Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia.

Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis.

One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation.

A comparative study of immunodepletion and equalization methods for aortic stenosis human plasma.

Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. Until now, aortic stenosis (AS) was thought to result from aging and "wear and tear" of the aortic valve, but nowadays, it is known that it presents the same risk factors as atherosclerosis and cardiovascular diseases.A proteomic analysis of plasma could permit to identify the changes in protein expression induced by AS in this biological sample.

Secretome of human aortic valves.

One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Between different proteomics studies, the secretome is a valuable tool in the search for biomarkers locally released by a studied tissue and remains particularly important while working with vascular tissues, since the secreted proteins would be probably shed to the blood. In this chapter, we described a method to validate the origin of secreted proteins in human aortic valves, demonstrating their synthesis and release by the tissue and ruling out blood origin.

Multiple reaction monitoring (MRM) of plasma proteins in cardiovascular proteomics.

Different methodologies have been used through years to discover new potential biomarkers related with cardiovascular risk. The conventional proteomic strategy involves a discovery phase that requires the use of mass spectrometry (MS) and a validation phase, usually on an alternative platform such as immunoassays that can be further implemented in clinical practice. This approach is suitable for a single biomarker, but when large panels of biomarkers must be validated, the process becomes inefficient and costly.

Secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis.

Aims: Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development.

Inside human aortic stenosis: a proteomic analysis of plasma.

Valvular aortic stenosis (AS) produces a slowly progressive obstruction in left ventricular outflow track. For this reason, aortic valve replacement is warranted when the valvular stenosis is hemodinamically significant, becoming the most common worldwide cause of aortic valve surgery. Recent epidemiologic studies have revealed an association between degenerative AS and cardiovascular risk factors for atherosclerosis, althought reducing the exposure to such factors and statin therapies both fail to delay or reverse the pathology.

Analysis of the plasma proteome associated with acute coronary syndrome: does a permanent protein signature exist in the plasma of ACS patients?

Acute coronary syndrome (ACS) is triggered by the occlusion of a coronary artery usually due to the thrombosis caused by an atherosclerotic plaque. The identification of proteins directly involved in the pathophysiological events underlying ACS will enable more precise diagnoses and a more accurate prognosis to be determined. Accordingly, we have performed a longitudinal study of the plasma proteome in ACS patients by 2-DE and DIGE.

Valvular aortic stenosis: a proteomic insight.

Unlabelled: Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible and effective method to carry out proteomic analysis of stenotic human valves by conventional 2-DE and 2D-DIGE, minimizing the interference due to high calcium concentrations. Furthermore, the protocol permits the aortic stenosis proteome to be analysed, advancing our knowledge in this area.

A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE.

With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE, paying particular attention to hemoglobin removal.

Atorvastatin modifies the protein profile of circulating human monocytes after an acute coronary syndrome.

Aggressive treatment with high-dose atorvastatin reduces more effectively the incidence of cardiovascular events than moderate statin therapy. The mechanism of this benefit has not been fully elucidated. In order to know the potential effects of statin treatment on the protein expression of circulating monocytes in acute coronary syndrome (ACS) patients, a proteomic analysis of these cells was carried out by 2-DE and MS.

Vascular proteomics.

The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach.

Circulating human monocytes in the acute coronary syndrome express a characteristic proteomic profile.

We examined the proteome of circulating monocytes of patients with acute coronary syndrome at different times in comparison to that of patients with stable coronary artery disease. On admission, the expression of 18 spot proteins was altered, 10 of which were totally absent. This pattern changed progressively, and at 6 months, there were no differences with the monocyte proteome of stable patients.

Depletion of high-abundance proteins in plasma by immunoaffinity subtraction for two-dimensional difference gel electrophoresis analysis.

Blood plasma is believed the most complex human-derived proteome, containing other tissue proteome subsets. Almost all body cells communicate with the plasma, either directly or through tissues or biological fluids, and many of these cells release at least a part of their content into the plasma upon damage or death. A comprehensive, systematic characterization of the plasma proteome in the healthy and diseased states will greatly facilitate the development of biomarkers for early disease detection, clinical diagnosis, and therapy.

Characterization of circulating human monocytes by proteomic analysis.

We describe a simple method for isolation of human blood monocytes with the high purity required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with monoclonal antibodies coupled to paramagnetic beads. This method is compatible with flow cytometry, which was used to assess the purity of the cell population.

Characterization of the human atheroma plaque secretome by proteomic analysis.

Atherosclerosis is one of the most common causes of death in developed countries. Atherosclerosis is an inflammatory process that results in the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction and stroke.

Quest for novel cardiovascular biomarkers by proteomic analysis.

Atherosclerosis, and the resulting coronary heart disease and stroke, is the most common cause of death in developed countries. Atherosclerosis is an inflammatory process that results in the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction (MI) and stroke.