Natasha Barascuk - Academia.edu (original) (raw)

Papers by Natasha Barascuk

American Journal of Veterinary Research, 2000

Objective—To develop and validate an ELISA for quantitative analysis of feline trypsin-like immun... more Objective—To develop and validate an ELISA for quantitative analysis of feline trypsin-like immunoreactivity (fTLI). Sample Population—Purified feline cationic trypsin (fCT) and rabbit anti-fCT antiserum; blood samples from 63 healthy cats. Procedures—A sandwich capture ELISA was developed, using anti-fCT antiserum purified by affinity chromatography that underwent biotinylation. Purified fCT was used for standards. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. A reference range was established by assaying serum samples from the 63 healthy cats. Results—Sensitivity was 1.23 µg/L; working range was 2 to 567 µg/L. Ratios of observed versus expected results for 4 samples tested at various dilutions ranged from 90.0 to 120.7%. Ratios of observed versus expected results for 5 samples spiked with various concentrations of fCT ranged from 82.0 to 101.8%. Intra- and inter-assay coefficients of variability ranged ...

Endocrine Reviews

Measurement of natriuretic peptides (NPs) has proven its clinical value as biomarker, especially ... more Measurement of natriuretic peptides (NPs) has proven its clinical value as biomarker, especially in the context of heart failure (HF). In contrast, a state of partial NP deficiency appears integral to several conditions in which lower NP concentrations in plasma presage overt cardiometabolic disease. Here, obesity and type 2 diabetes have attracted considerable attention. Other factors—including age, sex, race, genetics, and diurnal regulation—affect the NP “armory” and may leave some individuals more prone to development of cardiovascular disease. The molecular maturation of NPs has also proven complex, with highly variable O-glycosylation within the biosynthetic precursors. The relevance of this regulatory step in post-translational propeptide maturation has recently become recognized in biomarker measurement/interpretation and cardiovascular pathophysiology. An important proportion of people appear to have reduced effective net NP bioactivity in terms of receptor activation and p...

Atherosclerosis

Background and aims: Clinical interventions targeting nonlipid risk factors are needed given the ... more Background and aims: Clinical interventions targeting nonlipid risk factors are needed given the high residual risk of atherothrombotic events despite effective control of dyslipidemia. Dickkopf-1 (DKK1) plays a lipidindependent role in vascular pathophysiology but its involvement in atherosclerosis development and its therapeutic attractiveness remain to be established. Methods: Patient data, in vitro studies and pharmacological intervention in murine models of atherosclerosis were utilized. Results: In patients' material (n = 127 late stage plaque specimens and n = 10 control vessels), DKK1 mRNA was found to be higher in atherosclerotic plaques versus control arteries. DKK1 protein was detected in the luminal intimal area and in the necrotic core of plaques. DKK1 was released from isolated primary human platelets (~12-21-fold) and endothelial cells (~1.4-2.5-fold) upon stimulation with different pathophysiological stimuli. In ApoE − /− and Ldlr − /− mice, plasma DKK1 concentrations were similar to those observed in humans, whereas DKK1 expression in different atheroprone arterial segments was very low/absent. Chronic treatment with a neutralizing DKK1 antibody effectively reduced plasma concentrations, however, plaque lesion area was not reduced in ApoE − /− and Ldlr − /− mice fed a western diet for 14 and 16 weeks. Anti-DKK1 treatment increased bone volume and bone mineral content. Conclusions: Functional inhibition of DKK1 with an antibody does not alter atherosclerosis progression in classical murine models. This may reflect the absence of DKK1 expression in plaques and more advanced animal disease models could be needed to evaluate the role and therapeutic attractiveness of DKK1 in late stage complications such as plaque destabilization, calcification, rupture and thrombosis.

Background: The current study utilized a Bleomycin-induced model of skin fibrosis to investigate ... more Background: The current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies. Methods: Skin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels. Results: CO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of

Journal of Molecular and Cellular Cardiology, 2018

Highlights  Plasma levels of CatK increase in patients with CHD particularly during AMI, compare... more Highlights  Plasma levels of CatK increase in patients with CHD particularly during AMI, compared to controls.  CatK expression is elevated in post-MI mouse hearts and localizes to cardiomyocytes, endothelial cells, fibroblasts, macrophages, and CD4 + T cells.  CatK-deficiency worsens post-MI cardiac function along with increased collagen deposition and fibrosis, enhanced cardiac cell death, and reduced cardiac cell proliferation.  CatK inhibition or deficiency increases cardiomyocyte death, but CatK inhibition suppresses CD4 + T-cell and macrophage death. Background-Extracellular matrix metabolism and cardiac cell death participate centrally in myocardial infarction (MI). This study tested the roles of collagenolytic cathepsin K (CatK) in post-MI left ventricular remodeling. Methods and Results-Patients with acute MI had higher plasma CatK levels (20.49±7.07 pmol/L, n=26) than those in subjects with stable angina pectoris (8.34±1.66 pmol/L, n=28, P=0.01) or those without coronary heart disease (6.63±0.84 pmol/L, n=93, P=0.01). CatK protein expression increases in mouse hearts at 7 and 28 days post-MI. Immunofluorescent staining localized CatK expression in cardiomyocytes, endothelial cells, fibroblasts, macrophages, and CD4 + T cells in infarcted mouse hearts at 7 days post-MI. To probe the direct participation of CatK in MI, we produced experimental MI in CatK-deficient mice (Ctsk-/-) and their wild-type (Ctsk +/+) littermates. CatK-deficiency yielded worsened cardiac function at 7 and 28 days post-MI, compared to Ctsk +/+ littermates (fractional shortening percentage: 5.01±0.68 vs. 8.62±1.04, P<0.01, 7 days post-MI; 4.32±0.52 vs. 7.60±0.82, P<0.01, 28 days post-MI). At 7 days post-MI, hearts from Ctsk-/mice contained less CatK-specific type-I collagen fragments (10.37±1.91 vs. 4.60±0.49 ng/mg tissue extract, P=0.003) and more fibrosis (1.67±0.93 vs. 0.69±0.20 type-III collagen positive area percentage, P=0.01; 14.25±4.12 vs. 6.59±0.79 smooth muscle actin-positive area percentage, P=0.016; and 0.82 ± 0.06 vs. 0.31 ± 0.08 CD90positive area percentage, P=0.008) than those of Ctsk +/+ mice. Immunostaining demonstrated that CatK-deficiency yielded elevated cardiac cell death but reduced cardiac cell proliferation. In vitro studies supported a role of CatK in cardiomyocyte survival. Conclusion-Plasma CatK levels are increased in MI patients. Heart CatK expression is also elevated post-MI, but CatK-deficiency impairs post-MI cardiac function in mice by increasing myocardial fibrosis and cardiomyocyte death.

Physiological Reports, 2016

To examine the effect on serum lipase activity and protein concentration of intravenous infusions... more To examine the effect on serum lipase activity and protein concentration of intravenous infusions of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY 3-36) and of an ad libitum meal in healthy overweight men. Twenty-five healthy, male subjects participated in this randomized, double-blinded, placebo-controlled 4-arm crossover study (Body Mass Index (BMI): 29 AE 3 kg/ m 2 , age: 33 AE 9 years). On separate days, the subjects received a 150-min intravenous infusion of either (1) 0.8 pmol/kg/min PYY 3-36 , (2) 1.0 pmol/kg/ min GLP-1, (3) 1 + 2, or (4) placebo. Samples were collected throughout the infusion and after intake of an ad libitum meal for measurement of serum lipase. Serum lipase levels measured by enzyme-linked immunosorbent assay (ELISA) following mono-infusions of GLP-1 and PYY 3-36 were comparable to serum lipase levels following placebo (P = 0.054 and P = 0.873, respectively). Following the co-infusion of GLP-1 and PYY 3-36 , serum lipase levels measured by ELISA decreased over time compared to placebo (P = 0.012). However, the between-group difference was not consistent when each time point was analyzed separately. On the placebo day, serum lipase levels measured by ELISA after an ad libitum meal rose slightly compared to the preprandial values (P = 0.003). There was strong correlation between serum lipase levels measured by ELISA and LIPC Lipase colorimetric assay (COBAS) (0.94 < r; <0.0001). Infusions of GLP-1 and PYY 3-36 , separately or in combination, did not increase serum lipase. However, a small increase in serum lipase may occur in response to a meal.

American journal of translational research, 2013

The hallmark of a variety of fibrotic diseases such as liver fibrosis, lung fibrosis, skin fibros... more The hallmark of a variety of fibrotic diseases such as liver fibrosis, lung fibrosis, skin fibrosis and atherosclerosis is extensive extracellular matrix remodeling (ECMr) of the disease affected tissue. Inflammation often leads to tissue disruption and destruction, upon which locally released battery of proteases such as matrix metalloproteinases and cysteine proteases degrade the surrounding matrix. The degradation products of ECM proteins, the co-called neoepitopes, are released into the systemic circulation. By recent development of Enzyme-Linked Immunosorbent Assays (ELISAs) detecting the pathological tissue turnover in atherosclerosis and liver fibrosis, we have introduced a novel class of biomarkers into the field of fibrotic diseases, which have been proved efficient in the early diagnosis. This work has resulted in identification of common mechanisms involving specific cell types, proteins and proteases as well as pathways shared among the fibrotic diseases. In this analysi...

International journal of clinical and experimental medicine, 2013

Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. ... more Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. Versican, an extracellular matrix proteoglycan present in normal and atherosclerotic arteries is a substrate for matrix metalloproteinases (MMPs) present in macrophage rich areas. The aim of the current study was to develop an immunoassay to detect a specific MMP-12 derived versican degradation fragment (VCANM) and assess its potential as a biomarker for extracellular matrix remodelling in atherosclerosis. A mouse monoclonal antibody raised against VCANM was used for the development of a competitive ELISA for detection of the fragment in plasma. VCANM was measured in plasma of patients with different levels of heart diseases. Patients experiencing I) acute coronary syndrome, II) stable ischemic heart disease and III) demonstrating high levels of coronary calcium deposits had significantly higher plasma levels of VCANM compared to a control group of individuals with no detectable coronary...

PLoS ONE, 2013

Objective: Alzheimer's disease (AD) is a devastating neurological disease characterized by pathol... more Objective: Alzheimer's disease (AD) is a devastating neurological disease characterized by pathological proteolytic cleavage of tau protein, which appears to initiate death of the neurons. The objective of this study was to investigate whether a proteolytic fragment of the tau protein could serve as blood-based biomarker of cognitive function in AD. Methods: We developed a highly sensitive ELISA assay specifically detecting an A Disintegrin and Metalloproteinase 10 (ADAM10)-generated fragment of tau (Tau-A). We characterized the assay in detail with to respect specificity and reactivity in healthy human serum. We used samples from the Tg4510 tau transgenic mice, which over-express the tau mutant P301L and exhibit a tauopathy with similarities to that observed in AD. We used serum samples from 21 well-characterized Alzheimer's patients, and we correlated the Tau-A levels to cognitive function. Results: The Tau-A ELISA specifically detected the cleavage sequence at the N-terminus of a fragment of tau generated by ADAM10 with no cross-reactivity to intact tau or brain extracts. In brain extracts from Tg4510 mice compared to wt controls we found 10-fold higher levels of Tau-A (p,0.001), which indicates a pathological relevance of this marker. In serum from healthy individuals we found robust and reproducible levels of Tau-A, indicating that the analyte is present in serum. In serum from AD patients an inverse correlation (R 2 = 0.46, p,0.001) between the cognitive assessment score (Mattis Dementia Rating Scale (MDRS)) and Tau-A levels was observed. Conclusion: Based on the hypothesis that tau is cleaved proteolytically and then released into the blood, we here provide evidence for the presence of an ADAM10-generated tau fragment (Tau-A) in serum. In addition, the levels of Tau-A showed an inverse correlation to cognitive function, which could indicate that this marker is a serum marker with pathological relevance for AD.

Liver International, 2010

Background: During fibrogenesis in the liver, in which excessive remodelling of the extracellular... more Background: During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via their activity in the proteolytic degradation of extracellular macromolecules such as collagens, resulting in the generation of specific cleavage fragments. These neo-epitopes may be used as markers of fibrosis. Aims: The current study investigated whether a novel enzyme-linked immunosorbent assay (ELISA) assay specifically measuring an MMP-9-cleaved sequence of type III collagen located at position 610 (CO3-610C) may be used as a marker of liver fibrosis. Material and methods: Bile duct ligation (BDL) was performed in 20 rats, with sham operations performed on another 20 rats. Serum levels of the neoepitope CO3-610C (MMP-mediated type III collagen degradation) were determined with an ELISA at 14 and 28 days post-surgery. Liver fibrosis was evaluated by quantitative digital image analysis of Sirius red-stained formalinfixed and paraffin-embedded sections. Western blot and densitometry were performed to confirm the CO3-610C ELISA data. Results: CO3-610C levels in serum increased significantly in BDL rats compared with those undergoing sham operations (% increase: 14 days = 153%, P o 0.0001; 28 days = 134%, P = 0.0014). This increase was confirmed by Western blot and densitometry of the identified bands. The CO3-610C levels correlated to liver fibrosis (R 2 = 0.23 and P = 0.01), as evaluated by quantitative digital histology. Discussion and conclusion: The data suggest that MMP-9-mediated CO3 turnover is a central event in the pathogenesis of fibrosis, and that the neo-epitope generated may be a novel biochemical marker.

Journal of Hepatology, 2011

POSTERS pregnancy. LS was measured by Fibroscan either using the M or XL probe. Besides basic gyn... more POSTERS pregnancy. LS was measured by Fibroscan either using the M or XL probe. Besides basic gynecological data, BMI and transaminases were obtained. Results: LS could be measured in all 103 women using the M probe except one case where the XL probe was required for reliable interquartile range. 17 women (16.5%) had a pathological LS higher than 8 kPa, four of them higher than 12.5 kPa which is regarded as cutoff value for F4 fibrosis. All women with increased LS were in the third trimester starting with week 31 while all women within the second trimester had normal LS <6 kPa. LS correlated slightly with duration of pregnancy (P < 0.05), but not with gain of weight or BMI. Primary biliary or hepatic causes of increased LS were excluded by blood tests and ultrasound. No abnormal liver function tests were observed in this study population except one HELLP syndrome with elevated LS. Increased LS completely normalized after delivery in the three patients studied. Further studies on narcotized German landrace pigs suggest that not an elevated intraabdominal pressure alone but an impaired hepatic venous outflow seems to cause the increased LS during pregnancy. Conclusion: LS is significantly increased in >20% of pregnant women in the third trimester probably due to hemodynamic reasons. Increased LS generally normalizes after delivery. Our data suggest that LS could be an important non-invasive predictor of hepatic complications during pregnancy.

Journal of Hepatology, 2012

The International Journal of Cardiovascular Imaging, 2010

The aim of this study is to investigate new methods for describing the progression of atheroscler... more The aim of this study is to investigate new methods for describing the progression of atherosclerosis based on novel information of the growth patterns of individual abdominal aortic calcifications (AACs) over time. Lateral X-ray images were used due to their low cost, fast examination time, and widespread use, which facilitates a large statistical model (n > 100) based on longitudinal data. The examined cohort consisted of 103 post-menopausal women aged 62.4 y (± 7.0 y) with an average number of AACs of 4.7 (± 8.0) at baseline. The subjects had X-ray images taken in 1992-93 (baseline) and again in 2000-01 (follow-up). The growth patterns of the individual AACs were derived based on registered baseline and follow-up images. Area, height, width, centre of mass position, and movement of the centre of mass, and upper and lower boundary of the matched AACs were measured. The AACs occurred first, mainly, on the posterior aortic wall. The AACs grew on average 41 % in the longitudinal direction and 21 % in the radial direction. A correlation of 0.48 (P < 0.001) between growth in width and height of the AACs was present. The centre of mass of the AACs moved 0.60 mm (P < 0.001) downstream in the aorta, on average. The growth patterns of AACs may give new insights into the progression of atherosclerosis. The downstream asymmetry in the growth patterns indicates variability in microscopic environments around the AACs.

Fibrogenesis & Tissue Repair, 2013

Background: The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragm... more Background: The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis. The scope of the present study was to develop a novel enzyme-linked immunosorbent assay (ELISA) for the measurement of a MMP-9 and MMP-12-generated biglycan neo-epitope and to test its biological validity in a rat model of RA and in two rat models of liver fibrosis, chosen as models of ECMR. Results: Biglycan was cleaved in vitro by MMP-9 and −12 and the 344 0 YWEVQPATFR 0 353 peptide (BGM) was chosen as a potential neo-epitope. A technically sound competitive ELISA for the measurement of BGM was generated and the assay was validated in a bovine cartilage explant culture (BEX), in a collagen induced model of rheumatoid arthritis (CIA) and in two different rat models of liver fibrosis: the carbon tetrachloride (CCL4)-induced fibrosis model, and the bile duct ligation (BDL) model. Significant elevation in serum BGM was found in CIA rats compared to controls, in rats treated with CCL4 for 16 weeks and 20 weeks compared to the control groups as well as in all groups of rats subject to BDL compared with sham operated groups. Furthermore, there was a significant correlation of serum BGM levels with the extent of liver fibrosis determined by the Sirius red staining of liver sections in the CCL4 model. Conclusion: We demonstrated that the specific tissue remodeling product of MMPs-degraded biglycan, namely the neo-epitope BGM, is correlated with pathological ECMR. This assay represents both a novel marker of ECM turnover and a potential new tool to elucidate biglycan role during the pathological processes associated with ECMR.

Fibrogenesis & Tissue Repair, 2011

Background: Hepatic fibrosis is characterized by intense tissue remodeling, mainly driven by matr... more Background: Hepatic fibrosis is characterized by intense tissue remodeling, mainly driven by matrix metalloproteinases. We previously identified CO3-610, a type III collagen neoepitope generated by matrix metalloproteinase (MMP)-9, and tested its performance as a fibrosis marker in rats with bile-duct ligation. In this study, we assessed whether CO3-610 could be used as a surrogate biomarker of liver fibrosis and portal hypertension in carbon tetrachloride-induced experimental fibrosis. Results: For this study, 68 Wistar rats were used. Serum CO3-610 was measured by ELISA. Liver fibrosis was quantified by Sirius red staining. Serum hyaluronic acid (HA) was measured with a binding-protein assay. Gene expression of collagens I and III, Mmp2 and Mmp9, and tissue inhibitors of matrix metalloproteinase 1 (Timp1) and 2(Timp2) was quantified by PCR. Hemodynamic measurements were taken in a subgroup of animals. A close direct relationship was found between serum CO3-610 and hepatic collagen content (r = 0.78; P < 0.001), superior to that found for serum HA (r = 0.49; P < 0.05). CO3-610 levels in rats with severe fibrosis (43.5 ± 3.3 ng/mL, P < 0.001) and cirrhosis (60.6 ± 4.3 ng/mL, P < 0.001) were significantly higher than those in control animals (26.6 ± 1.3 ng/ mL). Importantly, a highly significant relationship was found between serum CO3-610 and portal hypertension (r = 0.84; P < 0.001). Liver Mmp9 expression increased significantly in fibrotic animals but decreased to control levels in cirrhotic ones. Conclusions: Circulating CO3-610 behaves as a reliable indicator of hepatic remodeling and portal hypertension in experimental fibrosis. This peptide could ultimately be a useful marker for the management of liver disease in patients.

BMC Dermatology, 2011

BMC Cardiovascular Disorders, 2011

Background: Abdominal aortic calcifications (AAC) predict cardiovascular mortality. A new scoring... more Background: Abdominal aortic calcifications (AAC) predict cardiovascular mortality. A new scoring model for AAC, the Morphological Atherosclerotic Calcification Distribution (MACD) index may contribute with additional information to the commonly used Aortic Calcification Severity (AC24) score, when predicting death from cardiovascular disease (CVD). In this study we investigated associations of MACD and AC24 with traditional metabolic-syndrome associated risk factors at baseline and after 8.3 years follow-up, to identify biological parameters that may account for the differential performance of these indices. Methods: Three hundred and eight healthy women aged 48 to 76 years, were followed for 8.3 ± 0.3 years. AAC was quantified using lumbar radiographs. Baseline data included age, weight, blood pressure, blood lipids, and glucose levels. Pearson correlation coefficients were used to test for relationships. Results: At baseline and across all patients, MACD correlated with blood glucose (r 2 = 0.1, P< 0.001) and to a lesser, but significant extent with traditional risk factors (p < 0.01) of CVD. In the longitudinal analysis of correlations between baseline biological parameters and the follow-up calcification assessment using radiographs we found LDL-cholesterol, HDL/LDL, and the ApoB/ApoA ratio significantly associated with the MACD (P< 0.01). In a subset of patients presenting with calcification at both baseline and at follow-up, all cholesterol levels were significantly associated with the MACD (P< 0.01) index. AC24 index was not correlated with blood parameters. Conclusion: Patterns of calcification identified by the MACD, but not the AC24 index, appear to contain useful biological information perhaps explaining part of the improved identification of risk of cardiovascular death of the MACD index. Correlations of MACD but not the AC24 with glucose levels at baseline suggest that hyperglycemia may contribute to unique patterns of calcification indicated by the MACD.

BMC Cardiovascular Disorders, 2010

Background: Proteolytic degradation of Type I Collagen by proteases may play an important role in... more Background: Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture. The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular matrix (ECM) of atherosclerotic plaques by cathepsin K mediated processes. Methods: We 1) cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I) 2) investigated the presence of CTX-I in human coronary arteries and 3) finally investigated the clinical potential by measuring circulating CTX-I in women with and without radiographic evidence of aortic calcified atherosclerosis. Results: Immune-histochemistry of early and advanced lesions of coronary arteries demonstrated co-localization of Cathepsin-K and CTX-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women with aortic calcifications compared to those without. Conclusions: Human macrophage foam cells degrade the atherosclerotic plaques though cathepsin K mediated processes, resulting in increase in levels of CTX-I. Serum CTX-I was not elevated in women with aortic calcification, likely due to the contribution of CTX-I from osteoclastic bone resorption which involves Cathepsin-K. The human macrophage model system may be used to identify important pathway leading to excessive proteolytic plaque remodeling and plaque rupture.