Rationale: MicroRNAs (miRNAs) have been implicated in the epigenetic regulation of key metabolic, inflammatory, and antiangiogenic pathways in type 2 diabetes (DM) and may contribute to common disease complications.Objective: In this study, we explore plasma miRNA profiles in patients with DM. Methods and Results:

This study applied mass spectrometry-based lipidomics profiling to population-based cohorts and identified molecular lipid signatures for cardiovascular disease. Molecular lipid species constitute promising new biomarkers that outperform the conventional biochemical measurements of lipid classes currently used in clinics.

IntroductionNumerous studies have demonstrated that endothelial progenitor cells (EPCs) are present among peripheral blood mononuclear cells (PBMNCs) and represent a subset of circulating bone marrow mononuclear cells (BMCs), which have the capacity to differentiate into endothelial cells in vivo. 1 New concepts of stem cell-based therapies for myocardial regeneration resulted in a rapid translation into a clinical context. [2][3][4] Yet, key questions remain unanswered. Importantly, the nomenclature and the phenotype of EPCs are subject to ongoing controversy and there are currently no specific markers that unambiguously identify these cells. 5,6 Thus, a more comprehensive approach is needed to analyze their antigenic profiles.MPs are small membrane vesicles (0.2-1.0 m) that originate from the plasma membrane and are shed from the cell surface after activation and apoptosis. 7 Importantly, MPs retain membrane antigens specific for the parent cell they originate from. Thus, MPs represent an ideal subproteome to clarify the cellular progeny of EPC cultures and mass spectrometry is the instrument of choice for this kind of research. 8 In this study, we used a proteomic approach to identify membrane proteins present on MPs in EPC cultures. Methods EPC cultureThe study was approved by the ethics review board of J. W. Goethe University and King's College London. Peripheral blood was collected from healthy adult volunteers and informed consent was obtained in accordance with the Declaration of Helsinki. EPC cultures were performed as previously described. 9,10 In brief, PBMNCs from healthy volunteers were isolated by Lymphoprep (1.077 g/mL; Axis-Shield PoCAS) density barrier centrifugation. The low-density fraction (Ͻ 1.077 g/mL) was carefully removed from the interface and washed 3 times with PBS (Dulbecco phosphate-buffered saline; Sigma-Aldrich) containing 2% FBS (fetal bovine serum, filtered and heat inactivated; Gibco, Invitrogen). Immediately after isolation, the cells were counted and 8 ϫ 10 6 cells were plated on fibronectin-coated (10 g/mL fibronectin from human plasma; SigmaAldrich) 12-well plates containing 1 mL endothelial basal medium (EBM; Cambrex Bio Science) supplemented with 20% FBS, EGM SingleQuots (10 g/mL epidermal growth factor, 3 g/mL bovine brain extract, 50 g/mL gentamicine, 50 g/mL amphotericin-B, 1 g/mL hydrocortisone; Cambrex Bio Science) and 10 ng/mL human vascular endothelial growth factor 165 (hVEGF 165; R&D Systems). Before use, the medium was passed through a 0.2-m filter. After 3 days in culture, the nonadherent An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From cells were removed and fresh EBM medium was added. T...

The present study characterized the metabolic adaptation to persistent AF, unraveling a potential role for ketone bodies, and demonstrated that discordant metabolic alterations are evident in individuals susceptible to post-operative AF.

Rationale: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. Objective: To perform a proteomics comparison of miRNA effects on extracellular matrix secretion by cardiac fibroblasts. Methods and Results: Mouse cardiac fibroblasts were transfected with pre-/anti-miR of miR-29b and miR-30c, and their conditioned medium was analyzed by mass spectrometry. miR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor, insulin-like growth factor 1, and pentraxin 3, 3 predicted targets of miR-29b. miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-β. In contrast, miR-30c had little effect on extracellular matrix production but opposite effects regarding leukemia inhibitory factor and insulin-like growth factor 1. Both miRNAs indirectly affected cardiac myocytes. On transfection with pre–miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (α-actinin, cardiac myosin-binding protein C, and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre–miR-29 conditioned medium, whereas pre–miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c, and miR-30c, but not miR-29b, were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. Conclusions: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCdelta knockout mice and performed vein bypass grafts on these animals. PKCdelta(-/-) mice developed normally and were fertile. Vein segments from PKCdelta(-/-) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCdelta(+/+) vein grafts. Arteriosclerotic lesions in PKCdelta(-/-) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCdelta(-/-) mice. SMCs derived from PKCdelta(-/-) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCdelta(-/-) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCdelta maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.

Objective-Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E Ϫ/Ϫ mice on normal chow diet. Methods and Results-Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E Ϫ/Ϫ mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E Ϫ/Ϫ mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E Ϫ/Ϫ mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool. Conclusion-Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism. Key Words: animal model Ⅲ apolipoprotein E Ⅲ atherosclerosis Ⅲ metabolomics Ⅲ oxidative stress Ⅲ proteomics T he generation of apolipoprotein E-deficient (apolipoprotein E Ϫ/Ϫ ) mice 1,2 has been one of the most critical advancements in the elucidation of factors affecting atherogenesis. It is currently the most popular murine model in cardiovascular research and has revealed important insights into atherosclerosis. But despite a decade of research, there is still a need for sophisticated experimental techniques to obtain a more comprehensive understanding of the complex pathophysiology. 3 Previous studies have revealed apolipoprotein E-related alterations in the transcriptome. 4 However, simple deduction of protein expression from mRNA transcript analysis is insufficient 5 and, importantly, provides no information on post-translational modifications, which are known to be instrumental in many human diseases.We recently analyzed the proteomic profile of mouse arterial smooth muscle that was markedly influenced by mutational ablation of the protein kinase C delta gene. 6 Our proteomic findings were translated into a functional context by combining proteomics with metabolomic techniques, under in vivo 7,8 as well as in vitro conditions. 6 This new research strategy allows us to decipher dynamic alterations of cellular proteins and metabolites revealing multiple facets of a single pathogenesis. 9 In vascular research, proteomics and metabolomics are still in their infancies. Human umbilical cord endothelial cells and arterial and saphenous vein medial smooth muscle have been scantily characterized, but most attempts to apply proteomic techniques t...

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCdelta knockout mice and performed vein bypass grafts on these animals. PKCdelta(-/-) mice developed normally and were fertile. Vein segments from PKCdelta(-/-) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCdelta(+/+) vein grafts. Arteriosclerotic lesions in PKCdelta(-/-) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCdelta(-/-) mice. SMCs derived from PKCdelta(-/-) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCdelta(-/-) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCdelta maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.