[Show abstract][Hide abstract] ABSTRACT: To compare the basic proteomic composition of abdominal aortic aneurysm (AAA) wall tissue in patients with nonruptured and ruptured aneurysms.
A proteomic approach with two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS) was used to identify differentially expressed proteins in AAA tissue from nine patients with nonruptured and eight patients with ruptured AAA. Computerized image analysis was used to detect protein spots. Differentially expressed protein spots were in-gel digested and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Western blot analysis was used to confirm differential expression.
Seven differentially expressed proteins were detected among 745 protein spots, selecting spots whose average relative volumes differed more than twofold between the nonruptured and the ruptured group. Four protein spots were up-regulated in the ruptured group, and three were down-regulated. Five of the spots were identified. Among the upregulated spots, No. 605 was identified as peroxiredoxin-2. The up-regulation was confirmed by Western blotting. No. 381 was identified as an actin fragment. Two spots, Nos. 719 and 499, could not be identified. Among the down-regulated protein spots, No. 130 contained two peptides; one reliably determined peptide, FEDGVLDPDYPR, is found in vitronectin. Another peptide, QIDNPDYK, was borderline significant and found in calreticulin. The down-regulation of vitronectin was confirmed by Western blotting. Spot Nos. 193 and 199 both contained peptides from albumin with actin also present in No. 199.
The identified proteins suggest that the aortic wall of ruptured aneurysms responds to a stressful condition and that proteolytic degradation of the cytoskeleton and connective tissue may be part of the response.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 12/2008; 49(2):455-63. DOI:10.1016/j.jvs.2008.08.097 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thrombocytes express thrombospondin-1 (TSP1), as well as the CREC proteins, calumenin and reticulocalbin. TSP1 and calumenin are released upon stimulation with thrombin. Calumenin has recently been shown to influence the synthesis of certain coagulation factors. Calumenin is present in atherosclerotic lesions but not in normal vasculature [Coppinger et al. (Blood 103:2096-2104, 2004)] and is able to modulate the protein expression pattern as well as the cell cycle of fibroblasts [Østergaard et al. (Proteomics 6:3509-3519, 2006)]. We here show that calumenin in the presence of Ca(2+) binds to TSP1 with a dissociation constant K (d) around 0.4 muM. This interaction is specific with respect to the secreted calumenin as the closest relative among the CREC family members, the non-secreted reticulocalbin, does not form a similar complex. This further indicates that calumenin may be broadly involved in haemostasis and in the pathophysiology of thrombosis.
[Show abstract][Hide abstract] ABSTRACT: The maximal diameter of abdominal aortic aneurysms (AAAs) is the dominating indication for repair. However half of the AAAs repaired would never have ruptured if left unrepaired, although small AAAs occasionally rupture. Earlier surgery may be associated with a lower mortality. More precise indicators for surgery are warranted. This systematic review identifies potential systemic biomarkers for AAA rupture or expansion.
MEDLINE/PubMed and EMBASE (from 1985 trough May 2007) were searched with the medical subject heading abdominal aortic aneurysm and keywords "size", "progression" or "growth" or "expansion rate" or "rupture" on the basis of MESH tree and as a text search restricted to English, German, French and Italian. In addition, reference lists were studied and manual searches performed. Observational studies investigating the association of circulating biomarkers with AAA rupture, expansion or size were selected.
Two reviewers (SU and GU) independently extracted the following data: year of publication, study characteristics, duration of follow-up, circulating biomarker, AAA expansion rate or size or rupture.
699 papers were identified. After exclusion of thoracic aneurysms and cardiac studies (n=118), surgical or medical treatment studies (n=179), case reports and animal studies (n=87), as well as reviews or letters (n=66), 249 articles were selected. Also excluded were 230 papers that did not report AAA size, expansion rate or rupture. 39 papers were included. Several potential biomarkers were identified. The strongest association with AAA was obtained with serum elastin peptides (SEP) and plasmin-antiplasmin (PAP) complexes. Matrix-degrading metalloproteinase 9 (MMP9) and interferon-gamma (IFN-gamma) could have clinical potential while many putative biomarkers showed poor association.
Several circulating agents in peripheral blood may predict AAA size, expansion rate or rupture. Few of them have clinical potential for future use. Confirmative studies and development of multivariate models are needed, together with continuing search for new biomarkers using the discovery based sciences within proteomics and/or genomics.
European journal of vascular and endovascular surgery: the official journal of the European Society for Vascular Surgery 09/2008; 36(3):273-80; discussion 281-2. DOI:10.1016/j.ejvs.2008.05.009 · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensional gel electrophoresis (2D-PAGE).
Protein spots excised from 2D-PAGE gels, were subjected to in-gel tryptic digestion and identified by liquid chromatography - tandem mass spectrometry. A total of 47 protein spots were identified. Immunological verification was performed for several of the differentially regulated proteins where suitable antibodies could be obtained. Functional annotation clustering revealed similarities as well as differences between the tumours. Twelve proteins that changed similarly in both tumours included up-regulation of rho GDP-dissociation inhibitor 2, proteasome subunit alpha type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin beta-3 chain, a 25 kDa actin fragment, proteasome subunit beta type 9, cofilin-1 and glia maturation factor gamma.
Some of the commonly differentially expressed proteins are also differentially expressed in other tumours and may be putative diagnostic and/or prognostic markers for lymphomas.
[Show abstract][Hide abstract] ABSTRACT: In the search for potential limbal stem cell protein markers, the purpose of this study was to characterize differences in protein expression between human central and limbal corneal epithelium by a proteomic approach using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with mass spectrometry (LC-MS/MS). The results were subsequently confirmed by Western blotting and immunohistochemistry. We detected more than 1000 protein spots in each gel. Thirty-two spots were significantly over-expressed in the central part and 70 spots were significantly over-expressed in the limbal part. We identified 25 different proteins. Among these 11 proteins representing different cellular locations and functions were selected for further investigations. Most interestingly, superoxide dismutase 2 (SOD2), was expressed in clusters of cells in the basal limbal epithelium. Heat shock protein 70 protein 1 (HSP70.1) and annexin I were highly abundant in limbal epithelium, although they were also present in the central epithelium to a minor extent. Among the proteins primarily expressed in the limbal fraction we further identified cytokeratin (CK) 15, CK19 and alpha enolase, which have been reported previously to be related to the limbal basal epithelium. The basal limbal epithelium consists of clusters of slow cycling limbal stem cells and rapid cycling transient amplifying cells. Ideally, proteins exclusively expressed in the limbal part of the epithelium may serve as markers for the basal limbal cells. SOD2 and CK15 identify clusters of limbal basal cells and therefore they may serve as markers for limbal stem cells in conjunction with the earliest transient amplifying cells.
Experimental Eye Research 06/2008; 87(2):96-105. DOI:10.1016/j.exer.2008.05.001 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BackgroundKnockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from
two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type
thymocytes by two-dimensional gel electrophoresis (2D-PAGE).
ResultsProtein spots excised from 2D-PAGE gels, were subjected to in-gel tryptic digestion and identified by liquid chromatography – tandem mass spectrometry. A total of 47 protein spots were identified.
Immunological verification was performed for several of the differentially regulated proteins where suitable antibodies could
be obtained. Functional annotation clustering revealed similarities as well as differences between the tumours. Twelve proteins
that changed similarly in both tumours included up-regulation of rho GDP-dissociation inhibitor 2, proteasome subunit α type
3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid
binding protein and down-regulation of adenylosuccinate synthetase, tubulin β-3 chain, a 25 kDa actin fragment, proteasome
subunit β type 9, cofilin-1 and glia maturation factor γ.
ConclusionSome of the commonly differentially expressed proteins are also differentially expressed in other tumours and may be putative
diagnostic and/or prognostic markers for lymphomas.
[Show abstract][Hide abstract] ABSTRACT: To identify the pattern of protein expression in the retina from a patient with Leber's Congenital Amaurosis (LCA) secondary to a mutation in the AIPL1 gene. The retina from one eye of a patient with LCA and 7 control eyes were studied. The tissue was subjected to high resolution two-dimensional gel electrophoresis, image analysis and mass spectrometry, in an effort to identify differentially regulated proteins.
In the LCA retina seven protein spots were differentially expressed. Six proteins were significantly up-regulated of which three could be identified as: alphaA-crystallin, triosephophate isomerase, and an N-terminal fragment of the beta-chain of ATP synthase. One protein spot that was down-regulated in the LCA retina was identified as a C-terminal fragment of beta-tubulin.
Retinal tissue in LCA is characterised by an up-regulation of alphaA-crystallin, triosephosphate isomerase, and ATP synthase (beta-chain fragment) and down-regulation of a fragment of beta-tubulin. These proteins/protein fragments may play a crucial role for the retinal degeneration processes in LCA and other retinal dystrophies.
[Show abstract][Hide abstract] ABSTRACT: The gamma-subunit of Na-K-ATPase (FXYD2) and corticosteroid hormone-induced factor (CHIF; FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in the kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of gamma and CHIF in the medulla of control and sodium-depleted rats were analyzed by immunofluorescence and immunoelectron microscopy and semiquantitative Western blotting. The results showed that antibodies against the gamma-subunit COOH terminus and splice variant gamma(a), but not splice variant gamma(b), labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3), all principal cells exhibited distinct basolateral labeling for both the gamma-subunit COOH terminus, splice variant gamma(a), and CHIF. Splice variant gamma(b) was abundant in the inner stripe of the outer medulla but absent in the inner medulla (IM). Double labeling by high-resolution immunoelectron microscopy showed close structural association between CHIF and the Na-K-ATPase alpha(1)-subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of gamma and CHIF in the renal medulla and show a new gamma variant in the IM. Extensive NaCl depletion did not induce significant changes in the locations or abundances of the gamma-subunit COOH terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during NaCl depletion.
American journal of physiology. Renal physiology 12/2006; 291(5):F1033-44. DOI:10.1152/ajprenal.00086.2006 · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Antibodies against Chlamydia pneumoniae are associated with the progression of abdominal aortic aneurysms (AAA), but cross-react with immunoglobulins in AAA walls indicating an autoimmune reaction.
Of 82 men with a small AAA followed for 1–5 years, 17% (10–27%) had antibodies against immunoglobulin, 3.7% had antinuclear antibodies (ANA), 19.5% (11–30%) had antinuclear core antibodies (ANCA), 2.4% had anti-beta-2-gpI IgG and 3,7% antibodies against cardiolipin.
The presence of antibodies against immunoglobulin and ANCA were not correlated with expansion rate; 2.61 and 2.76 mm/year, respectively, compared to 2.40 and 2.39 mm/year annually among those without such antibodies.
Known autoantibodies are seldomly present in AAA and seem not to influence the progression of AAA.
EJVES Extra 10/2006; 12(2):13-14. DOI:10.1016/j.ejvsextra.2006.05.001
[Show abstract][Hide abstract] ABSTRACT: CArG box-binding factor-A (CBF-A) is a protein involved in transcriptional control and interacts specifically with the penta-decameric (pd) element in kappa promoters. We show here that CBF-A will also bind specifically to a second region in the kappa promoter that overlaps with an early B cell factor binding site. Furthermore, the same region is present in multiple Ig promoters and we show that CBF-A can bind to several of these. Mitogenic stimulation of untransformed B lymphocytes promoted nuclear localisation of CBF-A. Using enhanced GFP (EGFP)-tagged constructs and transfection into COS7 cells, a nuclear localisation signal was defined in the C terminus of CBF-A. Deletion of only 13 amino acids from the C terminus of CBF-A led to the accumulation of the protein in bright speckles at the nuclear/cytoplasmic border. We also identified the heterogeneous ribonucleoprotein H as a specific interaction partner of CBF-A, but this interaction could be detected in the cytoplasm only. Thus, CBF-A has the potential to regulate the expression of multiple Ig V genes and has a complex, mitogen-responsive regulation of its intracellular localisation.
European Journal of Immunology 08/2006; 36(8):2192-202. DOI:10.1002/eji.200535659 · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of beta- or gamma-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an autocrine or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study is to identify corneal proteins differentially expressed between keratoconus and normal epithelial samples. Proteins from the corneal epithelium were isolated from 6 keratoconus and 6 myopia patients (controls) and separated by 2D-gel electrophoresis. Six % and 12% SDS-PAGE gels were used to separate low and high molecular weight proteins. Gels were silver stained and protein spots were defined by Melanie II software. The proteins that were most altered in expression comparing keratoconus and controls were extracted, trypsin-digested, and identified by mass spectroscopy. Approximately 200-500 protein spots were detected on each gel. Nineteen spots were identified as differentially expressed between keratoconus and reference epithelium including cytokeratin 3 (< 7.8 fold), gelsolin (1.6 fold), S100A4 (1.9 fold), and enolase 1 (0.72 fold). Another identified protein found at very high levels was cytokeratin 12. Gelsolin, cytokeratin 3, and cytokeratin 12 have previously been described to be involved in other corneal diseases. Three proteins, gelsolin, alpha enolase, and S100A4 were identified to be differentially expressed in keratoconus compared to reference epithelium and thus may be involved in the pathogenesis.
Experimental Eye Research 02/2006; 82(2):201-9. DOI:10.1016/j.exer.2005.06.009 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this prospective study of patients undergoing repair of non-ruptured abdominal aortic aneurysm between 1999 and 2003 was to evaluate and compare risk factors for mortality after surgery, to determine a complex of informative factors for lethal outcome, and to define patient risk groups. Logistic regression analysis revealed a complex of informative factors, including female gender, previous myocardial infarction, age greater than 75 years, and clinical course of abdominal aortic aneurysm as important indicators for lethal outcome. A risk score model identified low-, moderate- and high-risk groups with mortality rates of 2.9%, 8.0% and 44.4%, respectively.
Current controlled trials in cardiovascular medicine 10/2005; 6(1):14. DOI:10.1186/1468-6708-6-14 · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was designed to determine the oxygen dependency for expression of the endogenous hypoxic markers carbonic anhydrase IX (protein: CAIX/gene: CA9), glucose transporter 1 (GLUT1/GLUT1), osteopontin (OPN/OPN) and lactate dehydrogenase A (LDH-A/LDHA), and how this expression was influenced by extracellular pH (pHe).
Human cervix squamous cell carcinoma (SiHa) cells were used in all experiments. These cells were gassed in an enclosed environment under either anoxia (95% N2+5% CO2) for various times (0-30 h) or under different oxygen concentrations (0-21% O2) for 24 h at normal pHe (7.4) or low pHe (6.3). Response to radiation (7 Gy) was estimated using a clonogenic assay. Gene expression was determined by real-time PCR (normalized to the housekeeping gene, TFRC) and protein expression by Western blots.
Under normal pHe conditions, CA9, GLUT1 and LDHA gene expression was upregulated within 1-3h of anoxia, reaching near maximal values by 6h. OPN showed a slow increase over 24 h. At 24 h the relative increase was 135, 12, 90 and 5 times for CA9, GLUT1, OPN and LDHA, respectively. No induction was seen with the EGF receptor (EGFR). Gassing cells with differing oxygen concentrations for 24h resulted in a maximum level of expression for CA9 at 1% oxygen, whereas with GLUT1 and LDHA maximal expression occurred at 0.01% oxygen, but at 0% oxygen with OPN. The oxygen dependency for radiation response was identical to that seen for GLUT1 and LDHA. Expression of CA9, GLUT1, OPN and LDHA was inhibited under hypoxic conditions when pHe was reduced to 6.3. Expression of CAIX protein mimicked the CA9 gene expression patterns.
The expression of all the endogenous markers were upregulated by hypoxia, but the timing and oxygen dependencies were different and their expression was influenced by low pHe. This raises concerns about the generalised use of these agents as markers for hypoxia.
Radiotherapy and Oncology 09/2005; 76(2):187-93. DOI:10.1016/j.radonc.2005.06.037 · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied whether dysfunction of human hibernating (HIB) and irreversibly dysfunctional myocardium (IRDM) are associated with altered levels of the sarcoplasmatic reticulum calcium handling proteins Ca2+-ATPase (SERCA2a) and its inhibitor phospholamban (PLB).
In 12 patients myocardial biopsies were taken during bypass surgery and analysed for contents of these proteins. We classified regions as control, HIB, or IRDM based on echocardiographic studies before and 6 months after surgery.
SERCA2a content (mean+/-SEM) was similar to control in HIB and IRDM (2.6 +/- 1.7, 3.8 +/- 2.0, and 3.4 +/- 1.9 units/g non-collagen protein (NCP), p = 0.40). PLB content was similar to control in HIB (2.6 +/- 0.4 and 3.5 +/- 0.5 units/microg NCP) but reduced in IRDM (0.9 +/- 0.2 units/microg NCP, p < 0.05). SERCA2a:PLB ratio, an indicator of SERCA2a activity, did not differ between control and HIB (1.2 +/- 0.3 and 1.4 +/- 0.4 units/microg NCP) but was increased in IRDM (5.1 +/- 1.7 units/microg NCP, p < 0.05).
Inappropriate SERCA2a activity due to suppressed PLB levels may represent a maladaptive mechanism in chronic ischemic myocardium being causally linked to irreversibility of left ventricular dysfunction.
[Show abstract][Hide abstract] ABSTRACT: To compare the basic proteomic composition of aqueous humour (AH) from patients with corneal rejection (patients) with AH from patients with cataract (controls).
Aqueous humour was analysed for total protein concentration using Bradford's method and for protein composition using two-dimensional (2D) gel electrophoresis. Image analysis was used to detect protein spots in 2D gels that were increased by more than factor 2 in patients as compared with controls. Increased spots were identified by immunoblotting and mass spectrometry.
Aqueous humour from patients contained significantly higher total protein concentration than did AH from controls. A total of 31 spots were significantly increased in 2D gels from patients. The spots were derived from albumin, alpha1-antitrypsin, apolipoprotein J, cytokeratin type II, serin proteinase inhibitor and transthyretin. After correction of spot volumes by total protein concentrations, 10 spots derived from albumin, cytokeratin type II and alpha1-antitrypsin remained significantly increased.
The proteomic composition of AH differed significantly between patients and controls. The identified proteins suggest that the changes in AH are due to at least three different mechanisms: breakdown of the aqueous-blood barrier, enzymatic degradation, and liberation of locally synthesized proteins.
[Show abstract][Hide abstract] ABSTRACT: In mammals circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a monosynaptic neuronal pathway, the retinohypothalamic tract, originating in a subset of retinal ganglion cells. The nerve terminals of these cells contain the classical neurotransmitter glutamate and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), and there is evidence that these two transmitters interact to mediate photoentrainment of the oscillator in the SCN. To elucidate light-provoked PACAP receptor signaling we used proteomic analysis. Wild-type mice and mice lacking the PAC1 receptor (PAC1-/-) were light stimulated at early night, and the SCN was examined for proteins that were differentially expressed using two-dimensional gel electrophoresis and identification by tandem mass spectrometry. The most striking finding, which was subsequently confirmed by Western blotting, was a significant reduction of calmodulin (CaM) in wild-type mice as compared with PAC1-/- mice. Analysis at the mRNA level by quantitative in situ hybridization histochemistry was inconclusive, indicating that a translational mechanism might be involved. The findings indicate that PAC1 receptor signaling in the SCN in response to light stimulation induces a down-regulation of CaM expression and that CaM is involved in the photic-entrainment mechanism.