Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.

N-Acetyl Galactosamine Targeting: Paving the Way for Clinical Application of Nucleotide Medicines in Cardiovascular Diseases.

While the promise of oligonucleotide therapeutics, such as (chemically modified) ASO (antisense oligonucleotides) and short interfering RNAs, is undisputed from their introduction onwards, their unfavorable pharmacokinetics and intrinsic capacity to mobilize innate immune responses, were limiting widespread clinical use. However, these major setbacks have been tackled by breakthroughs in chemistry, stability and delivery. When aiming an intervention hepatic targets, such as lipid and sugar metabolism, coagulation, not to mention cancer and virus infection, introduction of N-acetylgalactosamine aided targeting technology has advanced the field profoundly and by now a dozen of N-acetylgalactosamine therapeutics for these indications have been approved for clinical use or have progressed to clinical trial stage 2 to 3 testing. This technology, in combination with major advances in oligonucleotide stability allows safe and durable intervention in targets that were previously deemed undruggable, such as Lp(a) and PCSK9 (proprotein convertase subtilisin/kexin type 9), at high efficacy and specificity, often with as little as 2 doses per year. Their successful use even the most visionary would not have predicted 2 decades ago. Here, we will review the evolution of N-acetylgalactosamine technology. We shall outline their fundamental design principles and merits, and their application for the delivery of oligonucleotide therapeutics to the liver. Finally, we will discuss the perspectives of N-acetylgalactosamine technology and propose directions for future research in receptor targeted delivery of these gene medicines.

Novel Plaque Enriched Long Noncoding RNA in Atherosclerotic Macrophage Regulation (PELATON).

OBJECTIVE: Long noncoding RNAs (lncRNAs) are an emergent class of molecules with diverse functional roles, widely expressed in human physiology and disease. Although some lncRNAs have been identified in cardiovascular disease, their potential as novel targets in the prevention of atherosclerosis is unknown. We set out to discover important lncRNAs in unstable plaque and gain insight into their functional relevance. Approach and Results: Analysis of RNA sequencing previously performed on stable and unstable atherosclerotic plaque identified a panel of 47 differentially regulated lncRNAs. We focused on LINC01272, a lncRNA upregulated in unstable plaque previously detected in inflammatory bowel disease, which we termed PELATON (plaque enriched lncRNA in atherosclerotic and inflammatory bowel macrophage regulation). Here, we demonstrate that PELATON is highly monocyte- and macrophage-specific across vascular cell types, and almost entirely nuclear by cellular fractionation (90%-98%). In situ hybridization confirmed enrichment of PELATON in areas of plaque inflammation, colocalizing with macrophages around the shoulders and necrotic core of human plaque sections. Consistent with its nuclear localization, and despite containing a predicted open reading frame, PELATON did not demonstrate any protein-coding potential in vitro. Functionally, knockdown of PELATON significantly reduced phagocytosis, lipid uptake and reactive oxygen species production in high-content analysis, with a significant reduction in phagocytosis independently validated. Furthermore, CD36, a key mediator of phagocytic oxLDL (oxidized low-density lipoprotein) uptake was significantly reduced with PELATON knockdown. CONCLUSIONS: PELATON is a nuclear expressed, monocyte- and macrophage-specific lncRNA, upregulated in unstable atherosclerotic plaque. Knockdown of PELATON affects cellular functions associated with plaque progression.

Urocortin 2 Gene Transfer Improves Heart Function in Aged Mice.

Prevalence of left ventricular (LV) systolic and diastolic dysfunction increases with aging. We previously reported that urocortin 2 (Ucn2) gene transfer increases heart function in mice with heart failure with reduced ejection fraction. Here, we test the hypotheses that (1) Ucn2 gene transfer will increase LV function in aged mice and that (2) Ucn2 gene transfer given in early life will prevent age-related LV dysfunction. Nineteen-month-old (treatment study) and 3-month-old (prevention study) mice received Ucn2 gene transfer or saline. LV function was examined 3-4 months (treatment study) or 20 months (prevention study) after Ucn2 gene transfer or saline injection. In both the treatment and prevention strategies, Ucn2 gene transfer increased ejection fraction, reduced LV volume, increased LV peak -dP/dt and peak +dP/dt, and reduced global longitudinal strain. Ucn2 gene transfer-in both treatment and prevention strategies-was associated with higher levels of LV SERCA2a protein, reduced phosphorylation of LV CaMKIIa, and reduced LV α-skeletal actin mRNA expression (reflecting reduced cardiac stress). In conclusion, Ucn2 gene transfer restores normal cardiac function in mice with age-related LV dysfunction and prevents development of LV dysfunction.

MicroRNAs in Chronic Kidney Disease: Four Candidates for Clinical Application.

There are still major challenges regarding the early diagnosis and treatment of chronic kidney disease (CKD), which is in part due to the fact that its pathophysiology is very complex and not clarified in detail. The diagnosis of CKD commonly is made after kidney damage has occurred. This highlights the need for better mechanistic insight into CKD as well as improved clinical tools for both diagnosis and treatment. In the last decade, many studies have focused on microRNAs (miRs) as novel diagnostic tools or clinical targets. MiRs are small non-coding RNA molecules that are involved in post-transcriptional gene regulation and many have been studied in CKD. A wide array of pre-clinical and clinical studies have highlighted the potential role for miRs in the pathogenesis of hypertensive nephropathy, diabetic nephropathy, glomerulonephritis, kidney tubulointerstitial fibrosis, and some of the associated cardiovascular complications. In this review, we will provide an overview of the miRs studied in CKD, especially highlighting miR-103a-3p, miR-192-5p, the miR-29 family and miR-21-5p as these have the greatest potential to result in novel therapeutic and diagnostic strategies.

Prediction Power on Cardiovascular Disease of Neuroimmune Guidance Cues Expression by Peripheral Blood Monocytes Determined by Machine-Learning Methods.

Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.

Adipose tissue macrophages induce hepatic neutrophil recruitment and macrophage accumulation in mice.

OBJECTIVE: Obesity is a risk factor for non-alcoholic steatohepatitis (NASH). This risk has been attributed to visceral adipose tissue (vAT) expansion associated with increased proinflammatory mediators. Accumulation of CD11c+ proinflammatory adipose tissue macrophages (ATM) is an important driver of vAT inflammation. We investigated the role of ATMs in hepatic inflammation during NASH development. DESIGN: vAT isolated from lean, obese or ATM-depleted (using clodronate liposomes) obese mice was transplanted to lean ldlr-/- acceptor mice. Systemic and hepatic inflammation was assessed either after 2 weeks on standard chow or after 8 weeks on high cholesterol diet (HCD) to induce NASH. RESULTS: Transplanting donor vAT from obese mice increased HCD-induced hepatic macrophage content compared with lean-transplanted mice, worsening liver damage. ATM depletion prior to vAT transplantation reduced this increased hepatic macrophage accumulation. On chow, vAT transplantation induced a more pronounced increase in circulating and hepatic neutrophil numbers in obese-transplanted than lean-transplanted mice, while ATM depletion prior to vAT transplantation reversed this effect. Microarray analysis of fluorescence-activated cell sorting of CD11c+ and CD11c- macrophages isolated from donor adipose tissue showed that obesity resulted in enhanced expression of neutrophil chemotaxis genes specifically in CD11c+ ATMs. Involvement of the neutrophil chemotaxis proteins, CXCL14 and CXCL16, was confirmed by culturing vAT. In humans, CD11c expression in vAT of obese individuals correlated with vAT expression of neutrophil chemotactic genes and with hepatic expression of neutrophil and macrophage marker genes. CONCLUSION: ATMs from obese vAT induce hepatic macrophage accumulation during NASH development, possibly by enhancing neutrophil recruitment.

Macrophage complexity in human atherosclerosis: opportunities for treatment?

PURPOSE OF REVIEW: The pivotal role of macrophages in experimental atherosclerosis is firmly established, but their contribution to human disease is less well defined. In this review we have outlined the current insights on macrophage phenotypes and their presumed precursors, monocytes, in clinical atherosclerosis, and their association with disease progression. Moreover, we will assess major clinical modifiers of macrophage-mediated plaque inflammation and define the outstanding questions for further study. RECENT FINDINGS: Our survey indicates that macrophage accumulation and status in human plaques are linked with lesion progression and destabilization as well as with symptomatic coronary artery disease. Likewise, levels of their precursors, circulating monocytes were repeatedly seen to associate with atherosclerosis and to predict clinical outcome. Furthermore, the presence and phenotype of both macrophages and monocytes appears to be responsive to the traditional risk factors of atherosclerosis, including hypercholesterolemia, hypertension, and type 2 diabetes, and to treatment thereof, with clear repercussions on disease development. SUMMARY: Although plaque macrophages and their precursor cells do represent attractive targets for treating cardiovascular diseases, this therapeutic avenue requires much deeper understanding of the complexity of macrophage biology in human atherosclerosis than available at present.

Feasibility of in vivo 18F-florbetaben PET/MR imaging of human carotid amyloid-ß.

PURPOSE: Amyloid-beta (Aß) peptides are involved in the inflammatory pathology of atherosclerosis. 18F-Florbetaben is a PET tracer for clinical imaging of cerebral Aß plaques in Alzheimer's disease (AD). We sought to determine whether specific uptake of 18F-florbetaben in the carotid arteries can be identified using a fully integrated hybrid PET/MRI system and whether this uptake is associated with clinical cardiovascular disease (CVD) risk factors. METHODS: Carotid 18F-florbetaben uptake was quantified as the mean of the maximum target-to-background ratio (meanTBRmax) in 40 cognitively impaired subjects (age 68.2 ± 9.5 years) undergoing 18F-florbetaben PET/MRI to diagnose AD. Associations between carotid 18F-florbetaben uptake and several CVD risk factors were assessed by univariate analysis followed by a multivariate linear regression analysis. Furthermore, carotid 18F-florbetaben uptake was compared between patients with and without a positive cerebral Aß PET scan. RESULTS: 18F-Florbetaben uptake was clearly visualized in the carotid arteries. Values of meanTBRmax corrected for the blood pool activity of the tracer showed specific 18F-florbetaben uptake in the carotid wall. Male gender was associated with carotid 18F-florbetaben uptake in the univariate analysis, and was found to be an independent predictor of 18F-florbetaben uptake in the multivariate regression analysis (standardized regression coefficient ß = 0.407, p = 0.009). Carotid 18F-florbetaben meanTBRmax in patients with a positive cerebral Aß scan did not differ from that in patients without cerebral Aß deposits. CONCLUSION: Specific 18F-florbetaben uptake in human carotid arteries was detected. Male gender was identified as an independent clinical risk factor. Therefore, 18F-florbetaben PET/MRI might provide new insights into the pathophysiological process in atherosclerosis.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling.

OBJECTIVE: Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. APPROACH AND RESULTS: Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1ß, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. CONCLUSIONS: Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.

Deficiency of the oxygen sensor prolyl hydroxylase 1 attenuates hypercholesterolaemia, atherosclerosis, and hyperglycaemia.

AIMS: Normalization of hypercholesterolaemia, inflammation, hyperglycaemia, and obesity are main desired targets to prevent cardiovascular clinical events. Here we present a novel regulator of cholesterol metabolism, which simultaneously impacts on glucose intolerance and inflammation. METHODS AND RESULTS: Mice deficient for oxygen sensor HIF-prolyl hydroxylase 1 (PHD1) were backcrossed onto an atherogenic low-density lipoprotein receptor (LDLR) knockout background and atherosclerosis was studied upon 8 weeks of western-type diet. PHD1-/-LDLR-/- mice presented a sharp reduction in VLDL and LDL plasma cholesterol levels. In line, atherosclerotic plaque development, as measured by plaque area, necrotic core expansion and plaque stage was hampered in PHD1-/-LDLR-/- mice. Mechanistically, cholesterol-lowering in PHD1 deficient mice was a result of enhanced cholesterol excretion from blood to intestines and ultimately faeces. Additionally, flow cytometry of whole blood of these mice revealed significantly reduced counts of leucocytes and particularly of Ly6Chigh pro-inflammatory monocytes. In addition, when studying PHD1-/- in diet-induced obesity (14 weeks high-fat diet) mice were less glucose intolerant when compared with WT littermate controls. CONCLUSION: Overall, PHD1 knockout mice display a metabolic phenotype that generally is deemed protective for cardiovascular disease. Future studies should focus on the efficacy, safety, and gender-specific effects of PHD1 inhibition in humans, and unravel the molecular actors responsible for PHD1-driven, likely intestinal, and regulation of cholesterol metabolism.

Hck/Fgr Kinase Deficiency Reduces Plaque Growth and Stability by Blunting Monocyte Recruitment and Intraplaque Motility.

BACKGROUND: Leukocyte migration is critical for the infiltration of monocytes and accumulation of monocyte-derived macrophages in inflammation. Considering that Hck and Fgr are instrumental in this process, their impact on atherosclerosis and on lesion inflammation and stability was evaluated. METHODS AND RESULTS: Hematopoietic Hck/Fgr-deficient, LDLr(-/-) chimeras, obtained by bone marrow transplantation, had smaller but, paradoxically, less stable lesions with reduced macrophage content, overt cap thinning, and necrotic core expansion as the most prominent features. Despite a Ly6C(high)-skewed proinflammatory monocyte phenotype, Hck/Fgr deficiency led to disrupted adhesion of myeloid cells to and transmigration across endothelial monolayers in vitro and atherosclerotic plaques in vivo, as assessed by intravital microscopy, flow cytometry, and histological examination of atherosclerotic arteries. Moreover, Hck/Fgr-deficient macrophages showed blunted podosome formation and mesenchymal migration capacity. In consequence, transmigrated double-knockout macrophages were seen to accumulate in the fibrous cap, potentially promoting its focal erosion, as observed for double-knockout chimeras. CONCLUSIONS: The hematopoietic deficiency of Hck and Fgr led to attenuated atherosclerotic plaque formation by abrogating endothelial adhesion and transmigration; paradoxically, it also promoted plaque instability by causing monocyte subset imbalance and subendothelial accumulation, raising a note of caution regarding src kinase-targeted intervention in plaque inflammation.

Myeloid A disintegrin and metalloproteinase domain 10 deficiency modulates atherosclerotic plaque composition by shifting the balance from inflammation toward fibrosis.

A disintegrin and metalloproteinase domain 10 (ADAM10) is a metalloprotease involved in cleavage of various cell surface molecules, such as adhesion molecules, chemokines, and growth factor receptors. Although we have previously shown an association of ADAM10 expression with atherosclerotic plaque progression, a causal role of ADAM10 in atherosclerosis has not been investigated. Bone marrow from conditional knockout mice lacking Adam10 in the myeloid lineage or from littermate controls was transplanted into lethally irradiated low density lipoprotein receptor Ldlr(-/-) mice on an atherogenic diet. Myeloid Adam10 deficiency did not affect plaque size, but it increased plaque collagen content. Matrix metalloproteinase 9 and 13 expression and matrix metalloproteinase 2 gelatinase activity were significantly impaired in Adam10-deficient macrophages, whereas their capacity to stimulate collagen production was unchanged. Furthermore, relative macrophage content in advanced atherosclerotic lesions was decreased. In vitro, Adam10-deficient macrophages showed reduced migration toward monocyte chemoattractant protein-1 and transmigration through collagen. In addition, Adam10-deficient macrophages displayed increased anti-inflammatory phenotype with elevated IL-10, and reduced production of proinflammatory tumor necrosis factor, IL-12, and nitric oxide in response to lipopolysaccharide. These data suggest a critical role of Adam10 for leukocyte recruitment, inflammatory mediator production, and extracellular matrix degradation. Thereby, myeloid ADAM10 may play a causal role in modulating atherosclerotic plaque stability.

Leukocyte cathepsin C deficiency attenuates atherosclerotic lesion progression by selective tuning of innate and adaptive immune responses.

OBJECTIVE: The protein degrading activity of cathepsin C (CatC), combined with its role in leukocyte granule activation, suggests a contribution of this cystein protease in atherosclerosis. However, no experimental data are available to validate this concept. APPROACH AND RESULTS: CatC gene and protein expression were increased in ruptured versus advanced stable human carotid artery lesions. To assess causal involvement of CatC in plaque progression and stability, we generated LDLr(-/-)//CatC(-/-) chimeras by bone marrow transplantation. CatC(-/-) chimeras presented attenuated plaque burden in carotids, descending aorta, aortic arch and root, at both the early and advanced plaque stage. CatC was abundantly expressed by plaque macrophages and foam cells. CatC expression and activity were dramatically downregulated in plaques of CatC(-/-) chimeras, supporting a hematopoietic origin of plaque CatC. Our studies unveiled an unexpected feedback of CatC deficiency on macrophage activation programs and T helper cell differentiation in as much as that CatC expression was upregulated in M1 macrophages, whereas its deficiency led to combined M2 (in vitro) and Th2 polarization (in vivo). CONCLUSIONS: Our data implicate CatC has a role in the selective tuning of innate and adaptive immune responses, relevant to a chronic immune disease, such as atherosclerosis.

Reprogramming macrophages to an anti-inflammatory phenotype by helminth antigens reduces murine atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth-derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow-derived macrophages, SEAs induce anti-inflammatory macrophages, typified by high levels of IL-10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR(-/-) mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS-injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T-lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6C(high) monocytes, and macrophages showed high IL-10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA-treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF-α, monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth-derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.

Quaking, an RNA-binding protein, is a critical regulator of vascular smooth muscle cell phenotype.

RATIONALE: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.