Yvonne Baumer

MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis

Objective—Macrophage foam cell formation is a key feature of atherosclerosis. Recent studies have shown that specific microRNAs (miRs) are regulated in modified low-density lipoprotein–treated macrophages, which can affect the cellular cholesterol homeostasis. Undertaking a genome-wide screen of miRs regulated in primary macrophages by modified low-density lipoprotein, miR-302a emerged as a potential candidate that may play a key role in macrophage cholesterol homeostasis. Approach and Results—The objective of this study was to assess the involvement of miR-302a in macrophage lipid homeostasis and if it can influence circulating lipid levels and atherosclerotic development when it is inhibited in a murine atherosclerosis model. We found that transfection of primary macrophages with either miR-302a or anti–miR-302a regulated the expression of ATP-binding cassette (ABC) transporter ABCA1 mRNA and protein. Luciferase reporter assays showed that miR-302a repressed the 3′ untranslated regions (UTR) activity of mouse Abca1 by 48% and human ABCA1 by 45%. In addition, transfection of murine macrophages with miR-302a attenuated cholesterol efflux to apolipoprotein A-1 (apoA-1) by 38%. Long-term in vivo administration of anti–miR-302a to mice with low-density lipoprotein receptor deficiency (Ldlr−/−) fed an atherogenic diet led to an increase in ABCA1 in the liver and aorta as well as an increase in circulating plasma high-density lipoprotein levels by 35% compared with that of control mice. The anti–miR-302a–treated mice also displayed reduced atherosclerotic plaque size by ≈25% and a more stable plaque morphology with reduced signs of inflammation. Conclusions—These studies identify miR-302a as a novel modulator of cholesterol efflux and a potential therapeutic target for suppressing atherosclerosis.

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

Background— Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Extracellular RNA (eRNA) has recently been implicated to become enriched at sites of tissue damage and to act as a proinflammatory mediator. Here, we addressed the role of eRNA in high-fat diet–induced atherosclerosis and neointima formation after injury in atherosclerosis-prone mice. Methods and Results— The presence of eRNA was revealed in atherosclerotic lesions from high-fat diet–fed low-density lipoprotein receptor–deficient (Ldlr−/−) mice in a time-progressive fashion. RNase activity in plasma increased within the first 2 weeks (44±9 versus 70±7 mU/mg protein; P=0.0012), followed by a decrease to levels below baseline after 4 weeks of high-fat diet (44±9 versus 12±2 mU/mg protein; P<0.0001). Exposure of bone marrow–derived macrophages to eRNA resulted in a concentration-dependent upregulation of the proinflammatory mediators tumor necrosis factor-&agr;, arginase-2, interleukin-1&bgr;, interleukin-6, and interferon-&ggr;. In a model of accelerated atherosclerosis after arterial injury in apolipoprotein E–deficient (ApoE−/−) mice, treatment with RNase1 diminished the increased plasma level of eRNA evidenced after injury. Likewise, RNase1 administration reduced neointima formation in comparison with vehicle-treated ApoE−/− controls (25.0±6.2 versus 46.9±6.9×103 &mgr;m2, P=0.0339) and was associated with a significant decrease in plaque macrophage content. Functionally, RNase1 treatment impaired monocyte arrest on activated smooth muscle cells under flow conditions in vitro and inhibited leukocyte recruitment to injured carotid arteries in vivo. Conclusions— Because eRNA is associated with atherosclerotic lesions and contributes to inflammation-dependent plaque progression in atherosclerosis-prone mice, its targeting with RNase1 may serve as a new treatment option against atherosclerosis.

IL-32 Promotes Angiogenesis

IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVβ3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA–mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-β levels, but siIL-32–transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVβ3 inhibitor prevented this activity and reduced IL-32γ–induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVβ3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Selenoprotein K is required for palmitoylation of CD36 in macrophages: implications in foam cell formation and atherogenesis

Selk is an ER transmembrane protein important for calcium flux and macrophage activation, but its role in foam cell formation and atherosclerosis has not been evaluated. BMDMs from Selk−/− mice exhibited decreased uptake of modLDL and foam cell formation compared with WT controls, and the differences were eliminated with anti‐CD36 blocking antibody. CD36 expression was decreased in TNF‐α‐stimulated Selk−/− BMDMs compared with WT controls. Fluorescence microscopy revealed TNF‐α‐induced clustering of CD36 in WT BMDMs indicative of lipid raft localization, which was absent in Selk−/− BMDMs. Fractionation revealed lower levels of CD36 reaching lipid rafts in TNF‐α‐stimulated Selk−/− BMDMs. Immunoprecipitation showed that Selk−/− BMDMs have decreased CD36 palmitoylation, which occurs at the ER membrane and is crucial for stabilizing CD36 expression and directing its localization to lipid rafts. To assess if this phenomenon had a role in atherogenesis, a HFD was fed to irradiated Ldlr−/− mice reconstituted with BM from Selk−/− or WT mice. Selk was detected in aortic plaques of controls, particularly in macrophages. Selk−/− in immune cells led to reduction in atherosclerotic lesion formation without affecting leukocyte migration into the arterial wall. These findings suggest that Selk is important for stable, localized expression of CD36 in macrophages during inflammation, thereby contributing to foam cell formation and atherogenesis.

Association Between Skin and Aortic Vascular Inflammation in Patients With Psoriasis: A Case-Cohort Study Using Positron Emission Tomography/Computed Tomography

Importance Inflammation is critical in the development of atherosclerosis. Psoriasis is a chronic inflammatory skin disease that is associated with increased vascular inflammation by 18fluorodeoxyglucose positron emission tomography/computed tomography in vivo and future cardiovascular events. It provides a human model to understand the effect of treating inflammation in a target organ (eg, the skin) on vascular diseases. Objective To investigate the association between change in skin disease severity and change in vascular inflammation at 1 year and to characterize the impact of 1 year of anti–tumor necrosis factor therapy on vascular inflammation. Design, Setting, and Participants In this prospective cohort study, 220 participants from outpatient practices were recruited at the US National Institutes of Health. A total of 115 consecutively recruited patients with psoriasis were followed up at 1 year. The study was conducted from January 1, 2013, through October 31, 2016, with data analyzed in November 2016. Exposure Skin inflammation measured as Psoriasis Area and Severity Index (PASI) score. Main Outcomes and Measures Vascular inflammation assessed as target-to-background ratio by 18fluorodeoxyglucose positron emission tomography/computed tomography. Results Among the 115 patients, the mean (SD) age at 1-year follow-up was 50.8 (12.8) years and 68 were men (59%). The cohort had a low cardiovascular risk by Framingham risk score and mild-to-moderate psoriasis, with a median PASI score of 5.2 (interquartile range, 3.0-8.9). At follow-up, the total cohort had a median improvement in PASI score of 33%, with use of topical therapy (60%), biological therapy (66%, mostly anti–tumor necrosis factor) and phototherapy (15%) (P < .001). Moreover, improvement in PASI score was associated with improvement in target-to-background ratio of 6%, mainly driven by those with higher responses in PASI score (P < .001). This association persisted beyond traditional risk factors (&bgr; = 0.19; 95% CI, 0.012-0.375; P = .03) and was the strongest in those initiated with anti–tumor necrosis factor therapy (&bgr; = 0.79; 95% CI, 0.269-1.311; P = .03). Conclusions and Relevance Improvement in psoriasis skin disease severity was associated with improvement in aortic vascular inflammation by 18fluorodeoxyglucose positron emission tomography/computed tomography, with greater improvement in aortic vascular inflammation observed in those who had higher than 75% reduction in skin disease severity. These findings suggest that controlling remote target organ inflammation (eg, in the skin) may improve vascular diseases; however, randomized clinical trials are needed to confirm these findings.

Response to Letter Regarding Article “Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice”

We acknowledge the interest and comments of Dr Chen et al regarding our findings that self-extracellular RNA (eRNA) significantly contributes to atherogenesis (as demonstrated in 2 established animal models) by inducing a prominent inflammatory response in situ and in bone marrow–derived macrophages (BMDM), as well.1 In particular, Chen et al question whether eRNA-dependent effects may have been mediated by Toll-like receptor (TLR)–related signaling, because they recently reported that BMDM responses toward the RNA analogue poly(IC) were significantly dampened in TLR3-deficient cells.2 During the past decade, our laboratory has characterized a number of new functions of eRNA in inflammation and cardiovascular diseases. In the indicated study, we aimed to characterize a causal role of natural eRNA, which may also serve as a cell-injury marker, in the onset and progression of atherosclerosis. It …

Telomerase-based immortalization modifies the angiogenic/inflammatory responses of human coronary artery endothelial cells.

Telomerase reverse transcriptase (TERT) is fundamental in determining the life span by regulating telomere length of chromosomes. To address the question whether the enhancement of the proliferative potential hampers cell differentiation, we generated TERT-over-expressing endothelial cells (ECs) and analyzed in vitro their (1) barrier function; (2) low-density lipoprotein uptake; (3) expression pattern of six selected marker proteins; (4) angiogenic potential in four assays; and (5) inflammatory responses. In contrast to investigations with focus on other cell parameters, we demonstrate that immortalization of ECs by over-expression of TERT resulted in different angiogenic and inflammatory behavior in comparison to cells with low native telomerase levels.

IFN-γ and TNF-α synergism may provide a link between psoriasis and inflammatory atherogenesis

Chronic inflammation is a critical component of atherogenesis, however, reliable human translational models aimed at characterizing these mechanisms are lacking. Psoriasis, a chronic inflammatory skin disease associated with increased susceptibility to atherosclerosis, provides a clinical human model that can be utilized to investigate the links between chronic inflammation and atherosclerosis development. We sought to investigate key biological processes in psoriasis skin and human vascular tissue to identify biological components that may promote atherosclerosis in chronic inflammatory conditions. Using a bioinformatics approach of human skin and vascular tissue, we determined IFN-γ and TNF-α are the dominant pro-inflammatory signals linking atherosclerosis and psoriasis. We then stimulated primary aortic endothelial cells and ex-vivo atherosclerotic tissue with IFN-γ and TNF-α and found they synergistically increased monocyte and T-cell chemoattractants, expression of adhesion molecules on the endothelial cell surface, and decreased endothelial barrier integrity in vitro, therefore increasing permeability. Our data provide strong evidence of synergism between IFN-γ and TNF- α in inflammatory atherogenesis and provide rationale for dual cytokine antagonism in future studies.

Hyperlipidemia-induced cholesterol crystal production by endothelial cells promotes atherogenesis

Endothelial cells (EC) play a key role in atherosclerosis. Although EC are in constant contact with low density lipoproteins (LDL), how EC process LDL and whether this influences atherogenesis, is unclear. Here we show that EC take up and metabolize LDL, and when overburdened with intracellular cholesterol, generate cholesterol crystals (CC). The CC are deposited on the basolateral side, and compromise endothelial function. When hyperlipidemic mice are given a high fat diet, CC appear in aortic sinus within 1 week. Treatment with cAMP-enhancing agents, forskolin/rolipram (F/R), mitigates effects of CC on endothelial function by not only improving barrier function, but also inhibiting CC formation both in vitro and in vivo. A proof of principle study using F/R incorporated into liposomes, designed to target inflamed endothelium, shows reduced atherosclerosis and CC formation in ApoE−/− mice. Our findings highlight an important mechanism by which EC contribute to atherogenesis under hyperlipidemic conditions.Atherosclerosis is characterized by subendothelial lipid retention believed to be the result of endothelial trancytosis. Here, the authors show that endothelium can take up and process LDL, generating cholesterol crystals that are deposited on the basolateral side of the cells, causing their dysfunction that can be prevented by forskolin/rolipram treatment.

Chronic skin inflammation accelerates macrophage cholesterol crystal formation and atherosclerosis

Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12-/+/Srb1-/-/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis.