The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using two dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and nondilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared with the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and nondilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and nondilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Thoracic aortic aneurysm (TAA) 1 is a pathological widening of the aorta, resulting from degeneration of extra cellular

Although several risk factors such as infarct size have been identified, the progression of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of post-myocardial infarction (MI), we previously identified 45 proteins differentially modulated during HF by proteomic analysis. This study sought to identify microRNAs (miRNAs) able to regulate these proteins and to test their relevance as biomarkers for HF. In silico bioinformatical analysis selected 13 miRNAs related to the 45 proteins previously identified. These miRNAs were analyzed in the rat and in cohorts of patients phenotyped for left ventricular remodeling (LVR). We identified that 3 miRNAs, miR-21-5p, miR-23a-3p and miR-222-3p, and their target Mn superoxide dismutase (SOD2) were significantly increased in LV and plasma of HF-rats. We found by luciferase activity a direct interaction of miR-222-3p with 3′UTR of SOD2. Transfection of human cardiomyocytes with miR-222-3p mimic or inhibitor induced respectively a decrease and an increase of SOD2 expression. Circulating levels of the 3 miRNAs and their target SOD2 were associated with high LVR post-MI in REVE-2 patients. We demonstrated for the first time the potential of microRNAs regulating SOD2 as new circulating biomarkers of HF.

Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.

BackgroundRemote ischemic preconditioning (RIPC) has emerged as an attractive strategy in clinical settings. Despite convincing evidence of the critical role played by circulating humoral mediators, their actual identities remain unknown. In this study, we aimed to identify RIPC-induced humoral mediators using a proteomic approach.Methodsand Results Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5′) or 10-min (RIPC 10′) reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were analyzed using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). Three protein peaks were selected for their significant increase in RIPC 10′. They were identified and confirmed as apolipoprotein A-I (ApoA-I). Additional rats were exposed to myocardial ischemia-reperfusion (I/R) and assigned to one of the following groups RIPC+myocardial infarction (MI) (10-min limb ischemia followed by 10-min reperfusion initiated 20 minutes prior to myocardial I/R), ApoA-I+MI (10 mg/kg ApoA-I injection 10 minutes before myocardial I/R), and MI (no further intervention). In comparison with untreated MI rats, RIPC reduced infarct size (52.2±3.7% in RIPC+MI vs. 64.9±2.6% in MI; p<0.05). Similarly, ApoA-I injection decreased infarct size (50.9±3.8%; p<0.05 vs. MI).ConclusionsRIPC was associated with a plasmatic increase in ApoA-I. Furthermore, ApoA-I injection before myocardial I/R recapitulated the cardioprotection offered by RIPC in rats. This data suggests that ApoA-I may be a protective blood-borne factor involved in the RIPC mechanism.

ObjectiveLeft ventricular (LV) remodeling following myocardial infarction (MI) is characterized by progressive alterations of structure and function, named LV remodeling. Although several risk factors such as infarct size have been identified, LV remodeling remains difficult to predict in clinical practice. Changes within the extracellular matrix, involving matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are an integral part of left ventricular (LV) remodeling after myocardial infarction (MI). We investigated the temporal profile of circulating MMPs and TIMPs and their relations with LV remodeling at 1 year and clinical outcome at 3 years in post-MI patients.MethodsThis prospective multicentre study included 246 patients with a first anterior MI. Serial echocardiographic studies were performed at hospital discharge, 3 months, and 1 year after MI, and analysed at a core laboratory. LV remodeling was defined as the percent change in LV end-diastolic volume (EDV) from baseline to 1 year. Serum samples were obtained at hospital discharge, 1, 3, and 12 months. Multiplex technology was used for analysis of MMP-1, -2, -3, -8, -9, -13, and TIMP-1, -2, -3, -4 serum levels.ResultsBaseline levels of MMP-8 and MMP-9 were positively associated with changes in LVEDV (P = 0.01 and 0.02, respectively). When adjusted for major baseline characteristics, MMP-8 levels remained an independent predictor LV remodeling (P = 0.025). By univariate analysis, there were positive relations between cardiovascular death or hospitalization for heart failure during the 3-year follow-up and the baseline levels of MMP-2 (P = 0.03), MMP-8 (P = 0.002), and MMP-9 (P = 0.03). By multivariate analysis, MMP-8 was the only MMP remaining significantly associated with clinical outcome (P = 0.02).ConclusionBaseline serum MMP-8 is a significant predictor of LV remodeling and cardiovascular outcome after MI and may help to improve risk stratification.

BackgroundAbdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (>1.5 times normal) and loss of parallelism. This disease is responsible for 1–4% mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions.Methodology/Principal FindingsPooled human plasma samples of 17 AAA and 17 control patients were depleted of the most abundant proteins and compared using a data-independent shotgun proteomic strategy, Precursor Acquisition Independent From Ion Count (PAcIFIC), combined with spectral counting and isobaric tandem mass tags. Both quantitative methods collectively identified 80 proteins as statistically differentially abundant between AAA and control patients. Among differentially abundant proteins, a subgroup of 19 was selected according to Gene Ontology classification and implication in AAA for verification by Western blot (WB) in the same 34 individual plasma samples that comprised the pools. From the 19 proteins, 12 were detected by WB. Five of them were verified to be differentially up-regulated in individual plasma of AAA patients: adiponectin, extracellular superoxide dismutase, protein AMBP, kallistatin and carboxypeptidase B2.Conclusions/SignificancePlasma depletion of high abundance proteins combined with quantitative PAcIFIC analysis offered an efficient and sensitive tool for the screening of new potential biomarkers of AAA. However, WB analysis to verify the 19 PAcIFIC identified proteins of interest proved inconclusive save for five proteins. We discuss these five in terms of their potential relevance as biological markers for use in AAA screening of population at risk.

Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.