Mitochondrial fission inhibition protects against hypertension induced by angiotensin II.

Mitochondrial dysfunction has been implicated in various types of cardiovascular disease including hypertension. Mitochondrial fission fusion balance is critical to mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular disease. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. Here, we have tested the hypothesis that inhibition of mitochondrial fission attenuates the development of hypertension and associated vascular remodeling. In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor, mdivi1, significantly inhibited angiotensin II-induced development of hypertension assessed by radiotelemetry. Histological assessment of hearts and aortas showed that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both the endothelial and medial layers of aortas. Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight-to-body weight ratio as well as by echocardiography. In ex vivo experiments, mdivi1 inhibited vasoconstriction and abolished the enhanced vascular reactivity by angiotensin II in small mesenteric arteries. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature.

Differential regulation of lymphatic junctional morphology and the potential effects on cardiovascular diseases.

The lymphatic vasculature provides an essential route to drain fluid, macromolecules, and immune cells from the interstitium as lymph, returning it to the bloodstream where the thoracic duct meets the subclavian vein. To ensure functional lymphatic drainage, the lymphatic system contains a complex network of vessels which has differential regulation of unique cell-cell junctions. The lymphatic endothelial cells lining initial lymphatic vessels form permeable "button-like" junctions which allow substances to enter the vessel. Collecting lymphatic vessels form less permeable "zipper-like" junctions which retain lymph within the vessel and prevent leakage. Therefore, sections of the lymphatic bed are differentially permeable, regulated in part by its junctional morphology. In this review, we will discuss our current understanding of regulating lymphatic junctional morphology, highlighting how it relates to lymphatic permeability during development and disease. We will also discuss the effect of alterations in lymphatic permeability on efficient lymphatic flux in health and how it may affect cardiovascular diseases, with a focus on atherosclerosis.

FXR1 regulates vascular smooth muscle cell cytoskeleton, VSMC contractility, and blood pressure by multiple mechanisms.

Appropriate cytoskeletal organization is essential for vascular smooth muscle cell (VSMC) conditions such as hypertension. This study identifies FXR1 as a key protein linking cytoskeletal dynamics with mRNA stability. RNA immunoprecipitation sequencing (RIP-seq) in human VSMCs identifies that FXR1 binds to mRNA associated with cytoskeletal dynamics, and FXR1 depletion decreases their mRNA stability. FXR1 binds and regulates actin polymerization. Mass spectrometry identifies that FXR1 interacts with cytoskeletal proteins, particularly Arp2, a protein crucial for VSMC contraction, and CYFIP1, a WASP family verprolin-homologous protein (WAVE) regulatory complex (WRC) protein that links mRNA processing with actin polymerization. Depletion of FXR1 decreases the cytoskeletal processes of adhesion, migration, contraction, and GTPase activation. Using telemetry, conditional FXR1SMC/SMC mice have decreased blood pressure and an abundance of cytoskeletal-associated transcripts. This indicates that FXR1 is a muscle-enhanced WRC modulatory protein that regulates VSMC cytoskeletal dynamics by regulation of cytoskeletal mRNA stability and actin polymerization and cytoskeletal protein-protein interactions, which can regulate blood pressure.

Genetic Deletion of FXR1 Reduces Intimal Hyperplasia and Induces Senescence in Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis and other vascular diseases. Fragile-X related protein-1 (FXR1) is a muscle-enhanced RNA binding protein whose expression is increased in injured arteries. Previous studies suggest that FXR1 negatively regulates inflammation, but its causality in vascular disease is unknown. In the current study, RNA-sequencing of FXR1-depleted VSMC identified many transcripts with decreased abundance, most of which were associated with proliferation and cell division. mRNA abundance and stability of a number of these transcripts were decreased in FXR1-depleted hVSMC, as was proliferation (P < 0.05); however, increases in beta-galactosidase (P < 0.05) and γH2AX (P < 0.01), indicative of senescence, were noted. Further analysis showed increased abundance of senescence-associated genes with FXR1 depletion. A novel SMC-specific conditional knockout mouse (FXR1SMC/SMC) was developed for further analysis. In a carotid artery ligation model of intimal hyperplasia, FXR1SMC/SMC mice had significantly reduced neointima formation (P < 0.001) after ligation, as well as increases in senescence drivers p16, p21, and p53 compared with several controls. These results suggest that in addition to destabilization of inflammatory transcripts, FXR1 stabilized cell cycle-related genes in VSMC, and absence of FXR1 led to induction of a senescent phenotype, supporting the hypothesis that FXR1 may mediate vascular disease by regulating stability of proliferative mRNA in VSMC.

Deletion of LDLRAP1 Induces Atherosclerotic Plaque Formation, Insulin Resistance, and Dysregulated Insulin Response in Adipose Tissue.

Dyslipidemia, vascular inflammation, obesity, and insulin resistance often overlap and exacerbate each other. Mutations in low density lipoprotein receptor adaptor protein-1 (LDLRAP1) lead to LDLR malfunction and are associated with the autosomal recessive hypercholesterolemia disorder in humans. However, direct causality on atherogenesis in a defined preclinical model has not been reported. The objective of this study was to test the hypothesis that deletion of LDLRAP1 will lead to hypercholesteremia and atherosclerosis. LDLRAP1-/- mice fed a high-fat Western diet had significantly increased plasma cholesterol and triglyceride concentrations accompanied with significantly increased plaque burden compared with wild-type controls. Unexpectedly, LDLRAP1-/- mice gained significantly more weight compared with controls. Even on a chow diet, LDLRAP1-/- mice were insulin-resistant, and calorimetric studies suggested an altered metabolic profile. The study showed that LDLRAP1 is highly expressed in visceral adipose tissue, and LDLRAP1-/- adipocytes are significantly larger, have reduced glucose uptake and AKT phosphorylation, but have increased CD36 expression. Visceral adipose tissue from LDLRAP1-/- mice was hypoxic and had gene expression signatures of dysregulated lipid storage and energy homeostasis. These data are the first to indicate that lack of LDLRAP1 directly leads to atherosclerosis in mice and also plays an unanticipated metabolic regulatory role in adipose tissue. LDLRAP1 may link atherosclerosis and hypercholesterolemia with common comorbidities of obesity and insulin resistance.

Adipocyte Phenotype Flexibility and Lipid Dysregulation.

The prevalence of obesity and associated cardiometabolic diseases continues to rise, despite efforts to improve global health. The adipose tissue is now regarded as an endocrine organ since its multitude of secretions, lipids chief among them, regulate systemic functions. The loss of normal adipose tissue phenotypic flexibility, especially related to lipid homeostasis, appears to trigger cardiometabolic pathogenesis. The goal of this manuscript is to review lipid balance maintenance by the lean adipose tissue's propensity for phenotype switching, obese adipose tissue's narrower range of phenotype flexibility, and what initial factors account for the waning lipid regulatory capacity. Metabolic, hypoxic, and inflammatory factors contribute to the adipose tissue phenotype being made rigid. A better grasp of normal adipose tissue function provides the necessary context for recognizing the extent of obese adipose tissue dysfunction and gaining insight into how pathogenesis evolves.

Challenging the Paradigm: Anti-Inflammatory Interleukins and Angiogenesis.

Angiogenesis is a vital biological process, and neovascularization is essential for the development, wound repair, and perfusion of ischemic tissue. Neovascularization and inflammation are independent biological processes that are linked in response to injury and ischemia. While clear that pro-inflammatory factors drive angiogenesis, the role of anti-inflammatory interleukins in angiogenesis remains less defined. An interleukin with anti-inflammatory yet pro-angiogenic effects would hold great promise as a therapeutic modality to treat many disease states where inflammation needs to be limited, but revascularization and reperfusion still need to be supported. As immune modulators, interleukins can polarize macrophages to a pro-angiogenic and reparative phenotype, which indirectly influences angiogenesis. Interleukins could also potentially directly induce angiogenesis by binding and activating its receptor on endothelial cells. Although a great deal of attention is given to the negative effects of pro-inflammatory interleukins, less is described concerning the potential protective effects of anti-inflammatory interleukins on various disease processes. To focus this review, we will consider IL-4, IL-10, IL-13, IL-19, and IL-33 to be anti-inflammatory interleukins, all of which have recognized immunomodulatory effects. This review will summarize current research concerning anti-inflammatory interleukins as potential drivers of direct and indirect angiogenesis, emphasizing their role in future therapeutics.

Glucose consumption of vascular cell types in culture: toward optimization of experimental conditions.

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ∼275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Identification of Gm15441, a Txnip antisense lncRNA, as a critical regulator in liver metabolic homeostasis.

BACKGROUND: The majority of mammalian genome is composed of non-coding regions, where numerous long non-coding RNAs (lncRNAs) are transcribed. Although lncRNAs have been identified to regulate fundamental biological processes, most of their functions remain unknown, especially in metabolic homeostasis. Analysis of our recent genome-wide screen reveals that Gm15441, a thioredoxin-interacting protein (Txnip) antisense lncRNA, is the most robustly induced lncRNA in the fasting mouse liver. Antisense lncRNAs are known to regulate their sense gene expression. Given that Txnip is a critical metabolic regulator of the liver, we aimed to investigate the role of Gm15441 in the regulation of Txnip and liver metabolism. METHODS: We examined the response of Gm15441 and Txnip under in vivo metabolic signals such as fasting and refeeding, and in vitro signals such as insulin and key metabolic transcription factors. We investigated the regulation of Txnip expression by Gm15441 and the underlying mechanism in mouse hepatocytes. Using adenovirus-mediated liver-specific overexpression, we determined whether Gm15441 regulates Txnip in the mouse liver and modulates key aspects of liver metabolism. RESULTS: We found that the expression levels of Gm15441 and Txnip showed a similar response pattern to metabolic signals in vivo and in vitro, but that their functions were predicted to be opposite. Furthermore, we found that Gm15441 robustly reduced Txnip protein expression in vitro through sequence-specific regulation and translational inhibition. Lastly, we confirmed the Txnip inhibition by Gm15441 in vivo (mice) and found that Gm15441 liver-specific overexpression lowered plasma triglyceride and blood glucose levels and elevated plasma ketone body levels. CONCLUSIONS: Our data demonstrate that Gm15441 is a potent Txnip inhibitor and a critical metabolic regulator in the liver. This study reveals the therapeutic potential of Gm15441 in treating metabolic diseases.

Promyelocytic leukemia protein: an atherosclerosis suppressor protein?

As many as 70% of cells in atherosclerotic plaque are vascular smooth muscle cell (VSMC) in origin, and pathways and proteins which regulate VSMC migration, proliferation, and phenotype modulation represent novel targets for rational drug design to reduce atherosclerotic vascular disease. In this volume of Clinical Science, Karle et al. demonstrate that tumor suppressor, promyelocytic leukemia protein (PML) plays an important role in regulation of VSMC phenotype and response to inflammatory stimuli (Clin Sci (2021) 135(7), 887-905; DOI: 10.1042/CS20201399). This important work demonstrates that PML, previously unrecognized as a participant in development of atherosclerosis, may represent a novel target for anti-atherosclerotic therapeutic modalities.

Adipose tissue inflammation and metabolic dysfunction in obesity.

Several lines of preclinical and clinical research have confirmed that chronic low-grade inflammation of adipose tissue is mechanistically linked to metabolic disease and organ tissue complications in the overweight and obese organism. Despite this widely confirmed paradigm, numerous open questions and knowledge gaps remain to be investigated. This is mainly due to the intricately intertwined cross-talk of various pro- and anti-inflammatory signaling cascades involved in the immune response of expanding adipose depots, particularly the visceral adipose tissue. Adipose tissue inflammation is initiated and sustained over time by dysfunctional adipocytes that secrete inflammatory adipokines and by infiltration of bone marrow-derived immune cells that signal via production of cytokines and chemokines. Despite its low-grade nature, adipose tissue inflammation negatively impacts remote organ function, a phenomenon that is considered causative of the complications of obesity. The aim of this review is to broadly present an overview of adipose tissue inflammation by highlighting the most recent reports in the scientific literature and summarizing our overall understanding of the field. We also discuss key endogenous anti-inflammatory mediators and analyze their mechanistic role(s) in the pathogenesis and treatment of adipose tissue inflammation. In doing so, we hope to stimulate studies to uncover novel physiological, cellular, and molecular targets for the treatment of obesity.

Resolution of inflammation in immune and nonimmune cells by interleukin-19.

The inflammatory response is a complex, tightly regulated process activated by tissue wounding, foreign body invasion, and sterile inflammation. Over the decades, great progress has been made to advance our understanding of this process. One often overlooked aspect of inflammation is its sequel: resolution. We know that dysregulated resolution often results in numerous chronic degenerative diseases such as arthritis, cancer, and asthma. However, identification of components and mechanisms of resolving pathways lags behind those of proinflammatory processes, yet represents overlooked therapeutic opportunities. One approach is identification of endogenous, negative compensatory mechanisms, which are activated in response to inflammation for the purpose of resolution of that inflammatory stimuli. This review will focus on literature that describes expression and function of interleukin-19, a proposed anti-inflammatory cytokine, in numerous inflammatory diseases. The literature concerning IL-19 is complex, context-dependent, and often contradictory. The expression and function of IL-19 in the inflammatory response are in no way settled. We will attempt to clarify the role that this interesting and understudied cytokine plays in resolution of inflammation and discuss its mechanisms of action in different cell types. We will present a hypothesis that endogenous IL-19 expression in response to inflammatory stimuli is a cellular compensatory mechanism to dampen inflammation. We further present studies suggesting that while endogenously expressed IL-19 may be a response to inflammation, pharmacological levels may be necessary to effectively resolve the inflammatory cascade.

Angiotensin II, Hypercholesterolemia, and Vascular Smooth Muscle Cells: A Perfect Trio for Vascular Pathology.

Cardiovascular disease is the leading cause of morbidity and mortality in the Western and developing world, and the incidence of cardiovascular disease is increasing with the longer lifespan afforded by our modern lifestyle. Vascular diseases including coronary heart disease, high blood pressure, and stroke comprise the majority of cardiovascular diseases, and therefore represent a significant medical and socioeconomic burden on our society. It may not be surprising that these conditions overlap and potentiate each other when we consider the many cellular and molecular similarities between them. These intersecting points are manifested in clinical studies in which lipid lowering therapies reduce blood pressure, and anti-hypertensive medications reduce atherosclerotic plaque. At the molecular level, the vascular smooth muscle cell (VSMC) is the target, integrator, and effector cell of both atherogenic and the major effector protein of the hypertensive signal Angiotensin II (Ang II). Together, these signals can potentiate each other and prime the artery and exacerbate hypertension and atherosclerosis. Therefore, VSMCs are the fulcrum in progression of these diseases and, therefore, understanding the effects of atherogenic stimuli and Ang II on the VSMC is key to understanding and treating atherosclerosis and hypertension. In this review, we will examine studies in which hypertension and atherosclerosis intersect on the VSMC, and illustrate common pathways between these two diseases and vascular aging.

Regulation of Stress Granule Formation by Inflammation, Vascular Injury, and Atherosclerosis.

OBJECTIVE: Stress granules (SGs) are dynamic cytoplasmic aggregates containing mRNA, RNA-binding proteins, and translation factors that form in response to cellular stress. SGs have been shown to contribute to the pathogenesis of several human diseases, but their role in vascular diseases is unknown. This study shows that SGs accumulate in vascular smooth muscle cells (VSMCs) and macrophages during atherosclerosis. Approach and Results: Immunohistochemical analysis of atherosclerotic plaques from LDLR-/- mice revealed an increase in the stress granule-specific markers Ras-G3BP1 (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP1+ SGs were rapidly induced in VSMC and bone marrow-derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α (eukaryotic translation initiation factor 2-alpha) phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. CONCLUSIONS: These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.

Postprandial activation of leukocyte-endothelium interaction by fatty acids in the visceral adipose tissue microcirculation.

High-fat diet (HFD)-induced obesity is associated with accumulation of inflammatory cells predominantly in visceral adipose depots [visceral adipose tissue (VAT)] rather than in subcutaneous ones [subcutaneous adipose tissue (SAT)]. The cellular and molecular mechanisms responsible for this phenotypic difference remain poorly understood. Controversy also exists on the overall impact that adipose tissue inflammation has on metabolic health in diet-induced obesity. The endothelium of the microcirculation regulates both the transport of lipids and the trafficking of leukocytes into organ tissue. We hypothesized that the VAT and SAT microcirculations respond differently to postprandial processing of dietary fat. We also tested whether inhibition of endothelial postprandial responses to high-fat meals (HFMs) preserves metabolic health in chronic obesity. We demonstrate that administration of a single HFM or ad libitum access to a HFD for 24 h quickly induces a transient P-selectin-dependent inflammatory phenotype in the VAT but not the SAT microcirculation of lean wild-type mice. Studies in P-selectin-deficient mice confirmed a mechanistic role for P-selectin in the initiation of leukocyte trafficking, myeloperoxidase accumulation, and acute reduction in adiponectin mRNA expression by HFMs. Despite reduced VAT inflammation in response to HFMs, P-selectin-deficient mice still developed glucose intolerance and insulin resistance when chronically fed an HFD. Our data uncover a novel nutrient-sensing role of the vascular endothelium that instigates postprandial VAT inflammation. They also demonstrate that inhibition of this transient postprandial inflammatory response fails to correct metabolic dysfunction in diet-induced obesity.-Preston, K. J., Rom, I., Vrakas, C., Landesberg, G., Etwebe, Z., Muraoka, S., Autieri, M., Eguchi, S., Scalia, R. Postprandial activation of leukocyte-endothelium interaction by fatty acids in the visceral adipose tissue microcirculation.

Regulation of pro- and anti-atherogenic cytokines.

Despite advances in prevention and treatment, vascular diseases continue to account for significant morbidity and mortality in the developed world. Incidence is expected to worsen as the number of patients with common co-morbidities linked with atherosclerotic vascular disease, such as obesity and diabetes, continues to increase, reaching epidemic proportions. Atherosclerosis is a lipid-driven vascular inflammatory disease involving multiple cell types in various stages of inflammation, activation, apoptosis, and necrosis. One commonality among these cell types is that they are activated and communicate with each other in a paracrine fashion via a complex network of cytokines. Cytokines mediate atherogenesis by stimulating expression of numerous proteins necessary for induction of a host of cellular responses, including inflammation, extravasation, proliferation, apoptosis, and matrix production. Cytokine expression is regulated by a number of transcriptional and post-transcriptional mechanisms. In this context, proteins that control and fine-tune cytokine expression can be considered key players in development of atherosclerosis and also represent targets for rational drug therapy to combat this disease. This review will describe the cellular and molecular mechanisms that drive atherosclerotic plaque progression and present key cytokines that participate in this process. We will also describe RNA binding proteins that mediate cytokine mRNA stability and regulate cytokine abundance. Identification and characterization of the cytokines and proteins that regulate their abundance are essential to our ability to identify therapeutic approaches to ameliorate atherosclerotic vascular disease.

RNA stability protein ILF3 mediates cytokine-induced angiogenesis.

Interleukin enhancer-binding factor 3 (ILF3), an RNA-binding protein, is best known for its role in innate immunity by participation in cellular antiviral responses. A role for ILF3 in angiogenesis is unreported. ILF3 expression in CD31+ capillaries of hypoxic cardiac tissue was detected by immunohistochemistry. Proangiogenic stimuli induce ILF3 mRNA and protein expression in cultured human coronary artery endothelial cells (hCAECs). Angiogenic indices, including proliferation, migration, and tube formation, are all significantly reduced in hCAECs when ILF3 is knocked down using small interfering RNA (siRNA), but are significantly increased when ILF3 is overexpressed using adenovirus. Protein and mRNA abundance of several angiogenic factors including CXCL1, VEGF, and IL-8 are decreased when ILF3 is knocked down by siRNA. These factors are increased when ILF3 is overexpressed by adenovirus. ILF3 is phosphorylated and translocates from the nucleus to the cytoplasm in response to angiogenic stimuli. Proangiogenic transcripts containing adenine and uridine-rich elements were bound to ILF3 through RNA immunoprecipitation. ILF3 stabilizes proangiogenic transcripts including VEGF, CXCL1, and IL-8 in hCAECs. Together these data suggest that in endothelial cells, the RNA stability protein, ILF3, plays a novel and central role in angiogenesis. Our working hypothesis is that ILF3 promotes angiogenesis through cytokine-inducible mRNA stabilization of proangiogenic transcripts.-Vrakas, C. N., Herman, A. B., Ray, M., Kelemen, S. E., Scalia, R., Autieri, M. V. RNA stability protein ILF3 mediates cytokine-induced angiogenesis.

FXR1 Is an IL-19-Responsive RNA-Binding Protein that Destabilizes Pro-inflammatory Transcripts in Vascular Smooth Muscle Cells.

This work identifies the fragile-X-related protein (FXR1) as a reciprocal regulator of HuR target transcripts in vascular smooth muscle cells (VSMCs). FXR1 was identified as an HuR-interacting protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HuR-FXR1 interaction is abrogated in RNase-treated extracts, indicating that their association is tethered by mRNAs. FXR1 expression is induced in diseased but not normal arteries. siRNA knockdown of FXR1 increases the abundance and stability of inflammatory mRNAs, while overexpression of FXR1 reduces their abundance and stability. Conditioned media from FXR1 siRNA-treated VSMCs enhance activation of naive VSMCs. RNA EMSA and RIP demonstrate that FXR1 interacts with an ARE and an element in the 3' UTR of TNFα. FXR1 expression is increased in VSMCs challenged with the anti-inflammatory cytokine IL-19, and FXR1 is required for IL-19 reduction of HuR. This suggests that FXR1 is an anti-inflammation responsive, HuR counter-regulatory protein that reduces abundance of pro-inflammatory transcripts.

Genetic Deletion of IL-19 (Interleukin-19) Exacerbates Atherogenesis in Il19-/-×Ldlr-/- Double Knockout Mice by Dysregulation of mRNA Stability Protein HuR (Human Antigen R).

OBJECTIVE: To test the hypothesis that loss of IL-19 (interleukin-19) exacerbates atherosclerosis. APPROACH AND RESULTS: Il19-/- mice were crossed into Ldlr-/- (low-density lipoprotein receptor knock out) mice. Double knockout (dKO) mice had increased plaque burden in aortic arch and root compared with Ldlr-/- controls after 14 weeks of high-fat diet (HFD). dKO mice injected with 10 ng/g per day rmIL-19 had significantly less plaque compared with controls. qRT-PCR and Western blot analysis revealed dKO mice had increased systemic and intraplaque polarization of T cells and macrophages to proinflammatory Th1 and M1 phenotypes, and also significantly increased TNF (tumor necrosis factor)-α expression in spleen and aortic arch compared with Ldlr-/- controls. Bone marrow transplantation suggests that immune cells participate in IL-19 protection. Bone marrow-derived macrophages and vascular smooth muscle cells isolated from dKO mice had a significantly greater expression of inflammatory cytokine mRNA and protein compared with controls. Spleen and aortic arch from dKO mice had significantly increased expression of the mRNA stability protein HuR (human antigen R). Bone marrow-derived macrophage and vascular smooth muscle cell isolated from dKO mice also had greater HuR abundance. HuR stabilizes proinflammatory transcripts by binding AU-rich elements in the 3' untranslated region. Cytokine and HuR mRNA stability were increased in dKO bone marrow-derived macrophage and vascular smooth muscle cell, which was rescued by addition of IL-19 to these cells. IL-19-induced expression of miR133a, which targets and reduced HuR abundance; miR133a levels were lower in dKO mice compared with controls. CONCLUSIONS: These data indicate that IL-19 is an atheroprotective cytokine which decreases the abundance of HuR, leading to reduced inflammatory mRNA stability.