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.

Investigation of the terminal P4 domain in a series of D-phenylglycinamide-based factor Xa inhibitors.

Several P4 domain derivatives of the general d-phenylglycinamide-based scaffold (2) were synthesized and evaluated for their ability to bind to the serine protease factor Xa. Some of the more potent compounds were evaluated for their anticoagulant effects in vitro. A select subset containing various P1 indole constructs was further evaluated for their pharmacokinetic properties after oral administration to rats.

Immunohistochemical analysis of target proteins of Rho-kinase in a mouse model of accelerated atherosclerosis.

BACKGROUND: 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. OBJECTIVES: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

Inhibition of thrombin activatable fibrinolysis inhibitor by cysteine derivatives.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a basic carboxypeptidase that functions as a fibrinolysis inhibitor through the cleavage of C-terminal lysine on partially degraded fibrin. Modulation of TAFI activity provides a potential therapy for thrombosis complications by potentiating fibrinolysis. In our study, we identified three novel TAFI inhibitors containing a cysteine backbone. Three cysteine derivatives, guanidinyl-L-cysteine, glycyl-L-cysteine, and glycyl-glycyl-L-cysteine were tested in TAFI substrate assays and showed K(app)(i)=0.08, 0.14, and 0.99 microM, respectively. Subsequent fibrinolysis assays confirmed their TAFI inhibitory activities. Guanidinyl-L-cysteine showed inhibitory activity in a human plasma clot lysis assay (IC(50)=9.4 microM). Identification of these cysteine derivatives represents an opportunity to develop potent and specific TAFI inhibitors.

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 PPARalpha,gamma 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.

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-1beta, 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.

Investigation of factor Xa inhibitors containing non-amidine S1 elements.

Several non-amidino S1 derivatives of the 1,2-diaminobenzene-based scaffold (4) were synthesized and evaluated for their ability to bind to the active site and inhibit the human protease factor Xa. A subset of these compounds were also evaluated for their anticoagulant effects in human plasma as measured by prothrombin time (PT).