Judit Cubedo

Proteomic signature of Apolipoprotein J in the early phase of new-onset myocardial infarction.

Acute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide. Despite all the efforts, there is a lack of early markers for prevention, diagnosis, and treatment of ischemic syndromes. By applying a proteomic expression profiling approach to identify biomarkers of early stages of AMI, we have detected significant changes in Apolipoprotein J/clusterin (ApoJ) in patients with an acute new-onset myocardial infarction. ApoJ characterization by bidimensional electrophoresis (2-DE), followed by mass spectrometry (MALDI-TOF) depicted a cluster of 13 spots (pI, 4.5-5.0; M(w), 37.1-47.3 kDa) with a significantly different distribution between AMI-patients and controls. Specifically, spots 2, 3, 7, 10, and 13 showed a 2-fold increase in their intensity in AMI-patients (P = 0.001). Western-blot analysis (WB) for total serum ApoJ depicted two bands of 40-45 and 65-70 kDa. When only glycosylated forms were analyzed, the band of 65-70 kDa was the most predominant one. A 25% decrease (P = 0.05) of ApoJ glycosylated forms in AMI-patients was detected by 2-DE. Serum ApoJ levels, determined by a commercial ELISA, were significantly lower (P < 0.001) in AMI-patients (n = 39) immediately after the event than in controls (n = 60). In 60% of patients, the lowest ApoJ level was detected within 6 h after the onset of AMI. Between 72 and 96 h after admission, ApoJ values in AMI-patients had reached control levels. Our results demonstrate alterations in ApoJ proteomic profile, due to a differential glycosylation pattern, in AMI-patients within the first 6 h after the onset of the event. Therefore, the analysis of this isoform glycosylation shift in patients with AMI may be of better use to understand ApoJ function than the total serum levels of ApoJ and this isoform shift may become an early marker of AMI.

Changes in thrombus composition and profilin-1 release in acute myocardial infarction

AIM Thrombus formation is a dynamic process regulated by flow, blood cells, and plasma proteins. The present study was performed to investigate the characteristics of human coronary thrombus in ST-segment elevation myocardial infarction (STEMI). METHODS AND RESULTS Patients admitted with ST-elevation myocardial infarction, in which thrombectomy was performed, were included (n = 86). Intracoronary thrombi and blood from the culprit coronary site and the systemic circulation were obtained during percutaneous coronary intervention (PCI). Thrombi were categorized by onset-of-pain-to-PCI elapsed time in thrombus of <3 (T3) and more than 6 h of evolution (T6). Clinical, morphological, and proteomic variables were investigated. While T3 were mainly composed by platelets and fibrin(ogen), T6 were characterized by a reduced platelet content, increased leucocytes infiltration (including monocytes, neutrophils, T-cells, and B-cells), and appearance of undifferentiated progenitor cells. Significant differences between T3 and T6 were found in the cell cytoskeleton-associated proteome (beta-actin and tropomyosin 3 and 4). By discovery proteomics, we have identified profilin-1 (Pfn-1) in the coronary thrombi and detected higher levels in T3 than in T6. While plasma Pfn-1 levels were low in T3 patients, levels significantly increased in both coronary and peripheral circulation in T6 patients indicating release. In vitro platelet aggregation studies showed that platelets secrete Pfn-1 upon complete activation. CONCLUSION Coronary thrombi show rapid dynamic changes both in structure and cell composition as a function of elapsed onset-of-pain-to-PCI time. Aged ischaemic thrombi were more likely to have reduced Pfn-1 content releasing Pfn-1 to the circulation. Onset-of-pain-to-PCI elapsed time in STEMI patients and hence age of occlusive thrombus can be profiled by Pfn-1 levels found in the peripheral circulation.

Differential proteomic distribution of TTR (pre-albumin) forms in serum and HDL of patients with high cardiovascular risk

UNLABELLED Inflammation is a common condition contributing to cardiovascular disease progression which leads to clinical manifestations such as acute myocardial infarction (AMI). By applying a proteomic expression profiling approach we have investigated changes in transthyretin (TTR) in AMI-patients and its distribution patterns in HDL samples of patients with high cardiovascular risk, such as those with familiar hypercholesterolemia (FH). METHODS AND RESULTS The characterization by bidimensional electrophoresis (2-DE), followed by mass-spectrometry (MALDI-TOF) of serum samples revealed changes in the intensity of the TTR spot with a pI of 5.6 and a Mw of 42kDa (tTTR) between AMI-patients in association to diabetic dyslipemia. Serum TTR levels, determined by commercial ELISA, were significantly lower (p<0.0001) in AMI-patients (n=39) and FH-patients (n=100) than in healthy controls (n=60). Western blot and 2-DE analysis showed a differential distribution profile of TTR forms between serum, where 3 TTR forms of 42 (tTTR), 28 (dTTR), and 14kDa (mTTR) were detected, and HDL samples, where only mTTR was present. CONCLUSIONS Our results demonstrate alterations in TTR proteomic profile in relation to the clustering of risk factors which seems to highlight the implication of TTR in cardiovascular risk. The significant differences in TTR between serum (tTTR) and HDL (mTTR) underscore the importance of TTR-forms in the circulation and deserve further investigation to understand their function.

Reperfusion-triggered stress protein response in the myocardium is blocked by post-conditioning. Systems biology pathway analysis highlights the key role of the canonical aryl-hydrocarbon receptor pathway

AIMS Ischaemic post-conditioning (IPost-Co) exerts cardioprotection by diminishing ischaemia/reperfusion injury. Yet, the mechanisms involved in such protection remain largely unknown. We have investigated the effects of IPost-Co in cardiac cells and in heart performance using molecular, proteomic and functional approaches. METHODS AND RESULTS Pigs underwent 1.5 h mid-left anterior descending balloon occlusion and then were sacrificed without reperfusion (ischaemia; n= 7), subjected to 2.5 h of cardiac reperfusion and sacrificed (n= 5); or subjected to IPost-Co before reperfusion and sacrificed 0.5 h (n= 4) and 2.5 h (n= 5) afterwards. A sham-operated group was included (n= 4). Ischaemic and non-ischaemic myocardium was obtained for molecular/histological analysis. Proteomic analysis was performed by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-time-of-flight identification. Potential protein networks involved were identified by bioinformatics and Ingenuity Pathway Analysis (IPA). Cardiac function was assessed by echocardiography. IPost-Co diminished (up to 2.5 h) reperfusion-induced apoptosis of both the intrinsic and extrinsic pathways whereas it did not affect reperfusion-induced Akt/mammalian target of rapamycin (mTOR)/P70S6K activation. Proteomic studies showed that IPost-Co reverted 43% of cardiac cytoplasmic protein changes observed during ischaemia and ischaemia + reperfusion. Systems biology assessment revealed significant changes in the aryl-hydrocarbon receptor (AhR) pathway (cell damage related). Bioinformatic data were confirmed since the expression of HSP90, AhR, ANRT, and β-tubulin (involved in AhR-signalling transduction) were accordingly modified after IPost-Co. IPost-Co rescued 52% of the left ventricle-at-risk compared with reperfusion alone and resulted in a ≈30% relative improvement in left ventricular ejection fraction (P <0.05). CONCLUSION IPost-Co improves cardiac function post-myocardial infarction and reduces reperfusion-induced cell damage by down-regulation of the AhR-signalling transduction pathway ultimately leading to infarct size reduction.

Retinol-binding protein 4 levels and susceptibility to ischaemic events in men.

Many efforts in cardiovascular medicine have been focused in the identification of patients at risk of developing an acute ischaemic event. Biomarker discovery studies have become an essential research area, being proteomic technologies an excellent tool for biomarker identification. By applying proteomic approaches, we have detected changes in retinol‐binding protein 4 (RBP4) in acute new‐onset myocardial infarction patients (AMI) and in high‐risk patients with heterozygous familial hypercholesterolaemia (FH).

Intake of cooked tomato sauce preserves coronary endothelial function and improves apolipoprotein A-I and apolipoprotein J protein profile in high-density lipoproteins

Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remains unknown whether tomato intake exerts protective effects on the vasculature. The aim of this study was to determine whether medium-term supplementation with cooked tomato sauce (CTS) Mediterranean style (sofrito) attenuates diet-induced coronary endothelial dysfunction in an animal model with clinical impact and explore the mechanisms behind the effects. Pigs (N = 18) were fed a 10-day hypercholesterolemic diet. Half of the animals were given a supplement of 100 g/d of CTS (21.5 mg lycopene per day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide synthase [eNOS] inhibitor) were measured using flow Doppler. In the coronary arteries, we investigated eNOS gene expression and activation, monocyte chemoattractant protein 1 (MCP-1) expression, and oxidative DNA damage. In the circulation, we investigated lipoprotein resistance to oxidation and the differential proteomic protein profile. In dyslipidemic animals, CTS intake prevented diet-induced impairment of receptor-operated and nonreceptor-operated endothelial-dependent coronary vasodilation. These beneficial effects were associated with enhanced eNOS transcription and activation and diminished DNA damage in the coronary arteries. CTS-fed animals showed lower lipid peroxidation, higher high-density lipoprotein (HDL) antioxidant potential and plasma lycopene levels of 0.16 mg/L. Interestingly, improved HDL functionality was associated with protein profile changes in apolipoprotein A-I and apolipoprotein J. Lipids levels and MCP-1 expression were not affected by CTS. We report that CTS intake protects against low-density lipoprotein-induced coronary endothelial dysfunction by reducing oxidative damage, enhancing eNOS expression and activity, and improving HDL functionality.

Coordinated proteomic signature changes in immune response and complement proteins in acute myocardial infarction: The implication of serum amyloid P-component☆

BACKGROUND Patient outcomes after acute myocardial infarction (AMI) are influenced by the combined activation of several distinct interrelated signaling pathways. Specifically acute-phase response proteins can neutralize inflammatory agents, minimize the extent of tissue damage, and participate in tissue repair and regeneration. We have investigated the evolution of immune response and complement system-related proteins in the early and late phase post-AMI and their potential association with tissue damage after the ischemic event. METHODS Serum was analyzed by 2D-electrophoresis and mass-spectrometry (MALDI-TOF/TOF) in controls and de novo AMI-patients within the first 6h after onset (admission time), and 3 days after. RESULTS Among differential proteins two functional groups showed coordinated changes: immune response-inflammation (alpha-1B-glycoprotein, fetuin-A, serum amyloid P-component (SAP), and complex-forming glycoprotein HC) and complement system (C1r, C3 and factor-B). Immune-related proteins showed a decrease 3 days after admission except for SAP that depicted a progressive increase from the early phase. Specifically, fetuin-A decrease was associated with the extent of myocardial necrosis. C1r and C3 were increased in the early phase with a subsequent decrease 3 days after, being C1r levels at admission correlated with necrosis and troponin-T and GPT levels. Contrarily, factor-B was decreased in the early and late phase post-AMI. CONCLUSIONS Our results demonstrate that in the late phase post-AMI there is a coordinated decrease in immune response-inflammation proteins, except for SAP which showed an increase related to the specific activation of the classical complement pathway. Changes in immunity and complement-related proteins affect the severity of organ damage influencing prognosis after AMI.

High-molecular-weight kininogen and the intrinsic coagulation pathway in patients with de novo acute myocardial infarction

After an acute ischaemic event serum proteins may change reflecting the ischaemic damage. Proteomic studies could provide new insights into potential biomarkers in the evolution of ischaemic syndromes. In this study we have investigated the coordinated changes in coagulation-related proteins in the evolution after an acute myocardial infarction (AMI). Serum proteome (2D-electrophoresis and MALDI-TOF/TOF) of AMI-patients within the first 6 hours after event onset (admission-time) and 3 days after were compared to controls. Systems biology and bioinformatic analysis were performed to identify the differentially expressed canonical pathways. In silico analysis of differential proteins revealed changes in the intrinsic coagulation pathway in the early phase post-AMI. The two identified high-molecular weight kininogen (HMWK) clusters were inversely correlated in AMI patients at admission, being the intensity of the low-molecular-weight form inversely related to myocardial necrosis (p<0.05). Factor XI (FXI) levels were decreased in AMI patients at admission and normalised 3 days after (p<0.05). There was an early increase in fibrinogen gamma and D-dimer at admission, followed by a decrease in fibrinogen turnover 3 days after (p<0.05). The influence of elapsed time of ischaemia on fibrinogen distribution changes was validated in coronary thrombi retrieved by thromboaspiration. In conclusion, our results demonstrate an active exchange between HMWK forms and a decrease in FXI indicative of intrinsic pathway activation, together with an increase in fibrinogen gamma turnover and D-dimer formation in the early phase post-AMI. Moreover, coronary thrombi showed a dynamic evolution in fibrinogen composition depending on the duration of ischaemia influencing serum fibrinogen-related products content.

Position paper of the European Society of Cardiology–working group of coronary pathophysiology and microcirculation: obesity and heart disease

Cardiovascular Research Center (CSIC-ICCC), CIBERCV, and Biomedical Research Institute Sant Pau (IIB-Sant Pau), c/Sant Antoni MClaret 167, 08025 Barcelona, Spain; Cardiovascular Research Chair UAB, Barcelona, Spain; Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via Giuseppe Massarenti 9, 40138 Bologna, Italy; Cardiology Department, University of Medicine and Pharmacy “Carol Davila” of Bucharest, Emergency Clinical Hospital of Bucharest, 8, Calea Floreasca, Sector 1, 014461 Bucureşti, Romania; Division of Experimental Cardiology, Department of Cardiology, Thoraxcenter, Cardiovascular Research Institute COEUR, Erasmus University Medical Center, PO Box 1738, 3000 DR, Rotterdam, The Netherlands; Department of Internal Medicine, Hospital Clinic, IDIBAPS, University of Barcelona, Villarroel, 170, 08036 Barcelona, Spain; CIBER Obesity and Nutrition, Instituto de Salud Carlos III, Spain; Department for Cardiovascular Diseases, University Hospital Center Zagreb, University of Zagreb, Kispaticeva 12, HR-10000 Zagreb, Croatia; First Department of Cardiology, Hippokration Hospital, University of Athens Medical School, Vasilissis Sofias 114, TK 115 28 Athens, Greece; Clinical Center of Serbia, University of Belgrade, Pasterova 2, 11000 Belgrade, Serbia; Institut für Physiologie, Universit€at zu Lübeck and Deutsches Zentrumfür Herz-Kreislauf-Forschung (DZHK) e.V., partner site: Hamburg/Kiel/Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; Institute of Natural Sciences, University of Physical Education, Alkotas street, 44, 1123 Budapest, Hungary; and Department of Physiology, New York Medical College, Valhalla, NY 10595, USA

ApoL1 levels in high density lipoprotein and cardiovascular event presentation in patients with familial hypercholesterolemia

HDL composition rather than HDL-cholesterol (HDL-C) levels seems to be a key determinant of HDL-induced atheroprotection. Heterozygous familial hypercholesterolemia (FH) patients, with lifelong exposure to high LDL levels, show a high prevalence of premature coronary artery disease. We hypothesized that HDL of FH patients might have a modified protein composition and investigated the proteomic signature of HDL obtained from FH patients and their unaffected relatives. HDLs were characterized by 2D electrophoresis/MS in 10 families from the SAFEHEART cohort (3 individuals/family: 2 with genetic FH diagnosis and 1 non-FH relative) clinically characterized and treated as per guidelines. FH patients had lower apoA-I levels and a differential HDL distribution profile of apoL1 and apoA-IV. ELISA validation revealed decreased apoL1 serum levels in FH patients. ApoL1 levels were able to predict presentation of an ischemic cardiac event, and apoL1/HDL-C ratio was associated with the survival rate after the event. FH patients who died because of a fatal cardiac event had lower apoL1 and LCAT content in HDL3 an average of 3.5 years before the event than those who survived. Changes in HDL protein composition could affect patients’ prognosis. The proteomic profile of apoL1 is modified in HDLs of high cardiovascular risk patients, and apoL1 plasma levels are significantly lower in serum and in HDL3 of patients that will suffer an adverse cardiac event within 3 years.