Plasma Cholesterol-Induced Lesion Networks Activated before Regression of Early, Mature, and Advanced Atherosclerosis

Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (>= 80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-)Apob(100/100)Mttp(flox/flox)Mx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions. Author Summary The main underlying cause of heart attacks and strokes is atherosclerosis. One strategy to prevent these often deadly clinical events is therefore either to slow atherosclerosis progression or better, induce regression of atherosclerotic plaques making them more stable. Plasma cholesterol lowering (PCL) is the most efficient way to induce atherosclerosis regression but sometimes fails to do so. In our study, we used a mouse model with elevated LDL cholesterol levels, similar to humans who develop early atherosclerosis, and a genetic switch to lower plasma cholesterol at any time during atherosclerosis progression. In this model, we examined atherosclerosis gene expression and regression in response to PCL at three different stages of atherosclerosis progression. PCL led to complete regression in mice with early lesions but was incomplete in mice with mature and advanced lesions, indicating that early prevention with PCL in individuals with increased risk for heart attack or stroke would be particularly useful. In addition, by inferring PCL-responsive gene networks in early, mature and advanced atherosclerotic lesions, we identified key drivers specific for regression of early (PPARG), mature (MLL5) and advanced (SRSF10/XRN2) atherosclerosis. These key drivers should be interesting therapeutic targets to enhance PCL-mediated regression of atherosclerosis

Rectal INdomethacin, oral TacROlimus, or their combination for the prevention of post-ERCP pancreatitis (INTRO Trial): Protocol for a randomized, controlled, double-blinded trial.

BACKGROUND: Acute pancreatitis remains the most common and morbid complication of endoscopic retrograde cholangiopancreatography (ERCP). The use of rectal indomethacin and pancreatic duct stenting has been shown to reduce the incidence and severity of post-ERCP pancreatitis (PEP), but these interventions have limitations. Recent clinical and translational evidence suggests a role for calcineurin inhibitors in the prevention of pancreatitis, with multiple retrospective case series showing a reduction in PEP rates in tacrolimus users. METHODS: The INTRO trial is a multicenter, international, randomized, double-blinded, controlled trial. A total of 4,874 patients undergoing ERCP will be randomized to receive either oral tacrolimus (5 mg) or oral placebo 1-2 h before ERCP, and followed for 30 days post-procedure. Blood and pancreatic aspirate samples will also be collected in a subset of patients to quantify tacrolimus levels. The primary outcome of the study is the incidence of PEP. Secondary endpoints include the severity of PEP, ERCP-related complications, adverse drug events, length of hospital stay, cost-effectiveness, and the pharmacokinetics, pharmacodynamics, and pharmacogenomics of tacrolimus immune modulation in the pancreas. CONCLUSIONS: The INTRO trial will assess the role of calcineurin inhibitors in PEP prophylaxis and develop a foundation for the clinical optimization of this therapeutic strategy from a pharmacologic and economic standpoint. With this clinical trial, we hope to demonstrate a novel approach to PEP prophylaxis using a widely available and well-characterized class of drugs. TRIAL REGISTRATION: NCT05252754, registered on February 14, 2022

Transcriptional profiling during regression of aortic atherosclerotic lesions in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^flox/flox and <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice over time.

<p>Differential expression analyses was used to define sets of genes causally and reactively related to atherosclerosis regression in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice. RNA for the transcriptional profiling was isolated from the atherosclerotic aortic arch. Narrow and bold arrows indicate times of PCL and sacrifice, respectively. Colored horizontal lines indicate time frame of transcriptional profiles used for differential expression analysis to define gene sets. Colors indicate when PCL was started: green, 30 weeks; yellow, 40 weeks; red, 50 weeks. (A) To define the PCL-responsive gene sets, we compared transcriptional profiles (4–6 per time point) of PBS-treated, high-cholesterol littermate controls sacrificed at 30, 40 and 50 weeks with those immediately after PCL. (B) To define the regression-reactive gene sets, we compared transcriptional profiles (3–6 per time point) immediately after PCL with those at 10 weeks after PCL (10 per time point).</p

Plasma cholesterol, triglyceride and glucose concentrations in the study mice at sacrifice.

<p>Values are mean ± SD.</p><p>*<i>P</i><0.05,</p><p>***<i>P</i><0.001 vs. before PCL.</p

Top hubs in the causal TF-regulatory co-expression networks inferred in human macrophages.

<p>TF, transcription factor. Common hubs >5 connections.</p

Atherosclerosis progression in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^flox/flox mice and regression in <i>Ldlr^−/−Apob</i>^100/100<i>Mttp</i>^Δ/Δ mice.

<p>(A) Atherosclerosis progression and regression curves. Values are surface lesion area (mean ± SD), assessed by Sudan IV staining, as a percentage of the total area of pinned-out aortas. n = 4–10 per time point. Lesion development in controls without PCL (•) (<i>P</i><0.001 vs. 30 weeks) and in mice after PCL started at week 30 (▴), 40 (▪), or 50 (). Changes in lesion area between 10 and 20 weeks of low plasma cholesterol were significant only in mice with early lesions (PCL at 30 weeks, <i>P</i> = 0.05). *<i>P</i> = 0.05, ***<i>P</i><0.001. (B) Representative aortic trees (above) with magnified arches (below) stained with Sudan IV before and 10 and 20 weeks after PCL at 30, 40 and 50 weeks. Graphs indicate degree of regression at that PCL time-point (red).</p

Lim Domain Binding 2: A Key Driver of Transendothelial Migration of Leukocytes and Atherosclerosis.

Using a multitissue, genome-wide gene expression approach, we recently identified a gene module linked to the extent of human atherosclerosis. This atherosclerosis module was enriched with inherited risk for coronary and carotid artery disease (CAD) and overlapped with genes in the transendothelial migration of leukocyte (TEML) pathway. Among the atherosclerosis module genes, the transcription cofactor Lim domain binding 2 (LDB2) was the most connected in a CAD vascular wall regulatory gene network. Here, we used human genomics and atherosclerosis-prone mice to evaluate the possible role of LDB2 in TEML and atherosclerosis