Clinical Hemorheology and Microcirculation - Volume 46, issue 2-3

Authors: Bahramsoltani, Mahtab | Spiegelaere, Ward De | Janczyk, Pawel | Hiebl, Bernhard | Cornillie, Pieter | Plendl, Johanna

Article Type: Research Article

Abstract: Angiogenic therapy is considered to be a promising tool for treatment of ischemic diseases. Many in vivo and in vitro assays have been developed to identify potential proangiogenic drugs and to investigate their mode of action. However, until now no validated system exists that would allow quantitation of angiogenesis in vitro in only one assay. Here, a previously established all-in-one in vitro assay based on staging of the angiogenic cascade was validated by quantitation of the effects of the known proangiogenic factors VEGF-A and FGF-2. Both growth factors were applied separately or in combination to human endothelial cell cultures derived …from the heart and the foreskin, and angiogenesis was quantitated over 30 days of culture. Additionally, gene expression of VEGFR-1, VEGFR-2 and FGFR-1 at 3, 10, 20 or 40 days of cultivation was quantitated by RT-qPCR. In both cultures, VEGF-A as well as FGF-2 induced a run through all defined stages of angiogenesis in vitro. Application of VEGF-A only led to formation of irregular globular endothelial structures, while FGF-2 resulted in development of regular capillary-like structures. Quantitation of the angiogenic effects of VEGF-A and transcripts of VEGFR-1 and VEGFR-2 showed that a high VEGFR-1/VEGFR-2 ratio evoked deceleration of angiogenesis. Show more

Keywords: Angiogenesis, quantitative in vitro assay, VEGF-A, FGF-2

DOI: 10.3233/CH-2010-1345

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 189-202, 2010

Authors: Urschel, Katharina | Wörner, Anja | Daniel, Werner G. | Garlichs, Christoph D. | Cicha, Iwona

Article Type: Research Article

Abstract: Background: Atherosclerotic plaques develop at arterial regions subjected to non-uniform shear stress, and are initiated by increased leukocyte-endothelial interactions. In this study, we investigated the effects of distinct shear stress patterns on endothelial recruitment of monocytic cells. Methods: Human umbilical vein endothelial cells (ECs) were exposed to laminar or non-uniform shear stress in bifurcating flow-through slides, followed by 2 h stimulation with TNF-α. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells for 1 h. Endothelial protein expression was determined by immunofluorescence. Results: Exposure to non-uniform shear stress and TNF-α lead to progressive induction of adhesion …molecules and increase in monocytic cell adhesion observed over 0.5–3 h. To investigate the relative role of the shear stress patterns in monocytic cell recruitment, ECs were exposed to reduced levels of shear stress, resulting in a reduced gradient steepness in the non-uniform shear stress regions. Lowering the shear stress from 10 to 5 and 2 dyne/cm2 resulted in increased monocytic cell adhesion under laminar shear stress. However, in these conditions, adherent monocytic cells under non-uniform shear stress were strongly reduced. Moreover, in the region exposed to shear stress gradient parallel to flow direction, monocytic cell adhesion was significantly lower than in the region of non-uniform shear stress, characterized by transversal gradient. Conclusion: Exposure to non-uniform shear stress results in progressive induction of adhesion molecules and monocytic cell recruitment in response to circulating TNF-α. Enhanced monocytic cell recruitment at bifurcations is affected not only by the magnitude and steepness of shear stress gradient, but also by its direction in relation to the flow. Show more

Keywords: Shear stress, monocyte adhesion, adhesion molecules, endothelial cell, bifurcations

DOI: 10.3233/CH-2010-1346

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 203-210, 2010

Authors: Hiebl, B. | Bog, S. | Mrowietz, C. | Jünger, M. | Jung, F. | Lendlein, A. | Franke, R.-P.

Article Type: Research Article

Abstract: Monocytes/macrophages are known to exhibit pro-angiogenic activities after VEGF stimulation. Recently, it was shown that VEGF stimulated macrophages can support growth of microvascular endothelial cells from the lung when both cell types were cocultured using a cell ratio of 1:1. However, endothelial cells can have different phenotypic characteristics and metabolism depending on the originating vascular bed and tissues, and only few data have been published regarding the regiospecific sensitivity of microvascular endothelial cells for angiogenic stimuli. Reports about differences in the microvascular bed of the lung and the skin motivated to investigate angiogenic effects of VEGF stimulated macrophages (mΦa) on …the doubling time and the cell growth behaviour of skin derived microvascular endothelial cells (HMVEC/S). During the study period of 60 days, mΦa supported growth and proliferation of the HMVEC/S, when mΦa and HMVEC/S were cocultured at a ratio of 0.5:1. However, these effects were not seen in a 1:1 coculture. This result indicates that there is a positive correlation between the pro-angiogenic effects of mΦa and the number of endothelial cells in the direct neighbourhood of the mΦa and also suggests a different sensitivity of microvascular endothelial cells to angiogenic stimuli depending on the tissue from which they were isolated. Show more

Keywords: Monocyte, microvascular endothelial cell, coculture, VEGF, angiogenesis

DOI: 10.3233/CH-2010-1347

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 211-216, 2010

Authors: Mayer, A. | Lee, S. | Jung, F. | Grütz, G. | Lendlein, A. | Hiebl, B.

Article Type: Research Article

Abstract: The establishment of a stable endothelial layer on a biomaterial suture is a well known strategy to achieve hemocompatibility. The endothelialisation is supported by factors as e.g. vascular endothelial growth factor (VEGF), which can be secreted by monocytes/macrophages (mo/mΦ) in an angiogenic milieu. In order to avoid detrimental inflammation triggered by these mo/mΦ, we established a protocol for the generation of alternatively activated macrophages and studied their response towards VEGF-A165 . We could generate sufficient amounts of these CD14+ CD163+ IL-10+ mo/mΦ from buffy coats(8.6 ± 4.7 × 105 cells/mlbuffy coat). Furthermore, we achieved a VEGF-A165 …secretion in the nanogram range. The VEGF-A165 secretion increased 2.1-fold within 14 days from 7.6 ± 2.2 to 16.1 ± 2.5ng/ml when the cells were grown with a VEGF-A165 supplemented (10ng/ml) cell culture medium. Within this time period the secretion levels of other pro-angiogenic growth factors (bFGF, PDGF-BB) and immunomodulatory cytokines (IL-10, IL-12, IL-1ra, TNFα, IFNγ) reached only the picogram range. These results suggest that angiogenically stimulated CD14+ CD163+ IL-10+ mo/mΦ might be useful as a cellular cytokine delivery system supporting endothelialisation of biomaterials without inducing pro-inflammatory effects. Show more

Keywords: Monocytes, CD14, CD163, IL-10, VEGF, cytokine delivery

DOI: 10.3233/CH-2010-1348

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 217-223, 2010

Authors: Franke, R.P. | Minkow, A. | Hiebl, B. | Fuhrmann, R. | Mrowietz, C. | Jung, F.

Article Type: Research Article

Abstract: The incubation of erythrocytes (RBC) or endothelial cells (HUVEC) in radiographic contrast media (RCM) could induce morphological alterations of or at the cell membranes, e.g. the generation of echinocytes or the formation of stress fibres coinciding with a massive buckling of HUVEC into the vascular lumen, as was demonstrated in several examinations in the recent years. The apposition or embedding of RCM at or in the cell membranes was discussed as possible causative mechanisms because the embedding of molecules into the internal leaflet of the cell membrane bilayer is expected to bulge the cell membrane to the outside, thus inducing …e.g. the generation of echinocytes. The examination presented here is based therefore on high resolution scanning electron microscopy (SEM) analyses if iodine as marker element of RCM molecules can be found near the inside of or in RBC membranes (co-localisation study). Morphological analyses exploited secondary electron images (SE) while the analysis of elements exploited either back scattered electrons (BSE) or energy dispersive X-ray analysis (EDX) or the areal display of elements in high lateral resolution in the Bit-map modus. Even at the highest convenient magnification (1:40,000) it was impossible to detect RBC membrane associated iodine (I) after RBC incubation in RCM (Iodixanol, Iopromide) in vitro. Neither in the birds view on the samples nor looking from the side on the freeze fractured samples carrying the RBC was it possible to detect either the signal cohorts typical of I in the sum spectra or the main Lα1-peak in trace analysis. Show more

Keywords: Radiographic contrast media, Iodixanol, Iopromid, echinocyte formation

DOI: 10.3233/CH-2010-1349

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 225-232, 2010

Authors: Hiebl, B. | Mrowietz, C. | Goers, J. | Bahramsoltani, M. | Plendl, J. | Kratz, K. | Lendlein, A. | Jung, F.

Article Type: Research Article

Abstract: Multiblock copolymers with shape-memory capability attracted tremendous interest as promising candidate materials for smart, degradable implants. In the present study the hen's egg-chorioallantoic membrane test (HET-CAM test) was used to investigate the angiogenic properties of a thermoplastic, biodegradable multiblock copolymer PDC composed of poly(p-dioxanone) hard segments (PPDO) and crystallizable poly(ε-caprolactone) switching segments (PCL), whereby PPDO and PCL homopolymers were investigated as controls. According to our HET-CAM test data, only PDC induced significant microvessel attraction and formation in the contact area of the test specimen after 48hours of incubation showing newly formed blood vessels along the outer edge of the material. …In contrast, no newly formed blood vessels were observed around the PPDO or PCL specimen after the same incubation period. These in vivo results indicate that the multiblock copolymer PDC possibly possesses an angiogenic effect and it can induce blood vessel formation in its direct vicinity when it is implanted in vivo. Show more

Keywords: Angiogenesis, resorbable biomaterials, HET-CAM test, multiblock copolymer

DOI: 10.3233/CH-2010-1350

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 233-238, 2010

Authors: Braune, S. | Lange, M. | Richau, K. | Lützow, K. | Weigel, T. | Jung, F. | Lendlein, A.

Article Type: Research Article

Abstract: The processing of polymers for blood contacting devices can have a major influence on surface properties. In this study, we fabricated poly(ether imide) (PEI) membranes and films to investigate the effects of the processing on physicochemical surface properties by atomic force microscopy (AFM), scanning electron microscopy, contact angle as well as zeta potential measurements. A static platelet adhesion test was performed to analyze the thrombogenicity of both devices. While contact angle measurements showed similar levels of hydrophobicity and zeta potential values were equivalent, mean surface roughness as well as surface energies in the dispersive part were found to be increased …for the PEI membrane. The static platelet adhesion test showed a significantly decreased number of adherent platelets per surface area on the PEI film (178.98 ± 102.70/45000 μm2 ) compared to the PEI membrane (504 ± 314.27/45000μm2 ) and, consequently, revealed evidence for higher thrombogenicity of the PEI membrane. This study shows that processing can have a significant effect on platelet adhesion to biomaterials, even though, molar weight was identical. Thrombogenicity of polymer-based cardiovascular devices, therefore, have to be evaluated at the final product level, following the entire processing procedure. Show more

DOI: 10.3233/CH-2010-1351

Citation: Clinical Hemorheology and Microcirculation, vol. 46, no. 2-3, pp. 239-250, 2010