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author:("richter, Mark")
1.  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 
PLoS ONE  2014;9(4):e93297.
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.
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.
PMCID: PMC3973691  PMID: 24695114
2.  Glucose Metabolic Trapping in Mouse Arteries: Nonradioactive Assay of Atherosclerotic Plaque Inflammation Applicable to Drug Discovery 
PLoS ONE  2012;7(11):e50349.
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.
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.
PMCID: PMC3508896  PMID: 23209718
3.  Protein Kinase C inhibition ameliorates functional endothelial insulin resistance and Vascular Smooth Muscle Cell hypersensitivity to insulin in diabetic hypertensive rats 
Insulin 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.
A 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.
The 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.
These 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.
PMCID: PMC3127756  PMID: 21635764
4.  Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice 
Cell metabolism  2010;11(5):379-389.
To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls, but reduced to below control values by a VCAM-1 blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications.
PMCID: PMC3020149  PMID: 20444418
5.  Circulating Markers Reflect Both Anti- and Pro-Atherogenic Drug Effects in ApoE-Deficient Mice 
Biomarker Insights  2008;3:147-157.
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.
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.
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.
PMCID: PMC2688353  PMID: 19578502
6.  Immunohistochemical analysis of target proteins of Rho-kinase in a mouse model of accelerated atherosclerosis 
The pleiotropic antiatherosclerotic effects of statins are believed to be associated with the inhibition of Rho-kinase. However, a systematic analysis of Rho-kinase activation in atherosclerotic lesions is missing.
To analyze the distribution and phosphorylation of target proteins of Rho-kinase, such as myosin light chain (MLC) and ezrin-radixin-moesin (ERM) proteins, in the apolipoprotein E (ApoE) knockout model of accelerated atherosclerosis, as well as the effects of treatment with the Rho-kinase inhibitor Y-27632.
Western diet-fed ApoE-deficient mice underwent carotid ligation and were sacrificed 14 days after surgery. One group of ligated mice was treated with the Rho-kinase inhibitor Y-27632. Nonligated C57Bl6/J mice on normal chow and ApoE-deficient mice on Western diet were used as controls. Lesion structure and size were analyzed using Masson-elastic stained cross-sections. The distribution and phosphorylation of Rho-kinase target proteins were studied immunohistochemically.
Two weeks after surgery, atherosclerotic plaque-like lesions developed in ligated carotids. Lesion development was inhibited by Y-27632. ERM was expressed ubiquitously, but in the intact arteries, it was phosphorylated exclusively in the endothelium and periadventitial adipocytes. In the atherosclerotic lesions, foamy macrophages also exhibited a strong phospho-ERM signal. Y-27632 inhibited ERM phosphorylation in the plaques. MLC and phospho-MLC were associated with smooth muscle cells and did not respond to the Y-27632 treatment.
A cell type-selective distribution and phosphorylation of target proteins of Rho-kinase were demonstrated in the carotid artery of the normal mouse model, as well as in the ApoE-knockout model of accelerated atherosclerosis. Various downstream targets of the same enzyme may be differentially involved in specific pathological processes in a cell type-specific manner.
PMCID: PMC2359611  PMID: 18651000
Atherosclerosis; Endothelium; Macrophages; Phosphorylation; Rho-kinase

Results 1-6 (6)