm associated antigen 5 (SPAG5) is closely associated with the classical autophagy pathway, PI3K/Akt/mTOR signaling pathway. This work attempted to investigate whether SPAG5 can affect AS development by regulating autophagy. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized-low density lipoprotein (ox-LDL) to induce cell damage. ApoE-/- mice were fed a Western diet to establish an AS mouse model. Haematoxylin and eosin (H&E) staining and Oil Red O staining evaluated the pathological changes and in lipid deposition in aortic tissues. CCK-8 and flow cytometry detected cell proliferation and apoptosis. Immunohistochemistry, Enzyme linked immunosorbent assay, qRT-PCR and western blotting assessed the levels of mRNA and proteins. RESULTS: Ox-LDL treatment elevated SPAG5 expression and the expression of autophagy-related proteins, LC3-I, LC3-II, Beclin-1, and p62, in HUVECs. GFP-LC3 dots were increased in ox-LDL-treated HUVECs and LPS-treated HUVECs. SPAG5 knockdown reversed both ox-LDL and LPS treatment-mediated inhibition of cell proliferation and promotion of apoptosis in HUVECs. SPAG5 silencing further elevated autophagy and repressed the expression of PI3K, p-Akt/Akt, and p-mTOR/mTOR in ox-LDL-treated HUVECs. 3-MA (autophagy inhibitor) treatment reversed SPAG5 silencing-mediated increase of cell proliferation and decrease of apoptosis in ox-LDL-treated HUVECs. In vivo, SPAG5 knockdown reduced atherosclerotic plaques in AS mice through activating autophagy and inhibiting PI3K/Akt/mTOR signaling pathway. CONCLUSION: This work demonstrated that SPAG5 knockdown alleviated AS development through activating autophagy. Thus, SPAG5 may be a potential target for AS therapy.

IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Macrophage-Derived GSDMD Plays an Essential Role in Atherosclerosis and Cross Talk Between Macrophages via the Mitochondria-STING-IRF3/NF-κB Axis.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE-/- and Gsdmd-/- ApoE-/- mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd-/- attenuates the atherosclerotic lesion area in high-fat diet-fed ApoE-/- mice. We performed single-cell RNA sequencing of ApoE-/- and Gsdmd-/- ApoE-/- mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation.

AIMS: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. METHODS AND RESULTS: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. CONCLUSION: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

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

BACKGROUND AND AIMS: Atherosclerosis 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. METHODS: We 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. RESULTS: Hpse-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. CONCLUSIONS: This 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.

l-thyroxine attenuates extracellular Hsp90α-induced vascular endothelial calcification in diabetes mellitus, as revealed by parallel metabolic profiles.

BACKGROUND AND AIMS: Diabetic atherosclerotic vascular disease is characterized by extensive vascular calcification. However, an elevated blood glucose level alone does not explain this pathogenesis. We investigated the metabolic markers underlying diabetic atherosclerosis and whether extracellular Hsp90α (eHsp90α) triggers vascular endothelial calcification in this particular metabolic environment. METHODS: A parallel human/animal model metabolomics approach was used. We analyzed 40 serum samples collected from 24 patients with atherosclerosis and from the STZ-induced ApoE-/- mouse model. A multivariate statistical analysis of the data was performed, and mouse aortic tissue was collected for the assessment of plaque formation. In vitro, the effects of eHsp90α on endothelial cell calcification were assessed by serum analysis, Western blotting and immunoelectron microscopy. RESULTS: Diabetic ApoE-/- mice showed more severe plaque lesions and calcification damage. Stearamide, oleamide, l-thyroxine, l-homocitrulline and l-citrulline are biomarkers of diabetic ASVD; l-thyroxine was downregulated in both groups, and the thyroid sensitivity index was correlated with serum Hsp90α concentration. In vitro studies showed that eHsp90α increased Runx2 expression in endothelial cells through the LRP1 receptor. l-thyroxine reduced the increase in Runx2 levels caused by eHsp90α and affected the distribution and expression of LRP1 through hydrogen bonding with glutamine at position 1054 in the extracellular segment of LRP1. CONCLUSIONS: This study provides a mechanistic link between characteristic serum metabolites and diabetic atherosclerosis and thus offers new insight into the role of extracellular Hsp90α in promoting vascular calcification.

Resident Memory T Cells in the Atherosclerotic Lesion Associate With Reduced Macrophage Content and Increased Lesion Stability.

BACKGROUND: Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis. METHODS: To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (recombination activating gene 1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. CONCLUSIONS: TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Multifaceted roles of Meg3 in cellular senescence and atherosclerosis.

BACKGROUND AND AIMS: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. METHODS AND RESULTS: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr-/- mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. CONCLUSIONS: Our data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.

Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

Spatial Metabolomics Identifies LPC(18:0) and LPA(18:1) in Advanced Atheroma With Translation to Plasma for Cardiovascular Risk Estimation.

BACKGROUND: The metabolic alterations occurring within the arterial architecture during atherosclerosis development remain poorly understood, let alone those particular to each arterial tunica. We aimed first to identify, in a spatially resolved manner, the specific metabolic changes in plaque, media, adventitia, and cardiac tissue between control and atherosclerotic murine aortas. Second, we assessed their translatability to human tissue and plasma for cardiovascular risk estimation. METHODS: In this observational study, mass spectrometry imaging (MSI) was applied to identify region-specific metabolic differences between atherosclerotic (n=11) and control (n=11) aortas from low-density lipoprotein receptor-deficient mice, via histology-guided virtual microdissection. Early and advanced plaques were compared within the same atherosclerotic animals. Progression metabolites were further analyzed by MSI in 9 human atherosclerotic carotids and by targeted mass spectrometry in human plasma from subjects with elective coronary artery bypass grafting (cardiovascular risk group, n=27) and a control group (n=27). RESULTS: MSI identified 362 local metabolic alterations in atherosclerotic mice (log2 fold-change ≥1.5; P≤0.05). The lipid composition of cardiac tissue is altered during atherosclerosis development and presents a generalized accumulation of glycerophospholipids, except for lysolipids. Lysolipids (among other glycerophospholipids) were found at elevated levels in all 3 arterial layers of atherosclerotic aortas. LPC(18:0) (lysophosphatidylcholine; P=0.024) and LPA(18:1) (lysophosphatidic acid; P=0.025) were found to be significantly elevated in advanced plaques as compared with mouse-matched early plaques. Higher levels of both lipid species were also observed in fibrosis-rich areas of advanced- versus early-stage human samples. They were found to be significantly reduced in human plasma from subjects with elective coronary artery bypass grafting (P<0.001 and P=0.031, respectively), with LPC(18:0) showing significant association with cardiovascular risk (odds ratio, 0.479 [95% CI, 0.225-0.883]; P=0.032) and diagnostic potential (area under the curve, 0.778 [95% CI, 0.638-0.917]). CONCLUSIONS: An altered phospholipid metabolism occurs in atherosclerosis, affecting both the aorta and the adjacent heart tissue. Plaque-progression lipids LPC(18:0) and LPA(18:1), as identified by MSI on tissue, reflect cardiovascular risk in human plasma.

Paraspeckle protein NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 transcription.

Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.

The Characteristics of Macrophage Heterogeneity in Atherosclerotic Aortas.

Macrophage is the main effector cell during atherosclerosis. We applied single-cell RNA sequencing (scRNA) data to investigate the role of macrophage subsets in atherosclerosis. Monocyte and macrophage clusters were divided into 6 subclusters. Each subcluster's markers were calculated and validated by immunofluorescence. Elevated macrophage subclusters in the WD group were subject to enrichment pathway analysis and exhibited different phenotypes. Pseudotime analysis shows the subclusters originate from monocytes. We cultured bone marrow-derived macrophages with CSF-1 and ox-LDL to simulate an atherosclerotic-like environment and detected the transformation of subclusters. Macrophage-Vegfa and Macrophage-C1qb increased in the WD group. Macrophage-Vegfa acquires the characteristics of phagocytosis and immune response, while Macrophage-C1qb is not involved in lipid metabolism. The two subclusters are both enriched in cell movement and migration pathways. Experimental verification proved Monocyte-Ly6C evolved into Macrophage-Vegfa and Macrophage-C1qb during atherosclerosis progression.

Itaconate suppresses atherosclerosis by activating a Nrf2-dependent antiinflammatory response in macrophages in mice.

Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as immune-responsive gene 1 [IRG1]), are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-octyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.

MYD88 and Proinflammatory Chemokines in Aortic Atheromatosis: Exploring Novel Statin Effects.

Atherosclerosis is driven by a diverse range of cellular and molecular processes. In the present study, we sought to better understand how statins mitigate proatherogenic inflammation. 48 male New Zealand rabbits were divided into eight groups, each including 6 animals. The control groups received normal chow for 90 and 120 days. Three groups underwent a hypercholesterolemic diet (HCD) for 30, 60, and 90 days. Another three groups underwent HCD for 3 months, followed by normal chow for one month, with or without rosuvastatin or fluvastatin. The cytokine and chemokine expressions were assessed in the samples of thoracic and abdominal aorta. Rosuvastatin significantly reduced MYD88, CCL4, CCL20, CCR2, TNF-α, IFN-ß, IL-1b, IL-2, IL-4, IL-8, and IL-10, both in the thoracic and abdominal aorta. Fluvastatin also downregulated MYD88, CCR2, IFN-ß, IFN-γ, IL-1b, IL-2, IL-4, and IL-10 in both aortic segments. Rosuvastatin curtailed the expression of CCL4, IFN-ß, IL-2, IL-4, and IL-10 more effectively than fluvastatin in both types of tissue. MYD88, TNF-α, IL-1b, and IL-8 showed a stronger downregulation with rosuvastatin compared to fluvastatin only in the thoracic aorta. The CCL20 and CCR2 levels reduced more extensively with rosuvastatin treatment only in abdominal aortic tissue. In conclusion, statin therapy can halt proatherogenic inflammation in hyperlipidemic animals. Rosuvastatin may be more effective in downregulating MYD88 in atherosclerotic thoracic aortas.

Single-cell profiling reveals age-associated immunity in atherosclerosis.

AIMS: Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr-/-) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans. METHODS AND RESULTS: Here, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr-/- mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leucocytes of young vs. aged Ldlr-/- mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK+CD8+ T-cells and previously in atherosclerosis undefined CD11b+CD11c+T-bet+ age-associated B-cells (ABCs). ABCs of Ldlr-/- mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood. CONCLUSIONS: Collectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease.

Biomechanical and transcriptional evidence that smooth muscle cell death drives an osteochondrogenic phenotype and severe proximal vascular disease in progeria.

Hutchinson-Gilford Progeria Syndrome results in rapid aging and severe cardiovascular sequelae that accelerate near end-of-life. We found a progressive disease process in proximal elastic arteries that was less evident in distal muscular arteries. Changes in aortic structure and function were then associated with changes in transcriptomics assessed via both bulk and single cell RNA sequencing, which suggested a novel sequence of progressive aortic disease: adverse extracellular matrix remodeling followed by mechanical stress-induced smooth muscle cell death, leading a subset of remnant smooth muscle cells to an osteochondrogenic phenotype that results in an accumulation of proteoglycans that thickens the aortic wall and increases pulse wave velocity, with late calcification exacerbating these effects. Increased central artery pulse wave velocity is known to drive left ventricular diastolic dysfunction, the primary diagnosis in progeria children. It appears that mechanical stresses above ~ 80 kPa initiate this progressive aortic disease process, explaining why elastic lamellar structures that are organized early in development under low wall stresses appear to be nearly normal whereas other medial constituents worsen progressively in adulthood. Mitigating early mechanical stress-driven smooth muscle cell loss/phenotypic modulation promises to have important cardiovascular implications in progeria patients.

Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol.

Patients with Marfan syndrome (MFS) develop thoracic aortic aneurysms as the aorta presents excessive elastin breaks, fibrosis, and vascular smooth muscle cell (vSMC) death due to mutations in the FBN1 gene. Despite elaborate vSMC to aortic endothelial cell (EC) signaling, the contribution of ECs to the development of aortic pathology remains largely unresolved. The aim of this study is to investigate the EC properties in Fbn1C1041G/+ MFS mice. Using en face immunofluorescence confocal microscopy, we showed that EC alignment with blood flow was reduced, EC roundness was increased, individual EC surface area was larger, and EC junctional linearity was decreased in aortae of Fbn1C1041G/+ MFS mice. This modified EC phenotype was most prominent in the ascending aorta and occurred before aortic dilatation. To reverse EC morphology, we performed treatment with resveratrol. This restored EC blood flow alignment, junctional linearity, phospho-eNOS expression, and improved the structural integrity of the internal elastic lamina of Fbn1C1041G/+ mice. In conclusion, these experiments identify the involvement of ECs and underlying internal elastic lamina in MFS aortic pathology, which could act as potential target for future MFS pharmacotherapies.

A Segmental Approach from Molecular Profiling to Medical Imaging to Study Bicuspid Aortic Valve Aortopathy.

Bicuspid aortic valve (BAV) patients develop ascending aortic (AAo) dilation. The pathogenesis of BAV aortopathy (genetic vs. haemodynamic) remains unclear. This study aims to identify regional changes around the AAo wall in BAV patients with aortopathy, integrating molecular data and clinical imaging. BAV patients with aortopathy (n = 15) were prospectively recruited to surgically collect aortic tissue and measure molecular markers across the AAo circumference. Dilated (anterior/right) vs. non-dilated (posterior/left) circumferential segments were profiled for whole-genomic microRNAs (next-generation RNA sequencing, miRCURY LNA PCR), protein content (tandem mass spectrometry), and elastin fragmentation and degeneration (histomorphometric analysis). Integrated bioinformatic analyses of RNA sequencing and proteomic datasets identified five microRNAs (miR-128-3p, miR-210-3p, miR-150-5p, miR-199b-5p, and miR-21-5p) differentially expressed across the AAo circumference. Among them, three miRNAs (miR-128-3p, miR-150-5p, and miR-199b-5p) were predicted to have an effect on eight common target genes, whose expression was dysregulated, according to proteomic analyses, and involved in the vascular-endothelial growth-factor signalling, Hippo signalling, and arachidonic acid pathways. Decreased elastic fibre levels and elastic layer thickness were observed in the dilated segments. Additionally, in a subset of patients n = 6/15, a four-dimensional cardiac magnetic resonance (CMR) scan was performed. Interestingly, an increase in wall shear stress (WSS) was observed at the anterior/right wall segments, concomitantly with the differentially expressed miRNAs and decreased elastic fibres. This study identified new miRNAs involved in the BAV aortic wall and revealed the concomitant expressional dysregulation of miRNAs, proteins, and elastic fibres on the anterior/right wall in dilated BAV patients, corresponding to regions of elevated WSS.

Sestrin2 Is Increased in Calcific Aortic Disease and Inhibits Osteoblastic Differentiation in Valvular Interstitial Cells via the Nuclear Factor E2-related Factor 2 Pathway.

ABSTRACT: Sestrin2 (Sesn2) is involved in the progression of cardiovascular diseases, such as hypertension and myocardial infarction. This study aimed to examine Sesn2 expression in human calcific aortic valve disease (CAVD) and explore its possible mechanisms by which Sesn2 participates in this process. CAVD and normal aortic valves were collected. Sesn2 expression and sources were examined, and the results showed that Sesn2 expression was increased in aortic valves from patients with CAVD and was mainly secreted by macrophages. Additionally, U937 macrophages were pretreated with si-Sesn2 or cDNA-Sesn2 and further treated with oxidized low-density lipoprotein (ox-LDL); M1 macrophages and their markers were measured, and we found that pretreatment with si-Sesn2 increased ox-LDL-induced M1 macrophage polarization and marker mRNA levels, whereas pretreatment with cDNA-Sesn2 had the opposite effects. In ox-LDL-treated U937 macrophages, oxidative stress levels were increased in the si-Sesn2 pretreatment group and further increased by si-Nrf2 treatment, whereas oxidative stress levels were decreased in the cDNA-Sesn2 pretreatment group and significantly reversed by ML385, a specific Nrf2 inhibitor. The effects of Sesn2 on ox-LDL-induced oxidative stress and the osteogenic differentiation of ox-LDL-induced valvular interstitial cells (VICs) was examined by down-regulating Nrf2 pathway. When U937 macrophages were co-cultured with VICs, downregulation of Sesn2 increased ox-LDL-induced osteogenic differentiation in VICs, whereas overexpression of Sesn2 exerted the opposite effects. Our study suggests that Sesn2 is increased in CAVD aortic valves and may participate in the development of CAVD by regulating oxidative stress via the Nrf2 pathway.