Inhaled nitric oxide suppresses neuroinflammation in experimental ischemic stroke.

Ischemic stroke is a major global health issue and characterized by acute vascular dysfunction and subsequent neuroinflammation. However, the relationship between these processes remains elusive. In the current study, we investigated whether alleviating vascular dysfunction by restoring vascular nitric oxide (NO) reduces post-stroke inflammation. Mice were subjected to experimental stroke and received inhaled NO (iNO; 50 ppm) after reperfusion. iNO normalized vascular cyclic guanosine monophosphate (cGMP) levels, reduced the elevated expression of intercellular adhesion molecule-1 (ICAM-1), and returned leukocyte adhesion to baseline levels. Reduction of vascular pathology significantly reduced the inflammatory cytokines interleukin-1ß (Il-1ß), interleukin-6 (Il-6), and tumor necrosis factor-α (TNF-α), within the brain parenchyma. These findings suggest that vascular dysfunction is responsible for leukocyte adhesion and that these processes drive parenchymal inflammation. Reversing vascular dysfunction may therefore emerge as a novel approach to diminish neuroinflammation after ischemic stroke and possibly other ischemic disorders.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates.

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-ß (Aß) peptides and formation of Aß deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aß pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aß1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aß deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

Whole-exome sequencing reveals a role of HTRA1 and EGFL8 in brain white matter hyperintensities.

White matter hyperintensities (WMH) are among the most common radiological abnormalities in the ageing population and an established risk factor for stroke and dementia. While common variant association studies have revealed multiple genetic loci with an influence on their volume, the contribution of rare variants to the WMH burden in the general population remains largely unexplored. We conducted a comprehensive analysis of this burden in the UK Biobank using publicly available whole-exome sequencing data (n up to 17 830) and found a splice-site variant in GBE1, encoding 1,4-alpha-glucan branching enzyme 1, to be associated with lower white matter burden on an exome-wide level [c.691+2T>C, ß = -0.74, standard error (SE) = 0.13, P = 9.7 × 10-9]. Applying whole-exome gene-based burden tests, we found damaging missense and loss-of-function variants in HTRA1 (frequency of 1 in 275 in the UK Biobank population) to associate with an increased WMH volume (P = 5.5 × 10-6, false discovery rate = 0.04). HTRA1 encodes a secreted serine protease implicated in familial forms of small vessel disease. Domain-specific burden tests revealed that the association with WMH volume was restricted to rare variants in the protease domain (amino acids 204-364; ß = 0.79, SE = 0.14, P = 9.4 × 10-8). The frequency of such variants in the UK Biobank population was 1 in 450. The WMH volume was brought forward by ∼11 years in carriers of a rare protease domain variant. A comparison with the effect size of established risk factors for WMH burden revealed that the presence of a rare variant in the HTRA1 protease domain corresponded to a larger effect than meeting the criteria for hypertension (ß = 0.26, SE = 0.02, P = 2.9 × 10-59) or being in the upper 99.8% percentile of the distribution of a polygenic risk score based on common genetic variants (ß = 0.44, SE = 0.14, P = 0.002). In biochemical experiments, most (6/9) of the identified protease domain variants resulted in markedly reduced protease activity. We further found EGFL8, which showed suggestive evidence for association with WMH volume (P = 1.5 × 10-4, false discovery rate = 0.22) in gene burden tests, to be a direct substrate of HTRA1 and to be preferentially expressed in cerebral arterioles and arteries. In a phenome-wide association study mapping ICD-10 diagnoses to 741 standardized Phecodes, rare variants in the HTRA1 protease domain were associated with multiple neurological and non-neurological conditions including migraine with aura (odds ratio = 12.24, 95%CI: 2.54-35.25; P = 8.3 × 10-5]. Collectively, these findings highlight an important role of rare genetic variation and the HTRA1 protease in determining WMH burden in the general population.

TMEM147 interacts with lamin B receptor, regulates its localization and levels, and affects cholesterol homeostasis.

The structurally and functionally complex endoplasmic reticulum (ER) hosts critical processes including lipid synthesis. Here, we focus on the functional characterization of transmembrane protein TMEM147, and report that it localizes at the ER and nuclear envelope in HeLa cells. Silencing of TMEM147 drastically reduces the level of lamin B receptor (LBR) at the inner nuclear membrane and results in mistargeting of LBR to the ER. LBR possesses a modular structure and corresponding bifunctionality, acting in heterochromatin organization via its N-terminus and in cholesterol biosynthesis via its sterol-reductase C-terminal domain. We show that TMEM147 physically interacts with LBR, and that the C-terminus of LBR is essential for their functional interaction. We find that TMEM147 also physically interacts with the key sterol reductase DHCR7, which is involved in cholesterol biosynthesis. Similar to what was seen for LBR, TMEM147 downregulation results in a sharp decline of DHCR protein levels and co-ordinate transcriptional decreases of LBR and DHCR7 expression. Consistent with this, lipidomic analysis upon TMEM147 silencing identified changes in cellular cholesterol levels, cholesteryl ester levels and profile, and in cellular cholesterol uptake, raising the possibility that TMEM147 is an important new regulator of cholesterol homeostasis in cells.This article has an associated First Person interview with the first author of the paper.

Histone Deacetylase 9 Activates IKK to Regulate Atherosclerotic Plaque Vulnerability.

RATIONALE: Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of HDAC (histone deacetylase)-9 in atherosclerosis and its clinical complications including stroke and myocardial infarction. OBJECTIVE: To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection. METHODS AND RESULTS: We studied the effects of Hdac9 on features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further used 2-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoietic Hdac9 deficiency reduces lesional macrophage content while increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKK (inhibitory kappa B kinase)-α and ß, resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting proinflammatory responses in macrophages. Transcriptional profiling using RNA sequencing revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKß. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL (interleukin)-1ß and IL-6. CONCLUSIONS: Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation. Graphical Abstract: A graphical abstract is available for this article.

Proteostasis in Cerebral Small Vessel Disease.

Maintaining the homeostasis of proteins (proteostasis) by controlling their synthesis, folding and degradation is a central task of cells and tissues. The gradual decline of the capacity of the various proteostasis machineries, frequently in combination with their overload through mutated, aggregation-prone proteins, is increasingly recognized as an important catalyst of age-dependent pathologies in the brain, most prominently neurodegenerative disorders. A dysfunctional proteostasis might also contribute to neurovascular disease as indicated by the occurrence of excessive protein accumulation or massive extracellular matrix expansion within vessel walls in conditions such as cerebral small vessel disease (SVD), a major cause of ischemic stroke, and cerebral amyloid angiopathy. Recent advances in brain vessel isolation techniques and mass spectrometry methodology have facilitated the analysis of cerebrovascular proteomes and fueled efforts to determine the proteomic signatures associated with neurovascular disease. In several studies in humans and mice considerable differences between healthy and diseased vessel proteomes were observed, emphasizing the critical contribution of an impaired proteostasis to disease pathogenesis. These findings highlight the important role of a balanced proteostasis for cerebrovascular health.

The Atherosclerosis Risk Variant rs2107595 Mediates Allele-Specific Transcriptional Regulation of HDAC9 via E2F3 and Rb1.

Background and Purpose- Genome-wide association studies have identified the HDAC9 (histone deacetylase 9) gene region as a major risk locus for atherosclerotic stroke and coronary artery disease in humans. Previous results suggest a role of altered HDAC9 expression levels as the underlying disease mechanism. rs2107595, the lead single nucleotide polymorphism for stroke and coronary artery disease resides in noncoding DNA and colocalizes with histone modification marks suggestive of enhancer elements. Methods- To determine the mechanisms by which genetic variation at rs2107595 regulates HDAC9 expression and thus vascular risk we employed targeted resequencing, proteome-wide search for allele-specific nuclear binding partners, chromatin immunoprecipitation, genome-editing, reporter assays, circularized chromosome conformation capture, and gain- and loss-of-function experiments in cultured human cell lines and primary immune cells. Results- Targeted resequencing of the HDAC9 locus in patients with atherosclerotic stroke and controls supported candidacy of rs2107595 as the causative single nucleotide polymorphism. A proteomic search for nuclear binding partners revealed preferential binding of the E2F3/TFDP1/Rb1 complex (E2F transcription factor 3/transcription factor Dp-1/Retinoblastoma 1) to the rs2107595 common allele, consistent with the disruption of an E2F3 consensus site by the risk allele. Gain- and loss-of-function studies showed a regulatory effect of E2F/Rb proteins on HDAC9 expression. Compared with the common allele, the rs2107595 risk allele exhibited higher transcriptional capacity in luciferase assays and was associated with higher HDAC9 mRNA levels in primary macrophages and genome-edited Jurkat cells. Circularized chromosome conformation capture revealed a genomic interaction of the rs2107595 region with the HDAC9 promoter, which was stronger for the common allele as was the in vivo interaction with E2F3 and Rb1 determined by chromatin immunoprecipitation. Gain-of-function experiments in isogenic Jurkat cells demonstrated a key role of E2F3 in mediating rs2107595-dependent transcriptional regulation of HDAC9. Conclusions- Collectively, our findings imply allele-specific transcriptional regulation of HDAC9 via E2F3 and Rb1 as a major mechanism mediating vascular risk at rs2107595.

Novel mutation in HTRA1 in a family with diffuse white matter lesions and inflammatory features.

OBJECTIVE: To investigate the possible involvement of germline mutations in a neurologic condition involving diffuse white matter lesions. METHODS: The patients were 3 siblings born to healthy parents. We performed homozygosity mapping, whole-exome sequencing, site-directed mutagenesis, and immunoblotting. RESULTS: All 3 patients showed clinical manifestations of ataxia, behavioral and mood changes, premature hair loss, memory loss, and lower back pain. In addition, they presented with inflammatory-like features and recurrent rhinitis. MRI showed abnormal diffuse demyelination lesions in the brain and myelitis in the spinal cord. We identified an insertion in high-temperature requirement A (HTRA1), which showed complete segregation in the pedigree. Functional analysis showed the mutation to affect stability and secretion of truncated protein. CONCLUSIONS: The patients' clinical manifestations are consistent with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL; OMIM #600142), which is known to be caused by HTRA1 mutations. Because some aspects of the clinical presentation deviate from those reported for CARASIL, our study expands the spectrum of clinical consequences of loss-of-function mutations in HTRA1.

CADASIL brain vessels show a HTRA1 loss-of-function profile.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and a phenotypically similar recessive condition (CARASIL) have emerged as important genetic model diseases for studying the molecular pathomechanisms of cerebral small vessel disease (SVD). CADASIL, the most frequent and intensely explored monogenic SVD, is characterized by a severe pathology in the cerebral vasculature including the mutation-induced aggregation of the Notch3 extracellular domain (Notch3ECD) and the formation of protein deposits of insufficiently determined composition in vessel walls. To identify key molecules and pathways involved in this process, we quantitatively determined the brain vessel proteome from CADASIL patient and control autopsy samples (n = 6 for each group), obtaining 95 proteins with significantly increased abundance. Intriguingly, high-temperature requirement protein A1 (HTRA1), the extracellular protease mutated in CARASIL, was found to be strongly enriched (4.9-fold, p = 1.6 × 10-3) and to colocalize with Notch3ECD deposits in patient vessels suggesting a sequestration process. Furthermore, the presence of increased levels of several HTRA1 substrates in the CADASIL proteome was compatible with their reduced degradation as consequence of a loss of HTRA1 activity. Indeed, a comparison with the brain vessel proteome of HTRA1 knockout mice (n = 5) revealed a highly significant overlap of 18 enriched proteins (p = 2.2 × 10-16), primarily representing secreted and extracellular matrix factors. Several of them were shown to be processed by HTRA1 in an in vitro proteolysis assay identifying them as novel substrates. Our study provides evidence for a loss of HTRA1 function as a critical step in the development of CADASIL pathology linking the molecular mechanisms of two distinct SVD forms.

Brain-released alarmins and stress response synergize in accelerating atherosclerosis progression after stroke.

Stroke induces a multiphasic systemic immune response, but the consequences of this response on atherosclerosis-a major source of recurrent vascular events-have not been thoroughly investigated. We show that stroke exacerbates atheroprogression via alarmin-mediated propagation of vascular inflammation. The prototypic brain-released alarmin high-mobility group box 1 protein induced monocyte and endothelial activation via the receptor for advanced glycation end products (RAGE)-signaling cascade and increased plaque load and vulnerability. Recruitment of activated monocytes via the CC-chemokine ligand 2-CC-chemokine receptor type 2 pathway was critical in stroke-induced vascular inflammation. Neutralization of circulating alarmins or knockdown of RAGE attenuated atheroprogression. Blockage of ß3-adrenoreceptors attenuated the egress of myeloid monocytes after stroke, whereas neutralization of circulating alarmins was required to reduce systemic monocyte activation and aortic invasion. Our findings identify a synergistic effect of the sympathetic stress response and alarmin-driven inflammation via RAGE as a critical mechanism of exacerbated atheroprogression after stroke.

The choroid plexus is a key cerebral invasion route for T cells after stroke.

Neuroinflammation contributes substantially to stroke pathophysiology. Cerebral invasion of peripheral leukocytes-particularly T cells-has been shown to be a key event promoting inflammatory tissue damage after stroke. While previous research has focused on the vascular invasion of T cells into the ischemic brain, the choroid plexus (ChP) as an alternative cerebral T-cell invasion route after stroke has not been investigated. We here report specific accumulation of T cells in the peri-infarct cortex and detection of T cells as the predominant population in the ipsilateral ChP in mice as well as in human post-stroke autopsy samples. T-cell migration from the ChP to the peri-infarct cortex was confirmed by in vivo cell tracking of photoactivated T cells. In turn, significantly less T cells invaded the ischemic brain after photothrombotic lesion of the ipsilateral ChP and in a stroke model encompassing ChP ischemia. We detected a gradient of CCR2 ligands as the potential driving force and characterized the neuroanatomical pathway for the intracerebral migration. In summary, our study demonstrates that the ChP is a key invasion route for post-stroke cerebral T-cell invasion and describes a CCR2-ligand gradient between cortex and ChP as the potential driving mechanism for this invasion route.

Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations. Transfection of these antisense oligonucleotides into CADASIL patient-derived cerebral vascular smooth muscle cells resulted in successful exon skipping, without abrogating NOTCH3 signalling. Combined, these data provide proof of concept for this novel application of exon skipping, and are a first step towards the development of a rational therapeutic approach applicable to up to 94% of CADASIL-causing mutations.

Genetic factors in cerebral small vessel disease and their impact on stroke and dementia.

Cerebral small vessel disease (SVD) is among the most frequent causes of both stroke and dementia. There is a growing list of genes known to be implicated in Mendelian forms of SVD. Also, genome-wide association studies have identified common variants at a number of genetic loci that are associated with manifestations of SVD, among them loci for white matter hyperintensities, small vessel stroke, and deep intracerebral hemorrhage. Driven by these discoveries and new animal models substantial progress has been made in elucidating the molecular, cellular, and physiologic mechanisms underlying SVD. A major theme emerging from these studies is the extracellular matrix (ECM). Recent findings include a role of structural constituents of the ECM such as type IV collagens in hereditary and sporadic SVD, the sequestration of proteins with a known role in ECM maintenance into aggregates of NOTCH3, and altered signaling through molecules known to interact with the ECM. Here, we review recent progress in the identification of genes involved in SVD and discuss mechanistic concepts with a particular focus on the ECM.

Heterozygous HTRA1 mutations are associated with autosomal dominant cerebral small vessel disease.

Cerebral small vessel disease represents a heterogeneous group of disorders leading to stroke and cognitive impairment. While most small vessel diseases appear sporadic and related to age and hypertension, several early-onset monogenic forms have also been reported. However, only a minority of patients with familial small vessel disease carry mutations in one of known small vessel disease genes. We used whole exome sequencing to identify candidate genes in an autosomal dominant small vessel disease family in which known small vessel disease genes had been excluded, and subsequently screened all candidate genes in 201 unrelated probands with a familial small vessel disease of unknown aetiology, using high throughput multiplex polymerase chain reaction and next generation sequencing. A heterozygous HTRA1 variant (R166L), absent from 1000 Genomes and Exome Variant Server databases and predicted to be deleterious by in silico tools, was identified in all affected members of the index family. Ten probands of 201 additional unrelated and affected probands (4.97%) harboured a heterozygous HTRA1 mutation predicted to be damaging. There was a highly significant difference in the number of likely deleterious variants in cases compared to controls (P = 4.2 × 10(-6); odds ratio = 15.4; 95% confidence interval = 4.9-45.5), strongly suggesting causality. Seven of these variants were located within or close to the HTRA1 protease domain, three were in the N-terminal domain of unknown function and one in the C-terminal PDZ domain. In vitro activity analysis of HTRA1 mutants demonstrated a loss of function effect. Clinical features of this autosomal dominant small vessel disease differ from those of CARASIL and CADASIL by a later age of onset and the absence of the typical extraneurological features of CARASIL. They are similar to those of sporadic small vessel disease, except for their familial nature. Our data demonstrate that heterozygous HTRA1 mutations are an important cause of familial small vessel disease, and that screening of HTRA1 should be considered in all patients with a hereditary small vessel disease of unknown aetiology.

Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro.

BACKGROUND AND PURPOSE: Mutations in NOTCH3 cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common monogenic cause of stroke and vascular dementia. Misfolding and aggregation of NOTCH3 proteins triggered by cysteine-affecting mutations are considered to be the key disease mechanisms. However, the significance of cysteine-sparing mutations is still debated. METHODS: We studied a family with inherited small vessel disease by standardized medical history, clinical examination, MRI, ultrastructural analysis of skin biopsies, and Sanger sequencing of all NOTCH3 exons. In addition, we performed in vitro characterization of NOTCH3 variants using recombinant protein fragments and a single-particle aggregation assay. RESULTS: We identified a novel cysteine-sparing NOTCH3 mutation (D80G) in 4 family members, which was absent in a healthy sibling. All mutation carriers exhibited a CADASIL typical brain imaging and clinical phenotype, whereas skin biopsy showed inconsistent results. In vitro aggregation behavior of the D80G mutant was similar compared with cysteine-affecting mutations. This was reproduced with cysteine-sparing mutations from previously reported families having a phenotype consistent with CADASIL. CONCLUSIONS: Our findings support the view that cysteine-sparing mutations, such as D80G, might cause CADASIL with a phenotype largely indistinguishable from cysteine mutations. The in vitro aggregation analysis of atypical NOTCH3 mutations offers novel insights into pathomechanisms and might represent a tool for estimating their clinical significance.

Deficiency of the stroke relevant HDAC9 gene attenuates atherosclerosis in accord with allele-specific effects at 7p21.1.

BACKGROUND AND PURPOSE: Recent genome-wide association studies identified the histone deacetylase 9 (HDAC9) gene region as a major risk locus for large-vessel stroke and coronary artery disease. However, the mechanisms linking variants at this locus to vascular risk are poorly understood. In this study, we investigated the candidacy and directionality of HDAC9 in atherosclerosis and analyzed associations between risk alleles at 7p21.1 and plaque characteristics. METHODS: Allele-dependent expression of HDAC9 was analyzed in human peripheral blood mononuclear cells of healthy donors. Effects of HDAC9 deficiency on atherosclerotic plaques were investigated in 18- and 28-week-old ApoE(-/-) mice by histology and immunohistochemistry. We further performed detailed plaque phenotyping and genotyping of rs2107595, the lead single-nucleotide polymorphism for large-vessel stroke, in carotid endarterectomy samples of 1858 subjects from the Athero-Express study. RESULTS: Gene expression studies in peripheral blood mononuclear cells revealed increased mRNA levels of HDAC9 but not of neighboring genes (TWIST1/FERD3L) in risk allele carriers of rs2107595. Compared with HDAC9(+/+)ApoE(-/-) mice, HDAC9(-/-)ApoE(-/-) mice exhibited markedly reduced lesion sizes throughout atherosclerotic aortas and significantly less advanced lesions. The proportion of Mac3-positive macrophages was higher in plaques from HDAC9(-/-)ApoE(-/-) mice, but this was largely because of a lower proportion of advanced lesions. Analysis of human atherosclerotic plaques revealed no association between rs2107595 and specific plaque characteristics. CONCLUSIONS: Our results suggest that HDAC9 represents the disease-relevant gene at the stroke and coronary artery disease risk locus on 7p21.1, and that risk alleles in this region mediate their effects through increased HDAC9 expression. Targeted inhibition of HDAC9 might be a viable strategy to prevent atherosclerosis.