Network-based prioritization and validation of regulators of vascular smooth muscle cell proliferation in disease.

Aberrant vascular smooth muscle cell (VSMC) homeostasis and proliferation characterize vascular diseases causing heart attack and stroke. Here we elucidate molecular determinants governing VSMC proliferation by reconstructing gene regulatory networks from single-cell transcriptomics and epigenetic profiling. We detect widespread activation of enhancers at disease-relevant loci in proliferation-predisposed VSMCs. We compared gene regulatory network rewiring between injury-responsive and nonresponsive VSMCs, which suggested shared transcription factors but differing target loci between VSMC states. Through in silico perturbation analysis, we identified and prioritized previously unrecognized regulators of proliferation, including RUNX1 and TIMP1. Moreover, we showed that the pioneer transcription factor RUNX1 increased VSMC responsiveness and that TIMP1 feeds back to promote VSMC proliferation through CD74-mediated STAT3 signaling. Both RUNX1 and the TIMP1-CD74 axis were expressed in human VSMCs, showing low levels in normal arteries and increased expression in disease, suggesting clinical relevance and potential as vascular disease targets.

Selenoprotein deficiency disorder predisposes to aortic aneurysm formation.

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient's cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity.

BACKGROUND AND PURPOSE: Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. EXPERIMENTAL APPROACH: Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. KEY RESULTS: On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. CONCLUSIONS AND IMPLICATIONS: In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes.

Somatostatin Receptor PET/MR Imaging of Inflammation in Patients With Large Vessel Vasculitis and Atherosclerosis.

BACKGROUND: Assessing inflammatory disease activity in large vessel vasculitis (LVV) can be challenging by conventional measures. OBJECTIVES: We aimed to investigate somatostatin receptor 2 (SST2) as a novel inflammation-specific molecular imaging target in LVV. METHODS: In a prospective, observational cohort study, in vivo arterial SST2 expression was assessed by positron emission tomography/magnetic resonance imaging (PET/MRI) using 68Ga-DOTATATE and 18F-FET-ßAG-TOCA. Ex vivo mapping of the imaging target was performed using immunofluorescence microscopy; imaging mass cytometry; and bulk, single-cell, and single-nucleus RNA sequencing. RESULTS: Sixty-one participants (LVV: n = 27; recent atherosclerotic myocardial infarction of ≤2 weeks: n = 25; control subjects with an oncologic indication for imaging: n = 9) were included. Index vessel SST2 maximum tissue-to-blood ratio was 61.8% (P < 0.0001) higher in active/grumbling LVV than inactive LVV and 34.6% (P = 0.0002) higher than myocardial infarction, with good diagnostic accuracy (area under the curve: ≥0.86; P < 0.001 for both). Arterial SST2 signal was not elevated in any of the control subjects. SST2 PET/MRI was generally consistent with 18F-fluorodeoxyglucose PET/computed tomography imaging in LVV patients with contemporaneous clinical scans but with very low background signal in the brain and heart, allowing for unimpeded assessment of nearby coronary, myocardial, and intracranial artery involvement. Clinically effective treatment for LVV was associated with a 0.49 ± 0.24 (standard error of the mean [SEM]) (P = 0.04; 22.3%) reduction in the SST2 maximum tissue-to-blood ratio after 9.3 ± 3.2 months. SST2 expression was localized to macrophages, pericytes, and perivascular adipocytes in vasculitis specimens, with specific receptor binding confirmed by autoradiography. SSTR2-expressing macrophages coexpressed proinflammatory markers. CONCLUSIONS: SST2 PET/MRI holds major promise for diagnosis and therapeutic monitoring in LVV. (PET Imaging of Giant Cell and Takayasu Arteritis [PITA], NCT04071691; Residual Inflammation and Plaque Progression Long-Term Evaluation [RIPPLE], NCT04073810).

Translational opportunities of single-cell biology in atherosclerosis.

The advent of single-cell biology opens a new chapter for understanding human biological processes and for diagnosing, monitoring, and treating disease. This revolution now reaches the field of cardiovascular disease (CVD). New technologies to interrogate CVD samples at single-cell resolution are allowing the identification of novel cell communities that are important in shaping disease development and direct towards new therapeutic strategies. These approaches have begun to revolutionize atherosclerosis pathology and redraw our understanding of disease development. This review discusses the state-of-the-art of single-cell analysis of atherosclerotic plaques, with a particular focus on human lesions, and presents the current resolution of cellular subpopulations and their heterogeneity and plasticity in relation to clinically relevant features. Opportunities and pitfalls of current technologies as well as the clinical impact of single-cell technologies in CVD patient care are highlighted, advocating for multidisciplinary and international collaborative efforts to join the cellular dots of CVD.

Cellular mechanisms of oligoclonal vascular smooth muscle cell expansion in cardiovascular disease.

AIMS: Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here, we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality. METHODS AND RESULTS: We investigate the dynamics of VSMC clone formation using confocal microscopy and single-cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency (<0.1%) of clonal VSMC proliferation is also observed in vitro. Single-cell RNA-sequencing revealed progressive cell state changes across a contiguous VSMC population at onset of injury-induced proliferation. Proliferating VSMCs mapped selectively to one of two distinct trajectories and were associated with cells showing extensive phenotypic switching. A proliferation-associated transitory state shared pronounced similarities with atypical SCA1+ VSMCs from uninjured mouse arteries and VSMCs in healthy human aorta. We show functionally that clonal expansion of SCA1+ VSMCs from healthy arteries occurs at higher rate and frequency compared with SCA1- cells. CONCLUSION: Our data suggest that activation of proliferation at low frequency is a general, cell-intrinsic feature of VSMCs. We show that rare VSMCs in healthy arteries display VSMC phenotypic switching akin to that observed in pathological vessel remodelling and that this is a conserved feature of mouse and human healthy arteries. The increased proliferation of modulated VSMCs from healthy arteries suggests that these cells respond more readily to disease-inducing cues and could drive oligoclonal VSMC expansion.

JAG1-NOTCH4 mechanosensing drives atherosclerosis.

Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 (Jag1ECKO) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.

Persistence and genetic adaptation of Pseudomonas aeruginosa in patients with chronic obstructive pulmonary disease.

OBJECTIVES: It is unclear whether recurrent sputum culture with Pseudomonas aeruginosa from patients with chronic obstructive pulmonary disease (COPD) is caused by intermittent airway carriage by different P. aeruginosa lineages or persistent carriage by the same lineage, and whether lineages genetically adapt during carriage. METHODS: Whole-genome sequencing was performed for P. aeruginosa isolates sampled longitudinally from sputum cultures in patients with COPD who were enrolled in an ongoing randomized controlled trial (clinicaltrials.gov: NCT03262142). RESULTS: A total of 153 P. aeruginosa isolates were sequenced for 23 patients during 365 days of follow-up. Recurrent presence of P. aeruginosa was seen in 19 patients (83%) and was caused by persistence of the same clonal lineage in all but one patient. We identified 38 genes mutated in parallel in two or more lineages, suggesting positive selection for adaptive mutations. Mutational enrichment analysis revealed genes important in antibiotic resistance and chronic infections to be more frequently mutated. DISCUSSION: Recurrent P. aeruginosa was common and carried for a prolonged time after initial detection in the airways of patients with COPD. Recurrence was caused by persistence of the same clonal lineage and was associated with genetic adaptation. Trial data on possible clinical benefits of attempting antibiotic eradication of P. aeruginosa in COPD are warranted.

Kinetics of SARS-CoV-2-Neutralising Antibodies of Residents of Long-Term Care Facilities.

INTRODUCTION: Elderly residents of nursing homes (NHs) and long-term care units (LTCUs) have been shown to have a high risk of mortality and morbidity in cases of SARS-CoV-2 infection. The objective of this study was to examine the kinetics of neutralizing antibodies (NAbs) directed against the SARS-CoV-2 virus in residents of the NH and LTCU units of our University Hospital who were identified with positive serology after the first epidemic outbreak. MATERIALS AND METHODS: The participants included were sampled every three months for qualitative serological testing, as well as quantitative testing by neutralization tests using retroviral particles containing the S glycoprotein of SARS-CoV-2. Vaccination using the Comirnaty (Pfizer BNT162b2) vaccine begun before the last serological follow-up. RESULTS: The median NAb titer in June 2020 was 80 [40; 60] versus 40 [40; 160] three months later, showing a statistically significant decline (p < 0.007), but remained stable between the three- and six-month timepoints (p = 0.867). By nine months after vaccination, we observed a significant difference between vaccinated residents known to have positive serology before vaccination (SERO+, Vacc+) and those vaccinated without having previously shown COVID-19 seroconversion (SERO-, Vacc+), the latter group showing similar titers to the SERO+, Vacc- participants (p=0.166). The median antibody titer in SERO+, Vacc+ patients increased 15-fold following vaccination. DISCUSSION: Humoral immunity against SARS-CoV-2 appears to be persistent in elderly institutionalized patients, with a good post-vaccination response by residents who had already shown seroconversion but a notably diminished response by those who were seronegative before vaccination. To evaluate immunity in its entirety and elaborate a sound vaccination strategy, the cellular immune response via T cells specific to SARS-CoV-2 merits analysis, as this response is susceptible to being affected by immunosenescence.

Mechanisms of vascular smooth muscle cell investment and phenotypic diversification in vascular diseases.

In contrast with the heart, the adult mammalian vasculature retains significant remodelling capacity, dysregulation of which is implicated in disease development. In particular, vascular smooth muscle cells (VSMCs) play major roles in the pathological vascular remodelling characteristic of atherosclerosis, restenosis, aneurysm and pulmonary arterial hypertension. Clonal lineage tracing revealed that the VSMC-contribution to disease results from the hyperproliferation of few pre-existing medial cells and suggested that VSMC-derived cells from the same clone can adopt diverse phenotypes. Studies harnessing the powerful combination of lineage tracing and single-cell transcriptomics have delineated the substantial diversity of VSMC-derived cells in vascular lesions, which are proposed to have both beneficial and detrimental effects on disease severity. Computational analyses further suggest that the pathway from contractile VSMCs in healthy arteries to phenotypically distinct lesional cells consists of multiple, potentially regulatable, steps. A better understanding of how individual steps are controlled could reveal effective therapeutic strategies to minimise VSMC functions that drive pathology whilst maintaining or enhancing their beneficial roles. Here we review current knowledge of VSMC plasticity and highlight important questions that should be addressed to understand how specific stages of VSMC investment and phenotypic diversification are controlled. Implications for developing therapeutic strategies in pathological vascular remodelling are discussed and we explore how cutting-edge approaches could be used to elucidate the molecular mechanisms underlying VSMC regulation.

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

Telomere damage promotes vascular smooth muscle cell senescence and immune cell recruitment after vessel injury.

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2T188A) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease.

Deep transfer learning can be used for the detection of hip joints in pelvis radiographs and the classification of their hip dysplasia status.

Reports of machine learning implementations in veterinary imaging are infrequent but changes in machine learning architecture and access to increased computing power will likely prompt increased interest. This diagnostic accuracy study describes a particular form of machine learning, a deep learning convolution neural network (ConvNet) for hip joint detection and classification of hip dysplasia from ventro-dorsal (VD) pelvis radiographs submitted for hip dysplasia screening. 11,759 pelvis images were available together with their Fédération Cynologique Internationale (FCI) scores. The dataset was dicotomized into images showing no signs of hip dysplasia (FCI grades "A" and "B", the "A-B" group) and hips showing signs of dysplasia (FCI grades "C", "D," and "E", the "C-E" group). In a transfer learning approach, an existing pretrained ConvNet was fine-tuned to provide models to recognize hip joints in VD pelvis images and to classify them according to their FCI score grouping. The results yielded two models. The first was successful in detecting hip joints in the VD pelvis images (intersection over union of 85%). The second yielded a sensitivity of 0.53, a specificity of 0.92, a positive predictive value of 0.91, and a negative predictive value of 0.81 for the classification of detected hip joints as being in the "C-E" group. ConvNets and transfer learning are applicable to veterinary imaging. The models obtained have potential to be a tool to aid in hip screening protocols if hip dysplasia classification performance was improved through access to more data and possibly by model optimization.

DNA glycosylase Neil3 regulates vascular smooth muscle cell biology during atherosclerosis development.

BACKGROUND AND AIMS: Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability. METHODS: Chow diet-fed atherosclerosis-prone Apoe-/- mice deficient in Neil3, and NEIL3-abrogated human primary aortic VSMCs were characterized by qPCR, and immunohistochemical and enzymatic-based assays; moreover, single-cell RNA sequencing, mRNA sequencing, and proteomics were used to map the molecular effects of Neil3/NEIL3 deficiency in the aortic VSMC phenotype. Furthermore, BrdU-based proliferation assays and Western blot were performed to elucidate the involvement of the Akt signaling pathway in the transdifferentiation of aortic VSMCs lacking Neil3/NEIL3. RESULTS: We show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs. CONCLUSIONS: Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.

Bone turnover biomarkers in COPD patients randomized to either a regular or shortened course of corticosteroids: a substudy of the randomized controlled CORTICO-COP trial.

BACKGROUND: Long-term treatment with corticosteroids causes loss of bone density, but the effects of using short-term high-dose systemic-corticosteroid therapy to treat acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are unclear. Our aim was to determine whether high-dose corticosteroid therapy affected bone turnover markers (BTMs) to a greater extent compared to low-dose corticosteroid therapy. METHODS: The CORTICO-COP trial (NCT02857842) showed that an eosinophil-guided corticosteroid intervention led to approximately 60% lower accumulated corticosteroid dose for hospitalized patients with AECOPD (low-dose group) compared with 5-day standard corticosteroid treatment (high-dose group). We compared the levels of BTMs C-terminal telopeptide of type 1 collagen (CTX) and procollagen type 1 N-terminal propeptide (P1NP) in 318 participants during AECOPD and at 1- and 3-month follow-up visits. RESULTS: CTX decreased and P1NP increased significantly over time in both treatment groups. There were no significant differences between the groups at 1- or 3-months follow-up for P1NP. A significant drop in CTX was seen at 3 months (down Δ24% from the baseline, p = 0.017) for the high dose group. CONCLUSION: Short-term, high-dose systemic corticosteroid treatment caused a rapid suppression of biomarkers of bone resorption. Corticosteroids did not suppress biomarkers of bone formation, regardless of patients receiving low or high doses of corticosteroids. This therapy was, therefore, harmless in terms of bone safety, in our prospective series of COPD patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02857842 . Submitted August 2nd, 2016.

Assessment of Image Quality in Digital Radiographs Submitted for Hip Dysplasia Screening.

Digital radiography is widely seen to be forgiving of poor exposure technique and to provide consistent high quality diagnostic images. Optimal quality images are however not universal; sub-optimal images are encountered. Evaluators on hip dysplasia schemes encounter images from multiple practices produced on equipment from multiple manufacturers. For images submitted to the Danish Kennel Club for hip dysplasia screening, a range of quality is seen and the evaluators are of the impression that variations in image quality area associated with particular equipment. This study was undertaken to test the hypothesis that there is an association between image quality in digital radiography and the manufacturer of the detector equipment, and to demonstrate the applicability of visual grading analysis (VGA) for image quality evaluation in veterinary practice. Data from 16,360 digital images submitted to the Danish Kennel Club were used to generate the hypothesis that there is an association between detector manufacturer and image quality and to create groups for VGA. Image quality in a subset of 90 images randomly chosen from 6 manufacturers to represent high and low quality images, was characterized using VGA and the results used to test for an association between image quality and system manufacturer. The range of possible scores in the VGA was -2 to +2 (higher scores are better). The range of the VGA scores for the images in the low image quality group (n = 45) was -1.73 to +0.67, (median -1.2). Images in the high image quality group (n = 44) ranged from -1.52 to +0.53, (median -0.53). This difference was statistically significant (p < 0.001). The study shows an association between VGA scores of image quality and detector manufacturer. Possible causes may be that imaging hardware and/or software are not equal in terms of quality, that the level of support sought and given differs between systems, or a combination of the two. Clinicians purchasing equipment should be mindful that image quality can differ across systems. VGA is practical for veterinarians to compare image quality between systems or within a system over time.

Epigenetic Regulation of Vascular Smooth Muscle Cells by Histone H3 Lysine 9 Dimethylation Attenuates Target Gene-Induction by Inflammatory Signaling.

OBJECTIVE: Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs (matrix metalloproteinases) and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response. Approach and Results: We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including MMP3, MMP9, MMP12, and IL6, in mouse and human VSMCs. Inhibition of G9A/GLP (G9A-like protein), the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK (mitogen-activated protein kinase) signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6. Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs. CONCLUSIONS: This study implicates H3K9me2 in regulating the proinflammatory VSMC phenotype. Our findings suggest that reduced H3K9me2 in disease enhance binding of NFκB and AP-1 (activator protein-1) transcription factors at specific inflammation-responsive genes to augment proinflammatory stimuli in VSMC. Therefore, H3K9me2-regulation could be targeted clinically to limit expression of MMPs and IL6, which are induced in vascular disease.