The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selection.

DNA aptamers are single-stranded oligonucleotides which can form various secondary and tertiary structures. They can recognize a broad range of targets ranging from small molecules, such as ions, vitamins, antibiotics, to high molecular weight structures, including enzymes and antibodies. DNA aptamers are extensively studied as a potential source of new pharmaceutical drugs due to their inexpensive synthesis, low immunogenicity, and high specificity. The commonly used aptamer selection procedure is systematic evolution of ligands by exponential enrichment (SELEX) where the target molecule is immobilized on an appropriate chromatography resin. For peptide/protein targets, immobilized metal affinity chromatography (IMAC) resins are frequently used. There is a broad range of commercially available resins which can be used for IMAC. They are characterized by different metal ions, linker types, and bead materials. In this study, we tested the impact of different IMAC resins on the DNA aptamer selection process during eight SELEX cycles. A histidine-tagged 29 amino acid peptide corresponding to the interdomain connecting loop of human proliferating cell nuclear antigen was used as a selection target. Different resin materials containing the same metal ion (Co(2+)) were tested. Simultaneously, agarose resins containing identical linkers, but different metal ions (Co(2+), Cu(2+), Ni(2+), and Zn(2+)) were analyzed. The results of this study clearly demonstrated the impact of the metal ion and resin material on the DNA aptamer selection progress. The presented data indicate that for successful IMAC resin-based SELEX, the determination of the optimal resin might be crucial.

[Individual risk screening and visualization as a central element of health days through the interactive electronic GloRiA program]. / Individuelles Risikoscreening und Visualisierung als zentrales Element von Gesundheitstagen. Einsatz des interaktiven elektronischen GloRiA-programms.

BACKGROUND: Health days are an established forum for prevention and health promotion for different groups in the general population. Through the use of modular questionnaires "Global Risk Assessment" (GloRiA) on computers (handheld and desktop), the recording of patient data and presentation of the results can be optimized. Possible applications include identification of risk factors, early detection of patients at risk, epidemiology and health services research, promotion of patient adherence by visualizations (e.g. risk scores). Up to 12 different question modules are available (e.g. risk for the occurrence of cardiovascularevents by Framingham score, forfuture riskof diabetes mellitus using FindRisk score, smoking, COPD, pain, comorbidities). METHODS AND RESULTS: During 57 health days in 2010 and 2011, data were collected from 3451 persons (53% women, mean age 59.6 +/- 15.4 years) using GloRiA. The percentage of former smokers was 32.7%, while that of current smokers was 14.7%. The average 10-year risk based on the Framingham score (calculated with 1739 persons) in 53.7% of respondents was at <10%, in 37.0% at 10-20%, and in 9.3% at > 20%. In men risk was higher than in women. Smoking cessation would theoretically reduce the mean 10-year risk from 10.9 +/- 9.2% to 7.4 +/- 6.6%. In 50.5% of participants blood pressure measurement revealed elevated values, and in 10% or 2%, respectively, a moderately high or high 10-year riskof incident diabetes mellitus according to FindRisk. CONCLUSION: The use of GloRiA for the consolidation of health data under the framework of health days provides new and sustained possibilities in early detection of cardiovascular disease. The calculation and visualization of risks and the impact of treatment decisions, e.g. reduction of cardiovascular risk by smoking cessation, were communicated directly to the participants. The individual health report facilitates the diagnostic procedures bya physician.

Long-term clopidogrel administration following severe coronary injury reduces proliferation and inflammation via inhibition of nuclear factor-kappaB and activator protein 1 activation in pigs.

BACKGROUND: The optimal duration of clopidogrel treatment following percutaneous coronary intervention (PCI) and the patient population that would benefit most are still unknown. In a porcine coronary injury model, we tested two different durations of clopidogrel treatment on severely or moderately injured arteries and examined the arterial response to injury. To understand the molecular mechanism, we also investigated the effects on transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1). MATERIALS AND METHODS: In 24 cross-bred pigs, one coronary artery was only moderately injured by percutaneous transluminal coronary angioplasty (PTCA) and one coronary artery was severely injured by PTCA and subsequent beta-irradiation (Brachy group). Animals received 325 mg aspirin daily for 3 months and 75 mg clopidogrel daily for either 28 days [short-term (ST) clopidogrel group] or 3 months [long-term (LT) clopidogrel group]. RESULTS: After 3 months, the number of proliferating cells per cross-section differed significantly between ST and LT in both injury groups (PTCA(ST) 90.2 +/- 10.3 vs. PTCA(LT )19.2 +/- 4.7, P < 0.05; Brachy(ST) 35.8 +/- 8.4 vs. Brachy(LT) 7.5 +/- 2.0, P < 0.05). Similar results were seen for inflammatory cells (CD3(+) cells): PTCA(ST) 23.5 +/- 3.55 vs. PTCA(LT )4.67 +/- 0.92, P < 0.05; Brachy(ST) 83.17 +/- 11.17 vs. Brachy(LT) 20 +/- 4.82, P < 0.05). Long-term administration also reduced the activity of NF-kappaB and AP-1 by 62-64% and 42-58%, respectively. However, the effects of different durations of clopidogrel administration on artery dimensions were not statistically significant. CONCLUSIONS: Regarding inflammation and transcription factor activity at the PCI site, long-term clopidogrel administration is superior to short-term administration, especially in severely injured arteries. Transferring our results to the human situation, patients with more severely diseased arteries may benefit from a prolonged clopidogrel medication after PCI.

Infusion of an antialpha4 integrin antibody is associated with less neoadventitial formation after balloon injury of porcine coronary arteries.

BACKGROUND: The alpha4beta1 (or very late antigen-4 [VLA-4]) integrin is thought to play a role in inflammatory processes, mediating mononuclear leukocyte infiltration. The adventitial response to balloon injury is an important determinant of neointimal formation and arterial remodelling. OBJECTIVES: To determine whether the monoclonal antibody hHP1/2 directed against the human alpha4-integrin subunit decreases neoadventitial formation and subsequent luminal narrowing following balloon injury. DESIGN: Randomized, double-blind, placebo controlled study. SETTING: Tertiary care, Canadian university hospital vascular biology laboratory. ANIMALS AND METHODS: In 16 pigs, two coronary arteries were injured with an oversized balloon, while a third coronary artery was designated as an uninjured control vessel. One hour before balloon injury, 5 mg/kg of hHP1/2 was administered to eight animals, while another eight animals received an infusion of a saline placebo. Animals were killed three and 14 days following balloon injury. MAIN RESULTS: Administration of hHP1/2 resulted in an immediate decrease in circulating monocyte and lymphocyte counts. These parameters returned to normal within three days. There was a decrease in neoadventitial formation 14 days after arterial injury in pigs treated with hHP1/2 compared with controls (2.26+/-0.77 versus 3.42+/-1.01 mm, respectively, P=0.04). There was a loss of lumen area between days 3 (4.33+/-1.09 mm2) and 14 (3.09+/-0.38 mm2, P=0.02) after balloon injury in pigs treated with saline, but not in the pigs treated with hHP1/2. CONCLUSIONS: Administration of an antibody to the alpha4-integrin subunit is associated with less neoadventitial formation and less lumenal narrowing after balloon injury. This novel therapy may play an important role in modulating arterial remodelling and thereby may reduce restenosis following percutaneous coronary interventions in humans.

Time course and importance of neoadventitial formation in arterial remodeling following balloon angioplasty of porcine coronary arteries.

OBJECTIVES: Arterial remodeling has been suggested as the predominant factor in restenosis. However, the time course and morphometric factors that determine whether remodeling occurs remain unclear. We hypothesized that arterial remodeling does not occur in all arteries following balloon injury and is dependent on neoadventitial formation. METHODS: Using single (SI) and double (DI) balloon injury of Yorkshire porcine coronary arteries we examined changes in morphometry 3, 7, 14, 28 days following balloon injury. RESULTS: In both SI and DI arteries, the neoadventitia (NAD) area expanded by day 3 and was the first compartment to increase following injury. In SI arteries lumen area (LA) decreased between day 3 and 14 while in DI arteries, there was significantly less loss in LA. In SI arteries, contracture of the area circumscribed by the external elastic lamina (EEL), which occurred predominantly between day 7 and 14, accounted for 67% of the loss of LA. CONCLUSIONS: Accumulation of NAD appears to be the earliest change in the vessel wall following balloon injury of normal or previously injured arteries and precedes the growth of the I + M (intima and media). The predominant mechanism for lumenal narrowing following single balloon injury of a normal artery is remodeling. In contrast, remodeling does not occur in DI arteries, possibly due to differences in the degree of adventitial fibrosis of normal and injured arteries.

Adventitial angiogenesis early after coronary angioplasty : correlation with arterial remodeling.

The spatial correlation between arterial wall microvessels and the accumulation of atherosclerotic plaque is well documented. The role of these microvessels in the development of primary and restenotic lesions is not known. To investigate the effect of interventional procedures on arterial wall microvessels, we studied the adventitial microvascularity of porcine coronary arteries subjected to angioplasty. Twenty-two juvenile domestic swine were subjected to single or repeated (double) balloon angioplasty of the coronary arteries, with the interval between the first and second injury being 14 days. The number, density, and size of adventitial microvessels were measured 1 hour as well as 3, 7, 14, and 28 days after injury. One hour after single balloon injury, there were very few intact adventitial microvessels. Adventitial microvessel number, microvessel area density, and microvessel size were maximal 3 days after single (SI) and double (DI) injury but subsequently underwent progressive regression. Adventitial endothelial cell replication, as assessed by the incorporation of bromodeoxyuridine, was very low for the majority of arteries. Maximal endothelial cell replication indices were observed 3 days after SI and DI (eg, 12.0+/-3.3%). Early after SI the central arterial lumen area transiently increased, then renarrowed. The lumen area did not change after DI. Arterial remodeling occurred, as the accumulation of intimal and medial mass was correlated with expansion of the external elastic lamina. Adventitial microvessel area density was correlated with central arterial luminal area (R=0.34, P=0.04). The adventitial microvessel area density and the microvessel size index were greater late after DI compared with SI. These data indicate that adventitial angiogenesis occurs within 3 days after balloon injury and that regression of adventitial microvessels after SI corresponds with arterial narrowing. Changes in the adventitial microvasculature may be a component of arterial remodeling after balloon angioplasty.

Arterial wall neovascularization--potential role in atherosclerosis and restenosis.

Neointimal formation and arterial wall remodeling are pivotal causes of luminal narrowing in atherogenesis and restenosis. Arterial remodeling refers to a series of dynamic structural changes that arteries may undergo in response to various stimuli, including changes in blood flow and pressure, and acute injury. The biological mechanisms involved in arterial remodeling are poorly understood and are currently a main target for research. We have recently focused on the role of the arterial wall microcirculation (ie, vasa vasorum) in arterial remodeling after injury. In the past, a correlation between arterial wall neovascularization and the accumulation of arterial plaque has been documented; however, the dynamic role of these microvessels in arterial repair and luminal narrowing has not been examined. The type of arterial injury, the nature of the lesion that develops, and the arterial compartment in which angiogenesis occurs may determine the role of the vasa vasorum in arterial narrowing. In this review, we highlight the data that link arterial wall neovascularization with lesion formation and the process of arterial remodeling.

Arterial expression of the plasminogen activator system early after cardiac transplantation.

OBJECTIVES: Recent studies suggest that alterations in tissue thrombolysis as well as the inward migration of cells may be specific events that contribute to coronary artery narrowing after cardiac transplantation. Plasminogen activators and inhibitors play a central role in governing not only tissue thrombolysis, but also vascular cell migration. The purpose of this study was to examine arterial wall expression of the plasminogen activation system in coronary arteries during graft vascular disease initiation and progression. METHODS: Using in situ hybridization and immunocytochemistry, the expression patterns of uPA and PAI-1 in coronary arteries from cardiac allografts were compared to those of young individuals without disease. RESULTS: Both PAI-1 and uPA were over-expressed early after transplantation and as late as 27 months post grafting. Over-expression of these molecules preceded morphological evidence of graft vascular disease. Of special note was the adventitial expression of uPA and PAI-1 in microvessels and myofibroblasts. In contrast, the expression of uPA and PAI-1 in normal coronary arteries was confined to endothelial cells of the central lumen, as well as low levels of expression in intimal and medial smooth muscle cells. CONCLUSIONS: Despite morphologic similarities between normal and transplant coronary arteries, differences were noted in the vascular expression pattern of uPA and PAI-1. The exact role of these molecules in graft vascular disease requires further study; however, it is intriguing to consider that a local imbalance in the plasminogen system may contribute to arterial wall thrombosis and/or excessive cell migration and the genesis of complex vascular lesions.

Activation of the fibrinolytic system in patients with coronary artery disease and hyperfibrinogenemia.

Elevated fibrinogen levels as well as an impaired activity of the fibrinolytic system are regarded as important cardiovascular risk factors. To elucidate a potential interrelation between fibrinogen as an indicator of a hypercoagulable state and the endogenous fibrinolytic function hemostatic and rheological as well as lipid parameters were determined in 224 consecutive patients, who underwent elective coronary angiography. In the selected study population of 81 men and 19 women with fibrinogen concentration either > or = 3.5 g/l (n = 70) or < or = 2.5 g/l (n = 30) hyperfibrinogenemia was found to be significantly associated with increased concentrations of plasmin-alpha 2-antiplasmin complex [PAP [median (25.-75. percentile)], 534 (361-680) micrograms/l vs. 289 (243-440) micrograms/l; p < 0.001] and tissue plasminogen activator (t-PA) antigen [9 (6-11) micrograms/l vs 8 (5-9) micrograms/l; p < 0.05] while this association was lost in the subgroup of patients with angiographically normal coronary arteries (n = 26). In addition to these findings fibrinogen was significantly correlated with PAP (r = 0.40, p < 0.001; n = 224) and t-PA antigen (r = 0.2, p < 0.01; n = 224) after adjustment for age, diabetes mellitus, lipid parameters and leucocyte counts. It can be argued that elevated fibrinogen levels in patients with coronary artery disease are concomitant with an activation of the fibrinolytic system.

[Mechanism of action of directed coronary atherectomy]. / Wirkungsmechanismen der gerichteten koronaren Atherektomie (DCA).

The mechanism of directional coronary atherectomy (DCA) results from tissue removal and mechanical dilation ("Dotter" effect, balloon inflation). To estimate the concomitant contribution of dilation to lumen enlargement by DCA, we determined the amount of retrieved tissue and the change of luminal volume (computer-based quantitative coronary artery analysis) and compared both. The first 25 patients undergoing DCA in 1992 were enrolled in this study. In all cases DCA was successful (tissue removal, residual stenosis < 50%, absence of major complications). The diameter stenosis was reduced from 68 +/- 11% to 10 +/- 24% (p < 0.001). Calculated luminal volume increased from 1.04 +/- 0.46 mm to 2.9 +/- 0.64 (p < 0.001). The comparative value of the adjacent reference segment was a luminal diameter of 3.23 +/- 0.56 mm. The increase of the calculated luminal volume was 23 +/- 13 mm3. The average weight of removed tissue was 11 +/- 7 mg. The calculated "atherectomy index" (ratio of tissue volume retrieved to change in luminal volume) revealed a significant correlation between both volumina and averaged 0.502 for the whole group. The results show that the mechanical component plays an important part in the mechanism of luminal enlargement in addition to tissue removal and therefore could be a possible explanation for the high rate of restenosis after DCA, which was documented in the recently published big DCA trials.