Lars Melholt Rasmussen

Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50u2009kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by Tm versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.

A novel assay for extracellular matrix remodeling associated with liver fibrosis: An enzyme-linked immunosorbent assay (ELISA) for a MMP-9 proteolytically revealed neo-epitope of type III collagen

OBJECTIVESnAccumulation of extracellular matrix (ECM) components and increased matrix-metalloprotease (MMPs) activity are hallmarks of fibrosis. We developed an ELISA for quantification of MMP-9 derived collagen type III (CO3) degradation.nnnDESIGN AND METHODSnA monoclonal antibody targeting a specific MMP-9 cleaved fragment of CO3 was used for development of a competitive ELISA. The assay was investigated in serum and tissues from bile duct ligated rats (BDL).nnnRESULTSnThe ELISA showed no cross-reaction with either intact CO3, or other collagens. The intra- and inter-assay CV were below 10%. Liver fibrosis was demonstrated in BDL animals by semi quantitative scoring (P<0.0001). Serum levels of CO3-610 increased 2.5 fold in BDL animals (P<0.001). The CO3-610 levels were 5 fold higher in ex vivo cultures of fibrotic livers compared to controls (P<0.001).nnnCONCLUSIONnWe have developed a novel ELISA for measuring a specific fragment CO3 generated by MMP-9 important in pathogenesis of liver fibrosis.

Plasma osteoprotegerin levels predict cardiovascular and all-cause mortality and deterioration of kidney function in type 1 diabetic patients with nephropathy

Aims/hypothesisThe bone-related peptide osteoprotegerin is produced by vascular cells and is involved in the process of vascular calcification. The aim of this study was to investigate the predictive value of plasma levels of osteoprotegerin in relation to mortality, cardiovascular events and deterioration in kidney function in patients with type 1 diabetes.MethodsThis prospective observational follow-up study included 397 type 1 diabetic patients with overt diabetic nephropathy (243 men; age [mean±SD] 42.1u2009±u200910.6xa0years, duration of diabetes 28.3u2009±u20099.9xa0years, GFR 67u2009±u200928xa0ml min−1 1.73xa0m2) and a group of 176 patients with longstanding type 1 diabetes and persistent normoalbuminuria (105 men; age 42.6u2009±u20099.7xa0years, duration of diabetes 27.6u2009±u20098.3xa0years).ResultsThe median (range) follow-up period was 11.3 (0.0–12.9) years. Among patients with diabetic nephropathy, individuals with high osteoprotegerin levels (fourth quartile) had significantly higher all-cause mortality than patients with low levels (first quartile) (covariate-adjusted hazard ratio [HR] 3.00 [1.24–7.27]). High osteoprotegerin levels also predicted cardiovascular mortality (covariate-adjusted HR 4.88 [1.57–15.14]). Furthermore, patients with high osteoprotegerin levels had significantly higher risk of progression to end-stage renal disease than patients with low levels (covariate-adjusted HR 4.32 [1.45–12.87]). In addition, patients with high levels of plasma osteoprotegerin had an elevated rate of decline in GFR.Conclusions/interpretationHigh levels of osteoprotegerin predict all-cause and cardiovascular mortality in patients with diabetic nephropathy. Furthermore, high levels of osteoprotegerin predict deterioration of kidney function towards end-stage renal disease.

Phenotype presentation of hypophosphatemic rickets in adults.

Hypophosphatemic rickets (HR) is a group of rare disorders caused by excessive renal phosphate wasting. The purpose of this cross-sectional study of 38 HR patients was to characterize the phenotype of adult HR patients. Moreover, skeletal and endodontic severity scores were defined to assess possible gender differences in disease severity in patients with genetically verified X-linked HR. Compared to normal reference data, i.e., zxa0=xa00, HR patients had significantly lower final height, with a mean difference in z-score of −1.9 (95% CI −2.4 to −1.4, Pxa0<xa00.001). Compared to paired z-scores of final height, z-scores of leg length were significantly lower and those of sitting height were significantly higher (Pxa0<xa00.001), resulting in disproportion as indicated by the significantly elevated sitting height ratio, mean difference in z-score of 2.6 (95% CI 2.1–3.1, Pxa0<xa00.001). Z-scores of head circumference (median 1.4, range −0.4 to 5.5, Pxa0<xa00.001) and z-scores of bone mineral density (BMD) of the lumbar spine (median 1.9, range −1.5 to 8.6, Pxa0<xa00.001) were significantly elevated compared to normal reference data. The relative risk (RR) of fracture was reduced (RRxa0=xa00.34, 95% CI 0.20–0.57, Pxa0<xa00.001). The skeletal severity score tended to be higher in males compared to females (Pxa0=xa00.07), and no gender difference in endodontic severity was found. In conclusion, adult HR patients were characterized by short stature and were disproportioned. They had elevated BMD of the lumbar spine and a reduced risk of fractures. We found a tendency for males to be more severely affected than females.

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer.

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

Plasma proteome profiling of atherosclerotic disease manifestations reveals elevated levels of the cytoskeletal protein vinculin.

UNLABELLEDnAtherosclerosis is a chronic disease of the arterial wall that is recognized as the leading cause of mortality and morbidity worldwide. There is an eminent need for better biomarkers that can aid in patient care before the onset of the first cardiovascular event. We used quantitative proteomics to identify proteins with altered concentrations in plasma samples from four groups: 1) Individuals without cardiovascular symptoms and without the presence of coronary calcium, 2) individuals without cardiovascular symptoms, but with high amounts of coronary calcium, 3) individuals operated because of atherosclerotic diseases, and 4) individuals with an acute coronary syndrome. Immunoassays and SRM-MS were used for single patient verification of candidate proteins. Proteins involved in cardiovascular diseases i.e. serum amyloid protein A (SAA), C-reactive protein (CRP), and apolipoprotein(a) [apo(a)] displayed an increased expression profile from groups 1 to 4. The top-most elevated protein, vinculin (Vcl) displayed a similar profile. Immunoassays confirmed the expression profile of apo(a) and CRP. A 5-plex SRM-MS assay for Vcl, SAA, CRP, apo(a) and thrombospondin-4 (TSP-4) was developed for multiplex verification in all 120 individual samples. The 5-plex SRM assay confirmed a statistically significant up-regulation of Vcl in the acute coronary syndrome group.nnnBIOLOGICAL SIGNIFICANCEnThe aim of this study was to identify new candidate plasma markers of atherosclerosis manifestations, which may develop into screening-, diagnostic- or monitoring biomarkers for risk stratification of cardiovascular disease (CVD). At present no studies have elucidated the proteomic changes that occur along with several stages and manifestations of atherosclerotic disease. By using 4-plex iTRAQ, we identified and quantified proteins with altered concentrations in pooled plasma samples from 120 individuals from four middle-aged groups. Proteins involved in cardiovascular diseases i.e. serum amyloid protein A (SAA), C-reactive protein (CRP), and apolipoprotein(a) [apo(a)] displayed an increased expression profile along with increased manifestations of CVD. A novel candidate marker was identified as vinculin (Vcl), a multi-protein linker that connects cell-matrix adhesions and cell-cell adhesions to the actin-based cytoskeleton. Immuno- and SRM-assays were used for single patient validation of candidate proteins. While further studies needs to address the role of Vcl in the development of atherosclerosis, the combined data provided in this report offers a catalog of the proteomic changes that occurs in plasma over several stages and manifestations of atherosclerotic disease.

No influence of OPG and its ligands, RANKL and TRAIL, on proliferation and regulation of the calcification process in primary human vascular smooth muscle cells.

The aim of this study was to examine the effects of the OPG-RANKL-TRAIL system on proliferation, regulation of calcification-associated genes and calcification of human vascular smooth muscle cells (HVSMCs). Small interfering (si)RNA-mediated knockdown of OPG was followed by treatment of HVSMCs with recombinant RANKL or TRAIL. Regulation of a calcification-associated gene set was assayed by pathway analysis of microarray results. The lack of OPG in HVSMCs or treatment with RANKL or TRAIL did not affect proliferation of HVSMCs. In addition, OPG, RANKL or TRAIL did not modify the regulation of a calcification-associated gene set. Finally, in the long term calcification assay, we found that cells isolated from seven different human donors showed a great variability in the response to RANKL and insulin. However, overall RANKL and/or insulin did not affect the development of calcification of HVSMCs. These studies indicate that OPG knockdown does not alter the calcification process in HVSMCs.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling.

OBJECTIVEnDegradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine.nnnDESIGN AND METHODSnA monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice.nnnRESULTSnThe lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice.nnnCONCLUSIONnWe have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.

Expression of osteoblast and osteoclast regulatory genes in the bone marrow microenvironment in multiple myeloma: only up-regulation of Wnt inhibitors SFRP3 and DKK1 is associated with lytic bone disease.

Abstract Multiple myeloma (MM) lytic bone disease (LBD) is caused by osteoclast activation and osteoblast inhibition. RANK/RANKL/OPG play central roles in osteoclast activation and Wnt inhibitor DKK1 in osteoblast inhibition. The role of other Wnt inhibitors is less clear. We evaluated gene expression of osteoclast regulators (RANK, RANKL, OPG, TRAIL, MIP1A), Wnt inhibitors (DKK1, SFRP2, SFRP3, sclerostin, WIF1) and osteoblast transcription factors (RUNX2, osterix) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the bone marrow (BM) microenvironment using snap-frozen BM biopsies, thereby achieving minimal post-sampling manipulation, and gene expression profiling (GEP) data, reflecting the in vivo situation. We analyzed 110 biopsies from newly diagnosed patients with MM and monoclonal gammopathy of unknown significance (MGUS) and healthy volunteers. LBD was evaluated using standard radiographs and the bone resorption marker CTX-1. Protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Among Wnt inhibitors, only SFRP3 and DKK1 were significantly overexpressed in advanced LBD, correlating with protein levels. SFRP3 correlated with CTX-1. Our findings support osteoblast inhibition as the driving force behind MM LBD.

Decorin is down-regulated in multiple myeloma and MGUS bone marrow plasma and inhibits HGF-induced myeloma plasma cell viability and migration†

Decorin is a stromal‐produced small leucine‐rich proteoglycan known to attenuate tumour pro‐survival, migration, proliferation and angiogenic signalling pathways. Recent studies have shown that decorin interacts with the hepatocyte growth factor (HGF) receptor c‐Met, a potential key pathway in multiple myeloma (MM).