Biomedical subjects

Ursula Mayr

Publications and source records attributed to Ursula Mayr.

10 recordsLinked to original sources

PubMed2005

Accelerated arteriosclerosis of vein grafts in inducible NO synthase(-/-) mice is related to decreased endothelial progenitor cell repair.

Inducible NO synthase (iNOS) is expressed by macrophages and smooth muscle cells in atherosclerotic lesions. Previously, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of iNOS in the development of vein graft arteriosclerosis in iNOS(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in iNOS(-/-) mice was increased 2-fold compared with that of wild-type controls. Neointimal lesions contained mainly MAC-1+ macrophages and alpha-actin+ smooth muscle cells (SMCs) in both vein grafts of iNOS(-/-) and iNOS(+/+) mice. Immunofluorescence analysis revealed that increased iNOS expression in neointimal macrophages and SMCs of wild-type, but not iNOS(-/-), mice coincided with increased vascular endothelial growth factor (VEGF) expression in vein grafts. When vein grafts were performed in iNOS(-/-)/TIE2-LacZ transgenic mice expressing LacZ gene only in endothelial cells, the number of beta-galactosidase+ cells in iNOS(-/-) vein grafts were significantly decreased. Furthermore, treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester resulted in delayed endothelial progenitor cell attachment, whereas L-arginine intake through drinking water enhanced endothelial repair. Interestingly, local application of VEGF to iNOS(-/-) vein grafts restored endothelial progenitor homing and reduced neointimal lesions, whereas the VEGF receptor inhibitor SU1498 increased the lesion formation. Additionally, iNOS-deficient SMCs showed a low level of VEGF production in response to interleukin 1beta stimulation. Thus, iNOS deficiency accelerates neointima formation by abrogating VEGF production and endothelial progenitor cell attachment and differentiation.

Animals↗

PubMed2005

Proteomic and metabolomic analyses of atherosclerotic vessels from apolipoprotein E-deficient mice reveal alterations in inflammation, oxidative stress, and energy metabolism.

OBJECTIVE: Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E(-/-) mice on normal chow diet. METHODS AND RESULTS: Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E(-/-) mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E(-/-) mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E(-/-) mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool. CONCLUSIONS: Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism.

Animals↗

PubMed2005

Proteomic dataset of Sca-1+ progenitor cells.

Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com.

Animals↗

PubMed2005

Proteomic dataset of mouse aortic smooth muscle cells.

In an accompanying study (in this issue, DOI 10.1002/pmic.200402044), we have characterised the proteome of Sca-1(+) progenitor cells, which may function as precursors of vascular smooth muscle cells (SMCs). In the present study, we have analysed and mapped protein expression in aortic SMCs of mice, using 2-DE, MALDI-TOF MS and MS/MS. The 2-D system comprised a non-linear immobilised pH 3-10 gradient in the first dimension (separating proteins with pI values of pH 3-10), and 12%T SDS-PAGE in the second dimension (separating proteins in the range 15,000-150,000 Da). Of the 2400 spots visualised, a subset of 267 protein spots was analysed, with 235 protein spots being identified corresponding to 154 unique proteins. The data presented here are the first map of aortic SMCs and the most extensive analysis of SMC proteins published so far. This valuable tool should provide a basis for comparative studies of protein expression in vascular smooth muscle of transgenic mice and is available on our website hhtp://www.vascular-proteomics.com.

Animals↗

PubMed2004

Proteomic and metabolomic analysis of vascular smooth muscle cells: role of PKCdelta.

Recent developments of proteomic and metabolomic techniques provide powerful tools for studying molecular mechanisms of cell function. Previously, we demonstrated that neointima formation was markedly increased in vein grafts of PKCdelta-deficient mice compared with wild-type controls. To clarify the underlying mechanism, we performed a proteomic and metabolomic analysis of cultured vascular smooth muscle cells (SMCs) derived from PKCdelta+/+ and PKCdelta-/- mice. Using 2-dimensional electrophoresis and mass spectrometry, we identified >30 protein species that were altered in PKCdelta-/- SMCs, including enzymes related to glucose and lipid metabolism, glutathione recycling, chaperones, and cytoskeletal proteins. Interestingly, nuclear magnetic resonance spectroscopy confirmed marked changes in glucose metabolism in PKCdelta-/- SMCs, which were associated with a significant increase in cellular glutathione levels resulting in resistance to cell death induced by oxidative stress. Furthermore, PKCdelta-/- SMCs overexpressed RhoGDIalpha, an endogenous inhibitor of Rho signaling pathways. Inhibition of Rho signaling was associated with a loss of stress fiber formation and decreased expression of SMC differentiation markers. Thus, we performed the first combined proteomic and metabolomic study in vascular SMCs and demonstrate that PKCdelta is crucial in regulating glucose and lipid metabolism, controlling the cellular redox state, and maintaining SMC differentiation.

Animals↗

PubMed2004

Thrombosis and neointima formation in vein grafts are inhibited by locally applied aspirin through endothelial protection.

Vein graft failure within the first month after bypass surgery is largely because of thrombosis. However, systemic study of thrombus formation in vein grafts is still lacking, and few effective techniques are available to prevent this event. Herein, we analyzed the kinetics of thrombosis and tested the effectiveness of locally applied aspirin on prevention of the disease in a mouse model. En face analysis of vein grafts revealed that 67+/-12% and 54+/-17% of the surface areas were covered by microthrombi at 1 and 3 days, respectively. Thrombus generation was also identified by labeling of platelets and fibrin, which occurred in 35 grafts examined at 1 and 3 days and 1, 2, 4, and 8 weeks. In a fifth of grafts, the thrombus occluded the vessel lumen by > or =1/4. Furthermore, a significant loss of endothelial cells was evidenced by beta-gal staining for vein grafts in transgenic mice expressing LacZ gene controlled by TIE2-endothelial specific gene promoter. Following thrombosis, neointimal lesions were significantly increased by 4-fold 2 weeks after the operation. When vein grafts were treated locally with aspirin in pluronic gel-127, the thrombus area was significantly reduced (P<0.005) at 1, 4, and 8 weeks. Interestingly, neointimal lesions were markedly reduced in the local, but not oral, aspirin-treated group at 4 and 8 weeks by 50% to 70% (P<0.005). The mechanism of reduced lesions by locally applied aspirin involved the protection of vein graft endothelium. Thus, we provide strong evidence that thrombus formation occurs before the development of neointimal lesions in vein grafts and that local aspirin treatment successfully reduces vein graft arteriosclerosis through endothelial protection, resulting in reduction of thrombosis.

Animals↗

PubMed2004

Vascular proteomics: linking proteomic and metabolomic changes.

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-translational modifications of the most abundant enzymes were displayed on 2-D gels while NMR spectroscopy revealed changes in the corresponding metabolites. Importantly, the simultaneous assessment of protein and metabolite changes translated purely descriptive proteomic and metabolomic profiles into a functional context and provided important insights into pathophysiological mechanisms that would not have been obtained by other techniques.

Animals↗

PubMed2004

Loss of PKC-delta alters cardiac metabolism.

PKC-delta is believed to play an essential role in cardiomyocyte growth. In the present study, we investigated the effect of PKC-delta on cardiac metabolism using PKC-delta knockout mice generated in our laboratories. Proteomic analysis of heart protein extracts revealed profound changes in enzymes related to energy metabolism: certain isoforms of glycolytic enzymes, e.g., lactate dehydrogenase and pyruvate kinase, were absent or decreased, whereas several enzymes involved in lipid metabolism, e.g., phosphorylated isoforms of acyl-CoA dehydrogenases, showed a marked increase in PKC-delta(-/-) hearts. Moreover, PKC-delta deficiency was associated with changes in antioxidants, namely, 1-Cys peroxiredoxin and selenium-binding protein 1, and posttranslational modifications of chaperones involved in cytoskeleton regulation, such as heat shock protein (HSP)20, HSP27, and the zeta-subunit of the cytosolic chaperone containing the T-complex polypeptide 1. High-resolution NMR analysis of cardiac metabolites confirmed a significant decrease in the ratio of glycolytic end products (alanine + lactate) to end products of lipid metabolism (acetate) in PKC-delta(-/-) hearts. Taken together, our data demonstrate that loss of PKC-delta causes a shift from glucose to lipid metabolism in murine hearts, and we provide a detailed description of the enzymatic changes on a proteomic level. The consequences of these metabolic alterations on sensitivity to myocardial ischemia are further explored in the accompanyingpaper (20).

Animals↗

PubMed2004

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice.

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.

Acetyltransferases↗

PubMed2002

Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice.

The transcription factor p53 is essentially involved in regulation of cell death and proliferation. Recently, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of p53 in the development of vein graft arteriosclerosis in p53(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in p53(-/-) mice was increased 2-fold compared with that of wild-type controls. Cell component analysis revealed that neointimal lesions in p53(-/-) mice consisted mainly of alpha-actin positive smooth muscle cells (SMCs), whereas the majority of cells in wild-type mice were MAC-1 (CD11b/18)-positive at 4 weeks. Importantly, SMC apoptosis as determined by TUNEL assay was significantly reduced in p53(-/-) vein grafts. TUNEL positive cells in wild-type vein grafts markedly increased from 0.5% to 6.4% of total cells 4 weeks postoperatively, but remained virtually unchanged in p53(-/-) grafts (0.8%). Immunofluorescence analysis revealed that increased p53 expression in neointimal SMCs of wild-type, but not p53(-/-), mice coincided with oxidative DNA damage in vein grafts. Interestingly, SMCs of p53(-/-) mice showed increased apoptosis in response to TNFalpha and decreased apoptosis in response to sodium nitroprusside. Additionally, p53-deficient SMCs showed a higher rate of proliferation and migration and expressed higher levels of matrix metalloproteinases. Thus, p53 deficiency accelerates neointima formation by facilitating SMC proliferation as well as abrogating cell apoptosis.

Actins↗