Patient outcomes after acute myocardial infarction (AMI) are influenced by the combined activatio... more 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. 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. 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. 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.
Many efforts in cardiovascular medicine have been focused in the identification of patients at ri... more 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). Differential serum proteome was analysed by two-dimensional electrophoresis and MALDI-TOF/TOF. Validation studies were performed by ELISA, and functional effects of RBP4 were tested in cell culture experiments. Retinol-binding protein 4 proteomic characterization depicted two spots (pI = 5·4;Mw = 23·01/22·78 kDa) with decreased intensity in AMI patients. Total serum RBP4 levels were decreased in AMI patients (N = 68) compared with controls (N = 132; P < 0·0001). RBP4 was also decreased in FH patients who had an ischaemic event 2 years (±0·3) after their inclusion compared with FH patients without any cardiovascular episode at follow-up (P < 0·001; N = 187). In both cases, changes were limited to men. RBP4 induced a significant increase in eNOS expression in human endothelial vascular cells and in prostaglandin I2 release in coronary vascular smooth muscle cells. We show decreased serum RBP4 levels in men in the acute phase of AMI, being this decrease already detected in men with FH previous to the presentation of an ischaemic event. The decrease in RBP4 levels could confer an increased susceptibility to the precipitation of an ischaemic event that could be mediated by the decrease in its vasculoprotective properties through NO and PGI2 .
After an acute ischaemic event serum proteins may change reflecting the ischaemic damage. Proteom... more 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.
Translational research : the journal of laboratory and clinical medicine, Jan 25, 2014
Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remai... more 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 damag...
Acute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide... more 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.
IntroductionAcute myocardial infarction (AMI) is one of the major causes of mortality and morbidi... more IntroductionAcute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide. Despite the efforts being made, there is a lack of early markers for the prevention, diagnosis and treatment of ischemic syndromes. Proteomic expression profiling technologies are a highly important tool for research into new serum biomarkers for the diagnosis and prognosis of acute coronary syndromes.
Drug-induced vascular injury (DIVI) is commonly associated with phosphodiesterase (PDE) inhibitor... more Drug-induced vascular injury (DIVI) is commonly associated with phosphodiesterase (PDE) inhibitors. Despite histological characterization, qualified biomarkers for DIVI detection are lacking. We investigated whether a single administration of roflumilast (PDE-IV inhibitor) induces vascular damage and identified novel surrogate biomarkers of acute vascular injury. Pigs received postoperative 250, 375, or 500 μg of roflumilast or placebo/control. After 1.5 hr, coronary reactivity was determined by catheter-based administration of acetylcholine and sodium nitroprusside (SNP) in the coronary sinus. Immunohistochemical analysis of vessel integrity (von Willebrand factor [vWF]) and fibrin(ogen) deposition was performed in the coronary artery and aorta. Peripheral blood was collected for differential proteomics and microparticles analysis. Circulating interleukin (IL)-6 was analyzed. Roflumilast-treated animals displayed higher vasodilation to acetylcholine and SNP versus controls (p < ...
Patient outcomes after acute myocardial infarction (AMI) are influenced by the combined activatio... more 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. 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. 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. 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.
Many efforts in cardiovascular medicine have been focused in the identification of patients at ri... more 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). Differential serum proteome was analysed by two-dimensional electrophoresis and MALDI-TOF/TOF. Validation studies were performed by ELISA, and functional effects of RBP4 were tested in cell culture experiments. Retinol-binding protein 4 proteomic characterization depicted two spots (pI = 5·4;Mw = 23·01/22·78 kDa) with decreased intensity in AMI patients. Total serum RBP4 levels were decreased in AMI patients (N = 68) compared with controls (N = 132; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0·0001). RBP4 was also decreased in FH patients who had an ischaemic event 2 years (±0·3) after their inclusion compared with FH patients without any cardiovascular episode at follow-up (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0·001; N = 187). In both cases, changes were limited to men. RBP4 induced a significant increase in eNOS expression in human endothelial vascular cells and in prostaglandin I2 release in coronary vascular smooth muscle cells. We show decreased serum RBP4 levels in men in the acute phase of AMI, being this decrease already detected in men with FH previous to the presentation of an ischaemic event. The decrease in RBP4 levels could confer an increased susceptibility to the precipitation of an ischaemic event that could be mediated by the decrease in its vasculoprotective properties through NO and PGI2 .
After an acute ischaemic event serum proteins may change reflecting the ischaemic damage. Proteom... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). Factor XI (FXI) levels were decreased in AMI patients at admission and normalised 3 days after (p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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.
Translational research : the journal of laboratory and clinical medicine, Jan 25, 2014
Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remai... more 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 damag...
Acute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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.
IntroductionAcute myocardial infarction (AMI) is one of the major causes of mortality and morbidi... more IntroductionAcute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide. Despite the efforts being made, there is a lack of early markers for the prevention, diagnosis and treatment of ischemic syndromes. Proteomic expression profiling technologies are a highly important tool for research into new serum biomarkers for the diagnosis and prognosis of acute coronary syndromes.
Drug-induced vascular injury (DIVI) is commonly associated with phosphodiesterase (PDE) inhibitor... more Drug-induced vascular injury (DIVI) is commonly associated with phosphodiesterase (PDE) inhibitors. Despite histological characterization, qualified biomarkers for DIVI detection are lacking. We investigated whether a single administration of roflumilast (PDE-IV inhibitor) induces vascular damage and identified novel surrogate biomarkers of acute vascular injury. Pigs received postoperative 250, 375, or 500 μg of roflumilast or placebo/control. After 1.5 hr, coronary reactivity was determined by catheter-based administration of acetylcholine and sodium nitroprusside (SNP) in the coronary sinus. Immunohistochemical analysis of vessel integrity (von Willebrand factor [vWF]) and fibrin(ogen) deposition was performed in the coronary artery and aorta. Peripheral blood was collected for differential proteomics and microparticles analysis. Circulating interleukin (IL)-6 was analyzed. Roflumilast-treated animals displayed higher vasodilation to acetylcholine and SNP versus controls (p < ...
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