Journal of Molecular and Cellular Cardiology, 2011
A transgenic mouse model for conditional induction of long-term hibernation via myocardium-specif... more A transgenic mouse model for conditional induction of long-term hibernation via myocardium-specific expression of a VEGF-sequestering soluble receptor allowed the dissection of the hibernation process into an initiation and a maintenance phase. The hypoxic initiation phase was characterized by peak levels of K(ATP) channel and glucose transporter 1 (GLUT1) expression. Glibenclamide, an inhibitor of K(ATP) channels, blocked GLUT1 induction. In the maintenance phase, tissue hypoxia and GLUT1 expression were reduced. Thus, we employed a combined "-omics" approach to resolve this cardioprotective adaptation process. Unguided bioinformatics analysis on the transcriptomic, proteomic and metabolomic datasets confirmed that anaerobic glycolysis was affected and that the observed enzymatic changes in cardiac metabolism were directly linked to hypoxia-inducible factor (HIF)-1 activation. Although metabolite concentrations were kept relatively constant, the combination of the proteomic and transcriptomic dataset improved the statistical confidence of the pathway analysis by 2 orders of magnitude. Importantly, proteomics revealed a reduced phosphorylation state of myosin light chain 2 and cardiac troponin I within the contractile apparatus of hibernating hearts in the absence of changes in protein abundance. Our study demonstrates how combining different "-omics" datasets aids in the identification of key biological pathways: chronic hypoxia resulted in a pronounced adaptive response at the transcript and the protein level to keep metabolite levels steady. This preservation of metabolic homeostasis is likely to contribute to the long-term survival of the hibernating myocardium.
which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the ... more which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the differentially expressed proteins were key enzymes in glucose metabolism, resulting in faster glucose consumption and a compensatory reduction in baseline interleukin-6 secretion. The latter was associated with a marked upregulation of insulin-like growth factor binding proteins (IGFBPs) 3 and 6. Notably, reconstitution of interleukin-6 to levels
Journal of Molecular and Cellular Cardiology, 2013
Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms... more Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, ... more Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, including elevated blood pressure, smoking, diabetes, and hypercholesterolemia directly or indirectly damage the vessel wall, eventually inducing arterial stiffness (arteriosclerosis) and lipid accumulation (atherosclerosis). However, the molecular mechanisms of atheroma formation are not yet fully clarified. While many investigators have used proteomic techniques to study cardiac diseases, vascular proteomics is still in its infancy. The present review highlights studies, in which proteomics has been successfully applied to study protein alterations in the vasculature. Furthermore, we will summarize our recent progress in combining proteomic and metabolomic techniques to reveal protein and metabolite alterations in the cardiovascular system: two-dimensional (2-D) gel electrophoresis proved to be highly complementary to nuclear magnetic resonance (NMR) spectroscopy, in that post-translat...
Microparticles (MPs) with procoagulant activity are present in human atherosclerosis, but no deta... more Microparticles (MPs) with procoagulant activity are present in human atherosclerosis, but no detailed information is available on their composition. To obtain insights into the role of MPs in atherogenesis, MP proteins were identified by tandem mass spectrometry, metabolite profiles were determined by high-resolution nuclear magnetic resonance spectroscopy, and antibody reactivity was assessed against combinatorial antigen libraries. Plaque MPs expressed surface antigens consistent with their leukocyte origin, including major histocompatibility complex classes I and II, and induced a dose-dependent stimulatory effect on T-cell proliferation. Notably, taurine, the most abundant free organic acid in human neutrophils, which scavenges myeloperoxidase-catalyzed free radicals, was highly enriched in plaque MPs. Moreover, fluorescent labeling of proteins on the MP surface suggested immunoglobulins to be trapped inside, which was confirmed by flow cytometry analysis on permeabilized and nonpermeabilized plaque MPs. Colabeling for CD14 and IgG established that more than 90% of the IgG containing MPs were CD14(+), indicating a macrophage origin. Screening against an antigen library revealed that the immunologic profiles of antibodies in MPs were similar to those found in plaques but differed profoundly from antibodies in plasma and unexpectedly, showed strong reactions with oligosaccharide antigens, in particular blood group antigen A. This study provides the first evidence that immunoglobulins are present within MPs derived from plaque macrophages, that the portfolio of plaque antibodies is different from circulating antibodies in plasma, and that anticarbohydrate antibodies are retained in human atherosclerotic lesions.
Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their cor... more Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.
In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discov... more In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung-common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the...
Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the firs... more Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. Objective: To investigate the regulation, and role, of MV biogenesis in VSMC calcification. Methods and Results: Alexa488-labelled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system and exocytosed from multivesicular bodies (MVBs) via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63 and CD81 and their release was regulated by sphingomyelin phosphodiesterase 3 (SMPD3). Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elev...
Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerot... more Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR-33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short-term anti-miR-33 treatment has been previously studied, the long-term effect of miR-33 antagonism in vivo remains to be elucidated. Here, we show that long-term therapeutic silencing of miR-33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high-fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR-33 increases the expression of genes involved in fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR-33 antisense oligonucleotides. We also report that anti-miR-33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR-33 in non-human primates before we can translate this therapy to humans.
Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, ... more Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, including elevated blood pressure, smoking, diabetes, and hypercholesterolemia directly or indirectly damage the vessel wall, eventually inducing arterial stiffness (arteriosclerosis) and lipid accumulation (atherosclerosis). However, the molecular mechanisms of atheroma formation are not yet fully clarified. While many investigators have used proteomic techniques to study cardiac diseases, vascular proteomics is still in its infancy. The present review highlights studies, in which proteomics has been successfully applied to study protein alterations in the vasculature. Furthermore, we will summarize our recent progress in combining proteomic and metabolomic techniques to reveal protein and metabolite alterations in the cardiovascular system: two-dimensional (2-D) gel electrophoresis proved to be highly complementary to nuclear magnetic resonance (NMR) spectroscopy, in that post-translat...
The mechanisms that determine whether the heart adapts to overload stress, or fails, are poorly u... more The mechanisms that determine whether the heart adapts to overload stress, or fails, are poorly understood. NADPH oxidase (NOX) proteins produce reactive oxygen species (ROS) involved in redox signalling, and our recent studies have found that an increase in Nox4 during pressure overload protects the heart against failure. We aimed to identify novel Nox4-driven cardioprotective mechanisms that promote adaptive cardiac remodelling. We first undertook a proteomic comparison of heart tissue from cardiac-targeted Nox4-overexpressing mice and controls. The Nox4 cardiac metabolome was then investigated by (1)H nuclear magnetic resonance (NMR) spectroscopy. Effects on cardiac metabolism were assessed by ex-vivo working heart perfusions and isolated mitochondrial respiration studies. Ex-vivo cardiac energetics were assessed by (31)P NMR. Alterations to cardiac fatty acid oxidation were explored in primary cardiomyocytes (extracellular flux analysis). Cardiac-targeted Nox4 overexpression profoundly remodelled the cardiac proteome in an isoform-specific manner, both in the unstressed and stressed heart. Glycolysis and fatty acid oxidation were identified as the most enriched pathways that were altered by Nox4. Metabolomic analysis showed a 2·2 times increase in acetylcarnitine concentrations (p=0·002). Ex-vivo heart perfusions demonstrated a profound increase in palmitate oxidation relative to wild-type hearts (3·6 times increase, p=0·01), with opposite findings observed in primary cardiomyocytes with a knockdown of Nox4. A preference for fatty acid oxidation in Nox4 hearts correlated with a better energetic state (phosphocreatine:ATP ratio) when subjected to increasing doses of isoprenaline stress under baseline and pressure-overload. In this study we identified a novel role for Nox4 in the regulation of cardiac fatty acid oxidation. Cardiomyocyte-targeted Nox4 hearts preferentially oxidised fatty acids for energy provision, improving myocardial energetics under stress. Enhancing fatty acid oxidation might have an adaptive role in the setting of pressure-overload hypertrophy. These data provide novel insights into ROS-dependent metabolic programming. UK Medical Research Council, British Heart Foundation.
Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 ... more Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E-deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E-deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.
The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel w... more The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.
Journal of Molecular and Cellular Cardiology, 2011
A transgenic mouse model for conditional induction of long-term hibernation via myocardium-specif... more A transgenic mouse model for conditional induction of long-term hibernation via myocardium-specific expression of a VEGF-sequestering soluble receptor allowed the dissection of the hibernation process into an initiation and a maintenance phase. The hypoxic initiation phase was characterized by peak levels of K(ATP) channel and glucose transporter 1 (GLUT1) expression. Glibenclamide, an inhibitor of K(ATP) channels, blocked GLUT1 induction. In the maintenance phase, tissue hypoxia and GLUT1 expression were reduced. Thus, we employed a combined "-omics" approach to resolve this cardioprotective adaptation process. Unguided bioinformatics analysis on the transcriptomic, proteomic and metabolomic datasets confirmed that anaerobic glycolysis was affected and that the observed enzymatic changes in cardiac metabolism were directly linked to hypoxia-inducible factor (HIF)-1 activation. Although metabolite concentrations were kept relatively constant, the combination of the proteomic and transcriptomic dataset improved the statistical confidence of the pathway analysis by 2 orders of magnitude. Importantly, proteomics revealed a reduced phosphorylation state of myosin light chain 2 and cardiac troponin I within the contractile apparatus of hibernating hearts in the absence of changes in protein abundance. Our study demonstrates how combining different "-omics" datasets aids in the identification of key biological pathways: chronic hypoxia resulted in a pronounced adaptive response at the transcript and the protein level to keep metabolite levels steady. This preservation of metabolic homeostasis is likely to contribute to the long-term survival of the hibernating myocardium.
which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the ... more which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the differentially expressed proteins were key enzymes in glucose metabolism, resulting in faster glucose consumption and a compensatory reduction in baseline interleukin-6 secretion. The latter was associated with a marked upregulation of insulin-like growth factor binding proteins (IGFBPs) 3 and 6. Notably, reconstitution of interleukin-6 to levels
Journal of Molecular and Cellular Cardiology, 2013
Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms... more Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, ... more Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, including elevated blood pressure, smoking, diabetes, and hypercholesterolemia directly or indirectly damage the vessel wall, eventually inducing arterial stiffness (arteriosclerosis) and lipid accumulation (atherosclerosis). However, the molecular mechanisms of atheroma formation are not yet fully clarified. While many investigators have used proteomic techniques to study cardiac diseases, vascular proteomics is still in its infancy. The present review highlights studies, in which proteomics has been successfully applied to study protein alterations in the vasculature. Furthermore, we will summarize our recent progress in combining proteomic and metabolomic techniques to reveal protein and metabolite alterations in the cardiovascular system: two-dimensional (2-D) gel electrophoresis proved to be highly complementary to nuclear magnetic resonance (NMR) spectroscopy, in that post-translat...
Microparticles (MPs) with procoagulant activity are present in human atherosclerosis, but no deta... more Microparticles (MPs) with procoagulant activity are present in human atherosclerosis, but no detailed information is available on their composition. To obtain insights into the role of MPs in atherogenesis, MP proteins were identified by tandem mass spectrometry, metabolite profiles were determined by high-resolution nuclear magnetic resonance spectroscopy, and antibody reactivity was assessed against combinatorial antigen libraries. Plaque MPs expressed surface antigens consistent with their leukocyte origin, including major histocompatibility complex classes I and II, and induced a dose-dependent stimulatory effect on T-cell proliferation. Notably, taurine, the most abundant free organic acid in human neutrophils, which scavenges myeloperoxidase-catalyzed free radicals, was highly enriched in plaque MPs. Moreover, fluorescent labeling of proteins on the MP surface suggested immunoglobulins to be trapped inside, which was confirmed by flow cytometry analysis on permeabilized and nonpermeabilized plaque MPs. Colabeling for CD14 and IgG established that more than 90% of the IgG containing MPs were CD14(+), indicating a macrophage origin. Screening against an antigen library revealed that the immunologic profiles of antibodies in MPs were similar to those found in plaques but differed profoundly from antibodies in plasma and unexpectedly, showed strong reactions with oligosaccharide antigens, in particular blood group antigen A. This study provides the first evidence that immunoglobulins are present within MPs derived from plaque macrophages, that the portfolio of plaque antibodies is different from circulating antibodies in plasma, and that anticarbohydrate antibodies are retained in human atherosclerotic lesions.
Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their cor... more Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.
In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discov... more In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung-common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the...
Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the firs... more Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. Objective: To investigate the regulation, and role, of MV biogenesis in VSMC calcification. Methods and Results: Alexa488-labelled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system and exocytosed from multivesicular bodies (MVBs) via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63 and CD81 and their release was regulated by sphingomyelin phosphodiesterase 3 (SMPD3). Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elev...
Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerot... more Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR-33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short-term anti-miR-33 treatment has been previously studied, the long-term effect of miR-33 antagonism in vivo remains to be elucidated. Here, we show that long-term therapeutic silencing of miR-33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high-fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR-33 increases the expression of genes involved in fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR-33 antisense oligonucleotides. We also report that anti-miR-33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR-33 in non-human primates before we can translate this therapy to humans.
Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, ... more Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, including elevated blood pressure, smoking, diabetes, and hypercholesterolemia directly or indirectly damage the vessel wall, eventually inducing arterial stiffness (arteriosclerosis) and lipid accumulation (atherosclerosis). However, the molecular mechanisms of atheroma formation are not yet fully clarified. While many investigators have used proteomic techniques to study cardiac diseases, vascular proteomics is still in its infancy. The present review highlights studies, in which proteomics has been successfully applied to study protein alterations in the vasculature. Furthermore, we will summarize our recent progress in combining proteomic and metabolomic techniques to reveal protein and metabolite alterations in the cardiovascular system: two-dimensional (2-D) gel electrophoresis proved to be highly complementary to nuclear magnetic resonance (NMR) spectroscopy, in that post-translat...
The mechanisms that determine whether the heart adapts to overload stress, or fails, are poorly u... more The mechanisms that determine whether the heart adapts to overload stress, or fails, are poorly understood. NADPH oxidase (NOX) proteins produce reactive oxygen species (ROS) involved in redox signalling, and our recent studies have found that an increase in Nox4 during pressure overload protects the heart against failure. We aimed to identify novel Nox4-driven cardioprotective mechanisms that promote adaptive cardiac remodelling. We first undertook a proteomic comparison of heart tissue from cardiac-targeted Nox4-overexpressing mice and controls. The Nox4 cardiac metabolome was then investigated by (1)H nuclear magnetic resonance (NMR) spectroscopy. Effects on cardiac metabolism were assessed by ex-vivo working heart perfusions and isolated mitochondrial respiration studies. Ex-vivo cardiac energetics were assessed by (31)P NMR. Alterations to cardiac fatty acid oxidation were explored in primary cardiomyocytes (extracellular flux analysis). Cardiac-targeted Nox4 overexpression profoundly remodelled the cardiac proteome in an isoform-specific manner, both in the unstressed and stressed heart. Glycolysis and fatty acid oxidation were identified as the most enriched pathways that were altered by Nox4. Metabolomic analysis showed a 2·2 times increase in acetylcarnitine concentrations (p=0·002). Ex-vivo heart perfusions demonstrated a profound increase in palmitate oxidation relative to wild-type hearts (3·6 times increase, p=0·01), with opposite findings observed in primary cardiomyocytes with a knockdown of Nox4. A preference for fatty acid oxidation in Nox4 hearts correlated with a better energetic state (phosphocreatine:ATP ratio) when subjected to increasing doses of isoprenaline stress under baseline and pressure-overload. In this study we identified a novel role for Nox4 in the regulation of cardiac fatty acid oxidation. Cardiomyocyte-targeted Nox4 hearts preferentially oxidised fatty acids for energy provision, improving myocardial energetics under stress. Enhancing fatty acid oxidation might have an adaptive role in the setting of pressure-overload hypertrophy. These data provide novel insights into ROS-dependent metabolic programming. UK Medical Research Council, British Heart Foundation.
Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 ... more Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E-deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E-deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.
The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel w... more The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.
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Papers by Xiaoke Yin