Plozasiran, an RNA Interference Agent Targeting APOC3, for Mixed Hyperlipidemia.

BACKGROUND: Persons with mixed hyperlipidemia are at risk for atherosclerotic cardiovascular disease due to an elevated non-high-density lipoprotein (HDL) cholesterol level, which is driven by remnant cholesterol in triglyceride-rich lipoproteins. The metabolism and clearance of triglyceride-rich lipoproteins are down-regulated through apolipoprotein C3 (APOC3)-mediated inhibition of lipoprotein lipase. METHODS: We carried out a 48-week, phase 2b, double-blind, randomized, placebo-controlled trial evaluating the safety and efficacy of plozasiran, a hepatocyte-targeted APOC3 small interfering RNA, in patients with mixed hyperlipidemia (i.e., a triglyceride level of 150 to 499 mg per deciliter and either a low-density lipoprotein [LDL] cholesterol level of ≥70 mg per deciliter or a non-HDL cholesterol level of ≥100 mg per deciliter). The participants were assigned in a 3:1 ratio to receive plozasiran or placebo within each of four cohorts. In the first three cohorts, the participants received a subcutaneous injection of plozasiran (10 mg, 25 mg, or 50 mg) or placebo on day 1 and at week 12 (quarterly doses). In the fourth cohort, participants received 50 mg of plozasiran or placebo on day 1 and at week 24 (half-yearly dose). The data from the participants who received placebo were pooled. The primary end point was the percent change in fasting triglyceride level at week 24. RESULTS: A total of 353 participants underwent randomization. At week 24, significant reductions in the fasting triglyceride level were observed with plozasiran, with differences, as compared with placebo, in the least-squares mean percent change from baseline of -49.8 percentage points (95% confidence interval [CI], -59.0 to -40.6) with the 10-mg-quarterly dose, -56.0 percentage points (95% CI, -65.1 to -46.8) with the 25-mg-quarterly dose, -62.4 percentage points (95% CI, -71.5 to -53.2) with the 50-mg-quarterly dose, and -44.2 percentage points (95% CI, -53.4 to -35.0) with the 50-mg-half-yearly dose (P<0.001 for all comparisons). Worsening glycemic control was observed in 10% of the participants receiving placebo, 12% of those receiving the 10-mg-quarterly dose, 7% of those receiving the 25-mg-quarterly dose, 20% of those receiving the 50-mg-quarterly dose, and 21% of those receiving the 50-mg-half-yearly dose. CONCLUSIONS: In this randomized, controlled trial involving participants with mixed hyperlipidemia, plozasiran, as compared with placebo, significantly reduced triglyceride levels at 24 weeks. A clinical outcomes trial is warranted. (Funded by Arrowhead Pharmaceuticals; MUIR ClinicalTrials.gov number NCT04998201.).

Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

Cancer Incidence After Diagnosis of Abdominal Aortic Aneurysm-Brief Report.

BACKGROUND: Epidemiological and mechanistic data support a potential causal link between cardiovascular disease (CVD) and cancer. Abdominal aortic aneurysms (AAAs) represent a common form of CVD with at least partially distinct genetic and biologic pathogenesis from other forms of CVD. The risk of cancer and how this risk differs compared with other forms of CVD, is unknown among AAA patients. We conducted a retrospective cohort study using the IBM MarketScan Research Database to test whether individuals with AAA have a higher cancer risk independent of traditional shared risk factors. METHODS: All individuals ≥18 years of age with ≥36 months of continuous coverage between 2008 and 2020 were enrolled. Those with potential Mendelian etiologies of AAA, aortic aneurysm with nonspecific anatomic location, or a cancer diagnosis before the start of follow-up were excluded. A subgroup analysis was performed of individuals having the Health Risk Assessment records including tobacco use and body mass index. The following groups of individuals were compared: (1) with AAA, (2) with non-AAA CVD, and (3) without any CVD. RESULTS: The propensity score-matched cohort included 58 993 individuals with AAA, 117 986 with non-AAA CVD, and 58 993 without CVD. The 5-year cumulative incidence of cancer was 13.1% (12.8%-13.5%) in participants with AAA, 10.1% (9.9%-10.3%) in participants with non-AAA CVD, and 9.6% (9.3%-9.9%) in participants without CVD. Multivariable-adjusted Cox proportional hazards regression models found that patients with AAA exhibited a higher cancer risk than either those with non-AAA CVD (hazard ratio, 1.28 [95% CI, 1.23-1.32]; P<0.001) or those without CVD (hazard ratio, 1.32 [95% CI, 1.26-1.38]; P<0.001). Results remained consistent after excluding common smoking-related cancers and when adjusting for tobacco use and body mass index. CONCLUSIONS: Patients with AAA may have a unique risk of cancer requiring further mechanistic study and investigation of the role of enhanced cancer screening.

Characterization of peripheral artery disease and associations with traditional risk factors, mobility, and biomarkers in the project baseline health study.

BACKGROUND: There is a dearth of research on immunophenotyping in peripheral artery disease (PAD). This study aimed to describe the baseline characteristics, immunophenotypic profile, and quality of life (QoL) of participants with PAD in the Project Baseline Health Study (PBHS). METHODS: The PBHS study is a prospective, multicenter, longitudinal cohort study that collected clinical, molecular, and biometric data from participants recruited between 2017 and 2018. In this analysis, baseline demographic, clinical, mobility, QoL, and flow cytometry data were stratified by the presence of PAD (ankle brachial index [ABI] ≤0.90). RESULTS: Of 2,209 participants, 58 (2.6%) had lower-extremity PAD, and only 2 (3.4%) had pre-existing PAD diagnosed prior to enrollment. Comorbid smoking (29.3% vs 14%, P < .001), hypertension (54% vs 30%, P < .001), diabetes (25% vs 14%, P = .031), and at least moderate coronary calcifications (Agatston score >100: 32% vs 17%, P = .01) were significantly higher in participants with PAD than in those with normal ABIs, as were high-sensitivity C-reactive protein levels (5.86 vs 2.83, P < .001). After adjusting for demographic and risk factors, participants with PAD had significantly fewer circulating CD56-high natural killer cells, IgM+ memory B cells, and CD10/CD27 double-positive B cells (P < .05 for all). CONCLUSIONS: This study reinforces existing evidence that a large proportion of PAD without claudication may be underdiagnosed, particularly in female and Black or African American participants. We describe a novel immunophenotypic profile of participants with PAD that could represent a potential future screening or diagnostic tool to facilitate earlier diagnosis of PAD. GOV IDENTIFIER: NCT03154346, https://clinicaltrials.gov/ct2/show/NCT03154346.

Leveraging Machine Learning and Artificial Intelligence to Improve Peripheral Artery Disease Detection, Treatment, and Outcomes.

Peripheral artery disease is an atherosclerotic disorder which, when present, portends poor patient outcomes. Low diagnosis rates perpetuate poor management, leading to limb loss and excess rates of cardiovascular morbidity and death. Machine learning algorithms and artificially intelligent systems have shown great promise in application to many areas in health care, such as accurately detecting disease, predicting patient outcomes, and automating image interpretation. Although the application of these technologies to peripheral artery disease are in their infancy, their promises are tremendous. In this review, we provide an introduction to important concepts in the fields of machine learning and artificial intelligence, detail the current state of how these technologies have been applied to peripheral artery disease, and discuss potential areas for future care enhancement with advanced analytics.

Bench-to-Bedside in Vascular Medicine: Optimizing the Translational Pipeline for Patients With Peripheral Artery Disease.

Peripheral arterial disease is a growing worldwide problem with a wide spectrum of clinical severity and is projected to consume >$21 billion per year in the United States alone. While vascular researchers have brought several therapies to the clinic in recent years, few of these approaches have leveraged advances in high-throughput discovery screens, novel translational models, or innovative trial designs. In the following review, we discuss recent advances in unbiased genomics and broader omics technology platforms, along with preclinical vascular models designed to enhance our understanding of disease pathobiology and prioritize targets for additional investigation. Furthermore, we summarize novel approaches to clinical studies in subjects with claudication and ischemic ulceration, with an emphasis on streamlining and accelerating bench-to-bedside translation. By providing a framework designed to enhance each aspect of future clinical development programs, we hope to enrich the pipeline of therapies that may prevent loss of life and limb for those with peripheral arterial disease.

2021 Jeffrey M. Hoeg Award Lecture: Defining the Role of Efferocytosis in Cardiovascular Disease: A Focus on the CD47 (Cluster of Differentiation 47) Axis.

A key feature of atherogenesis is the accumulation of diseased and dying cells within the lesional necrotic core. While the burden of intraplaque apoptotic cells may be driven in part by an increase in programmed cell death, mounting evidence suggests that their presence may primarily be dictated by a defect in programmed cell removal, or efferocytosis. In this brief review, we will summarize the evidence suggesting that inflammation-dependent changes within the plaque render target cells inedible and reduce the appetite of lesional phagocytes. We will present the genetic causation studies, which indicate these phenomena promote lesion expansion and plaque vulnerability, and the interventional data which suggest that these processes can be reversed. Particular emphasis is provided related to the antiphagocytic CD47 (cluster of differentiation 47) do not eat me axis, which has emerged as a novel antiatherosclerotic translational target that is predicted to provide benefit independent of traditional cardiovascular risk factors.

Macrophage LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Is Required for the Effect of CD47 Blockade on Efferocytosis and Atherogenesis-Brief Report.

OBJECTIVE: Antibody blockade of the "do not eat me" signal CD47 (cluster of differentiation 47) enhances efferocytosis and reduces lesion size and necrotic core formation in murine atherosclerosis. TNF (Tumor necrosis factor)-α expression directly enhances CD47 expression, and elevated TNF-α is observed in the absence of the proefferocytosis receptor LRP1 (low-density lipoprotein receptor-related protein 1), a regulator of atherogenesis and inflammation. Thus, we tested the hypothesis that CD47 blockade requires the presence of macrophage LRP1 to enhance efferocytosis, temper TNF-α-dependent inflammation, and limit atherosclerosis. Approach and Results: Mice lacking systemic apoE (apoE-/-), alone or in combination with the loss of macrophage LRP1 (double knockout), were fed a Western-type diet for 12 weeks while receiving anti-CD47 antibody (anti-CD47) or IgG every other day. In apoE-/- mice, treatment with anti-CD47 reduced lesion size by 25.4%, decreased necrotic core area by 34.5%, and decreased the ratio of free:macrophage-associated apoptotic bodies by 47.6% compared with IgG controls (P<0.05), confirming previous reports. Double knockout mice treated with anti-CD47 showed no differences in lesion size, necrotic core area, or the ratio of free:macrophage-associated apoptotic bodies compared with IgG controls. In vitro efferocytosis was 30% higher when apoE-/- phagocytes were incubated with anti-CD47 compared with IgG controls (P<0.05); however, anti-CD47 had no effect on efferocytosis in double knockout phagocytes. Analyses of mRNA and protein showed increased CD47 expression in anti-inflammatory IL (interleukin)-4 treated LRP1-/- macrophages compared with wild type, but no differences were observed in inflammatory lipopolysaccharide-treated macrophages. CONCLUSIONS: The proefferocytosis receptor LRP1 in macrophages is necessary for anti-CD47 blockade to enhance efferocytosis, limit atherogenesis, and decrease necrotic core formation in the apoE-/- model of atherosclerosis.

Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

BACKGROUND: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. METHODS: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). RESULTS: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. CONCLUSIONS: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Reduction in Acute Limb Ischemia With Rivaroxaban Versus Placebo in Peripheral Artery Disease After Lower Extremity Revascularization: Insights From VOYAGER PAD.

BACKGROUND: Patients with peripheral artery disease (PAD) are at heightened risk of acute limb ischemia (ALI), a thrombotic event associated with amputation, disability, and mortality. Previous lower extremity revascularization (LER) is associated with increased ALI risk in chronic PAD. However, the pattern of risk, clinical correlates, and outcomes after ALI early after LER are not well-studied, and effective therapies to reduce ALI post-LER are lacking. METHODS: The VOYAGER PAD trial (Vascular Outcomes Study of ASA [Acetylsalicylic Acid] Along With Rivaroxaban in Endovascular or Surgical Limb Revascularization for PAD; rNCT02504216) randomized patients with PAD undergoing LER to rivaroxaban 2.5 mg twice daily or placebo on a background of low-dose aspirin. The primary outcome was a composite of ALI, major amputation of vascular cause, myocardial infarction, ischemic stroke, or cardiovascular death. ALI was prospectively ascertained and adjudicated by a blinded committee. The cumulative incidence of ALI was calculated using Kaplan-Meier estimates, and Cox proportional hazards models were used to generate hazard ratios and associated CIs. Analyses were performed as intention-to-treat. RESULTS: Among 6564 patients followed for a median of 2.3 years, 382 (5.8%) had a total of 508 ALI events. In placebo patients, the 3-year cumulative incidence of ALI was 7.8%. After multivariable modeling, previous LER, baseline ankle-brachial index <0.50, surgical LER, and longer target lesion length were associated with increased risk of ALI. Incident ALI was associated with subsequent all-cause mortality (hazard ratio [HR], 2.59 [95% CI, 1.98-3.39]) and major amputation (HR, 24.87 [95% CI, 18.68-33.12]). Rivaroxaban reduced ALI relative to placebo by 33% (absolute risk reduction, 2.6% at 3 years; HR, 0.67 [95% CI, 0.55-0.82]; P=0.0001), with benefit starting early (HR, 0.45 [95% CI, 0.24-0.85]; P=0.0068 at 30 days). Benefit was present for severe ALI (associated with death, amputation, or prolonged hospitalization and intensive care unit stay, HR, 0.58 [95% CI, 0.40-0.83]; P=0.003) and regardless of LER type (surgical versus endovascular revascularization, P interaction=0.42) or clopidogrel use (P interaction=0.59). CONCLUSIONS: After LER for symptomatic PAD, ALI is frequent, particularly early after LER, and is associated with poor prognosis. Low-dose rivaroxaban plus aspirin reduces ALI after LER, including ALI events associated with the most severe outcomes. The benefit of rivaroxaban for ALI appears early, continues over time, and is consistent regardless of revascularization approach or clopidogrel use.

Development of a polygenic risk score to improve detection of peripheral artery disease.

INTRODUCTION: Peripheral artery disease (PAD) is a major cause of cardiovascular morbidity and mortality, yet timely diagnosis is elusive. Larger genome-wide association studies (GWAS) have now provided the ability to evaluate whether genetic data, in the form of genome-wide polygenic risk scores (PRS), can help improve our ability to identify patients at high risk of having PAD. METHODS: Using summary statistic data from the largest PAD GWAS from the Million Veteran Program, we developed PRSs with genome data from UK Biobank. We then evaluated the clinical utility of adding the best-performing PRS to a PAD clinical risk score. RESULTS: A total of 487,320 participants (5759 PAD cases) were included in our final genetic analysis. Compared to participants in the lowest 10% of PRS, those in the highest decile had 3.1 higher odds of having PAD (95% CI, 3.06-3.21). Additionally, a PAD PRS was associated with increased risk of having coronary artery disease, congestive heart failure, and cerebrovascular disease. The PRS significantly improved a clinical risk model (Net Reclassification Index = 0.07, p < 0.001), with most of the performance seen in downgrading risk of controls. Combining clinical and genetic data to detect risk of PAD resulted in a model with an area under the curve of 0.76 (95% CI, 0.75-0.77). CONCLUSION: We demonstrate that a genome-wide PRS can discriminate risk of PAD and other cardiovascular diseases. Adding a PAD PRS to clinical risk models may help improve detection of prevalent, but undiagnosed disease.

CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

Intradialytic Hypotension and Newly Recognized Peripheral Artery Disease in Patients Receiving Hemodialysis.

RATIONALE & OBJECTIVE: Intradialytic hypotension (IDH) may decrease systemic circulation to the legs, exacerbating symptoms of peripheral artery disease (PAD). We sought to evaluate the relationship between IDH and newly recognized lower extremity PAD among hemodialysis patients. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Linking data from the US Renal Data System to the electronic health records of a large dialysis provider, we identified adult patients (≥18 years of age) with Medicare Parts A and B who initiated dialysis (2006-2011) without previously recognized PAD. EXPOSURE: The time-varying proportion of hemodialysis sessions with IDH defined as the nadir intradialytic systolic blood pressure <90 mm Hg. We categorized the proportion of sessions with IDH within serial 30-day intervals as 0%, >0% to <15%, 15% to <30%, and ≥30%. OUTCOMES: Newly recognized PAD was ascertained using PAD diagnostic and procedure codes for amputation or revascularization, in serial 30-day intervals subsequent to each 30-day exposure interval. ANALYTICAL APPROACH: To account for the competing risks of death and kidney transplantation, we estimated unadjusted and adjusted subdistribution hazard ratios using the Kaplan-Meier multiple imputation method in combination with the extended Cox model to account for IDH as a time-varying exposure. RESULTS: Among 45,591 patients, those with more frequent baseline IDH had a higher prevalence of cardiovascular diseases. During 61,725 person-years of follow-up, 7,886 patients had newly recognized PAD. We found a graded, direct association between IDH and newly recognized PAD. For example, having IDH in ≥30% of dialysis sessions during a given 30-day interval (vs 0%) was associated with a 24% (95% CI, 17%-32%) higher hazard than having newly recognized PAD in the subsequent 30 days. LIMITATIONS: Unmeasured confounding; ascertainment of PAD from claims. CONCLUSIONS: Patients receiving hemodialysis who had more frequent IDH had higher rates of newly recognized PAD. Patients with frequent IDH may warrant careful examination for PAD.

Comparison of Pre-Amputation Evaluation in Patients with and without Chronic Kidney Disease.

INTRODUCTION: Patients with chronic kidney disease (CKD) and peripheral artery disease (PAD) are more likely to undergo lower extremity amputation than patients with preserved kidney function. We sought to determine whether patients with CKD were less likely to receive pre-amputation care in the 1-year prior to lower extremity amputation compared to patients without CKD. METHODS: We conducted a retrospective observational study of patients with PAD-related lower extremity amputation between January 2014 and December 2017 using a large commercial insurance database. The primary exposure was CKD identified using billing codes and laboratory values. The primary outcomes were receipt of pre-amputation care, defined as diagnostic evaluation (ankle-brachial index, duplex ultrasound, and computed tomographic angiography), specialty care (vascular surgery, cardiology, orthopedic surgery, and podiatry), and lower extremity revascularization in the 1-year prior to amputation. We conducted separate logistic regression models to estimate the adjusted odds ratio (aOR) and 95% confidence intervals (CIs) among patients with and without CKD. We assessed for effect modification by age, sex, Black race, and diabetes status. RESULTS: We identified 8,554 patients with PAD-related amputation. In fully adjusted models, patients with CKD were more likely to receive diagnostic evaluation (aOR 1.30; 95% CI 1.17-1.44) and specialty care (aOR 1.45, 95% CI 1.27-1.64) in the 1-year prior to amputation. There was no difference in odds of revascularization by CKD status (aOR 1.03, 0.90-1.19). Age, sex, Black race, and diabetes status did not modify these associations. DISCUSSION/CONCLUSION: Patients with CKD had higher odds of receiving diagnostic testing and specialty care and similar odds of lower extremity revascularization in the 1-year prior to amputation than patients without CKD. Disparities in access to pre-amputation care do not appear to explain the higher amputation rates seen among patients with CKD.

18F-Fluorodeoxyglucose-Positron Emission Tomography Imaging Detects Response to Therapeutic Intervention and Plaque Vulnerability in a Murine Model of Advanced Atherosclerotic Disease-Brief Report.

OBJECTIVE: This study sought to determine whether 18F-fluorodeoxyglucose-positron emission tomography/computed tomography could be applied to a murine model of advanced atherosclerotic plaque vulnerability to detect response to therapeutic intervention and changes in lesion stability. Approach and Results: To analyze plaques susceptible to rupture, we fed ApoE-/- mice a high-fat diet and induced vulnerable lesions by cast placement over the carotid artery. After 9 weeks of treatment with orthogonal therapeutic agents (including lipid-lowering and proefferocytic therapies), we assessed vascular inflammation and several features of plaque vulnerability by 18F-fluorodeoxyglucose-positron emission tomography/computed tomography and histopathology, respectively. We observed that 18F-fluorodeoxyglucose-positron emission tomography/computed tomography had the capacity to resolve histopathologically proven changes in plaque stability after treatment. Moreover, mean target-to-background ratios correlated with multiple characteristics of lesion instability, including the corrected vulnerability index. CONCLUSIONS: These results suggest that the application of noninvasive 18F-fluorodeoxyglucose-positron emission tomography/computed tomography to a murine model can allow for the identification of vulnerable atherosclerotic plaques and their response to therapeutic intervention. This approach may prove useful as a drug discovery and prioritization method.

Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus.

Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD.