Mark Rekhter

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation.

Inflammation is critically involved in atherogenesis. Signaling from innate immunity receptors TLR2 and 4, IL-1 and IL-18 is mediated by MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1. We hypothesized that IRAK4 kinase activity is critical for development of atherosclerosis. IRAK4 kinase-inactive knock-in mouse was crossed with the ApoE-/- mouse. Lesion development was stimulated by carotid ligation. IRAK4 functional deficiency was associated with down-regulation of several pro-inflammatory genes, inhibition of macrophage infiltration, smooth muscle cell and lipid accumulation in vascular lesions. Reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE-/- mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist thereby validating the model in the relevant pathway context. Thus, IRAK4 functional deficiency inhibits vascular lesion formation in ApoE-/- mice, which further unravels mechanisms of vascular inflammation and identifies IRAK4 as a potential therapeutic target.

Concordant Changes of Plasma and Kidney MicroRNA in the Early Stages of Acute Kidney Injury: Time Course in a Mouse Model of Bilateral Renal Ischemia-Reperfusion

Background Acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney excretory function and is strongly associated with increased early and long-term patient morbidity and mortality. Early diagnosis of AKI is challenging; therefore we profiled plasma microRNA in an effort to identify potential diagnostic circulating markers of renal failure. The goal of the present study was to investigate the dynamic relationship of circulating and renal microRNA profiles within the first 24 hours after bilateral ischemia-reperfusion kidney injury in mice. Methodology/Principal Findings Bilateral renal ischemia was induced in C57Bl/6 mice (n = 10 per group) by clamping the renal pedicle for 27 min. Ischemia-reperfusion caused highly reproducible, progressive, concordant elevation of miR-714, miR-1188, miR-1897-3p, miR-877*, and miR-1224 in plasma and kidneys at 3, 6 and 24 hours after acute kidney injury compared to the sham-operated mice (n = 5). These dynamics correlated with histologic findings of kidney injury and with a conventional plasma marker of renal dysfunction (creatinine). Pathway analysis revealed close association between miR-1897-3p and Nucks1 gene expression, which putative downstream targets include genes linked to renal injury, inflammation and apoptosis. Conclusions/Significance Systematic profiling of renal and plasma microRNAs in the early stages of experimental AKI provides the first step in advancing circulating microRNAs to the level of promising novel biomarkers.

Protein Kinase C inhibition ameliorates functional endothelial insulin resistance and Vascular Smooth Muscle Cell hypersensitivity to insulin in diabetic hypertensive rats

ObjectiveInsulin resistance, diabetes, and hypertension are considered elements of metabolic syndrome which is associated with vascular dysfunction. We investigated whether inhibition of protein kinase C (PKC) would affect vascular function in diabetic hypertensive (DH) rats.MethodsA combination of type 2 diabetes and arterial hypertension was produced in male Sprague Dawley rats by intrauterine protein deprivation (IUPD) followed by high salt diet. At the age of 32 weeks, DH rats were treated for 2 weeks with the angiotensin-converting enzyme inhibitor captopril (Capto, 30 mg/kg), PKC inhibitor ruboxistaurin (RBX, 50 mg/kg) or vehicle (n = 8 per group) and blood pressure was monitored using telemetry. At the end of experiments, femoral arteries were dissected, and vascular reactivity was evaluated with isovolumic myography.ResultsThe IUPD followed by high salt diet resulted in significant elevation of plasma glucose, plasma insulin, and blood pressure. Endothelium-dependent vascular relaxation in response to acetylcholine was blunted while vascular contraction in response to phenylephrine was enhanced in the DH rats. Neither Capto nor RBX restored endothelium-dependent vascular relaxation while both suppressed vascular contraction. Ex-vivo incubation of femoral arteries from control rats with insulin induced dose-response vasorelaxation while insulin failed to induce vasorelaxation in the DH rat arteries. In the control arteries treated with endothelial nitric oxide synthase inhibitor L-NAME, insulin induced vasoconstriction that was exacerbated in DH rats. Capto and RBX partially inhibited insulin-stimulated vascular contraction.ConclusionThese findings suggest that PKC inhibition ameliorates functional endothelial insulin resistance and smooth muscle cell hypersensitivity to insulin, but does not restore acetylcholine-activated endothelium-dependent vasodilation in DH rats.

Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE−/− mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARα,γ agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.

Glucose Metabolic Trapping in Mouse Arteries: Nonradioactive Assay of Atherosclerotic Plaque Inflammation Applicable to Drug Discovery

Background 18F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging. Methodology/Principal Findings Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity. Conclusions/Significance Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.

Sphingolipids in atherosclerosis: a metabolic underpinning of vascular disease

Although atherosclerotic plaque formation is driven primarily by cholesterol, new data suggest a proatherogenic role for sphingolipids. This role might be especially significant in the context of metabolic syndrome, where overabundance of long-chain saturated fatty acids stimulates sphingolipid synthesis and turnover. Increased levels of different sphingolipids in plasma and the vascular wall are associated strongly with atherosclerosis in patients and in experimental models. Inhibition of serine palmitoyl transferase, the key enzyme of sphingomyelin synthesis, reduced atherosclerosis in apolipoprotein E-deficient mice. Although the exact mechanisms of the antiatherosclerotic effect are still unknown, they likely involve reduction of both lipoprotein-associated and intracellular ceramide and sphingomyelin. In turn, they are involved in lipoprotein aggregation, retention, foam-cell formation, apoptosis and cholesterol synthesis. The ceramide derivative, sphingosine-1-phosphate may, in part, counterbalance ...

Factors associated with stroke, myocardial infarction, ischemic heart disease, unstable angina, or mortality in patients from real world clinical practice with newly-diagnosed type 2 diabetes and early glycemic control

Abstract Objectives: The objective of this study was to identify factors associated with stroke, myocardial infarction (MI), all-cause mortality, or a diagnosis of ischemic heart disease (IHD) or unstable angina (UA), among patients newly-diagnosed with type 2 diabetes (T2DM) with no recent history of cardiovascular (CV) events who rapidly achieve and maintain HbA1c ≤8.0%. Methods: Data were obtained from the Clinical Practice Research Datalink (CPRD) from January 1990 to December 2012. A nested case-control design was used with Cox proportional hazards analysis. Cases were identified by the first occurrence of stroke, MI, IHD, UA, or death within 5 years after HbA1c ≤ 8.0% was first reached (index date) following T2DM diagnosis. Controls were selected using a risk-set sampling approach and were matched 4:1 to cases using index date, exposure time, age, gender, and HbA1c at index date. Results: A total of 11,426 T2DM patients met the inclusion criteria for cases. Of these, 5,261 experienced a CV event. Stroke was the most frequent CV event (40%), followed by IHD (29%), MI (22%), and UA (9%). Mean HbA1c ≥7.0% over the length of exposure (vs 6.5 to <7.0%) was associated with an increased risk of stroke, MI, and IHD. The use of anti-platelet medications at baseline was also associated with increased risk of stroke (HR = 1.82 [CI = 1.60–2.06]), MI (HR = 1.67 [CI = 1.38–2.03]), and IHD (HR = 1.85 [CI = 1.57–2.17]). Mean HbA1c < 6.0% was associated with increased risk of stroke (HR = 1.29 [CI = 1.02–1.63]) and IHD (HR = 1.65 [CI = 1.25–2.19]). Use of nitrate medications at baseline was associated with increased risk of MI (HR = 2.83 [CI = 2.24–3.57]), IHD (HR = 4.32 [CI = 3.57–5.22]), and UA (HR = 10.38 [CI = 7.67–14.03]). Conclusions: Early and sustained HbA1c control between 6.5 and <7.0% appears to be an important modifiable factor that helps reduce CV risk in patients with newly-diagnosed T2DM in real-world clinical practice.

Circulating Markers Reflect Both Anti- and Pro-Atherogenic Drug Effects in ApoE-Deficient Mice

Background Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol. Results ApoE-/- mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro-and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1β, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis. Conclusion In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems.