Clinical Hemorheology and Microcirculation - Volume 58, issue 1

Authors: Dietze, Kathrin | Slosarek, Ilka | Fuhrmann-Selter, Tania | Hopperdietzel, Carsten | Plendl, Johanna | Kaessmeyer, Sabine

Article Type: Research Article

Abstract: Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine …horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis. Show more

Keywords: Equine macrovascular endothelial cells, life cell imaging, angiogenesis assay, microparticles

DOI: 10.3233/CH-141877

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 127-146, 2014

Authors: Roch, Toralf | Akymenko, Oksana | Krüger, Anne | Jung, Friedrich | Ma, Nan | Lendlein, Andreas

Article Type: Research Article

Abstract: The polarization behavior of macrophages determines the clinical outcome after implantation of biomaterials. Formation of classically activated macrophages (CAM) may result in cell fusion to form foreign body giant cells, which induce and support uncontrolled inflammatory responses and can cause undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration. The expression of matrix metalloproteinases (MMP) by the different macrophage subsets might play a crucial role for inflammatory and wound healing processes and may subsequently influence the implant integration. Therefore, it is of importance to characterize the MMP expression pattern …by the different macrophage subsets. This knowledge could support the design of biomaterials in which specific MMP cleavage sites are incorporated allowing a controlled cell-mediated degradation of the material. However, it needs to be considered that the pure expression levels may not correlate with the enzymatic activity of the MMP, which depends on a variety of different parameters such as additional co-factors. For this reason, the differential MMP expression levels and the overall enzymatic activity of in vitro generated human non-polarized macrophages (M0), CAM, and AAM are analyzed in this study. While MMP-1, MMP-3, and MMP-10 showed the highest expression levels in CAM, MMP-12 was most strongly expressed by AAM. Interestingly, although various MMP were expressed at high levels in CAM, the enzymatic MMP activity was increased in supernatants of AAM cultures. The data presented here illustrate the importance to combine the measurement of MMP expression levels with the analysis of the enzymatic activity. The observed MMP-12 expression in combination with the higher enzymatic activity detected in AAM supernatants might motivate the design of biomaterials, whose structure could be modified by MMP-12 catalyzed reactions leading to interactive polymers. Show more

Keywords: Biomaterials, macrophage polarization, matrix metalloproteinase

DOI: 10.3233/CH-141885

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 147-158, 2014

Authors: Braune, S. | Walter, M. | Schulze, F. | Lendlein, A. | Jung, F.

Article Type: Research Article

Abstract: For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and …twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5′-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL – 40 fL) decreased over time. Immediately after blood collection, no debris or platelet aggregates could be visualized microscopically. After four hours, first debris and very small aggregates occurred. After 24 hours, platelet aggregates and also debris progressively increased. In accordance to this, the CASY system revealed an increase of platelet aggregates (up to 90 μm diameter) with increasing storage time. The percentage of CD62P positive platelets and PF4 increased significantly with storage time in resting PRP. When soluble ADP was added to stored PRP samples, the number of activatable platelets decreased significantly over storage time. The present study reveals the importance of a consequent standardization in the preparation of WB and PRP. Platelet morphology and function, particularly platelet reactivity to adherent or soluble agonists in their surrounding milieu, changed rapidly outside the vascular system. This knowledge is of crucial interest, particularly in the field of biomaterial development for cardiovascular applications, and may help to define common standards in the in vitro hemocompatibility testing of biomaterials. Show more

Keywords: Platelet, platelet function, platelet rich plasma, whole blood, platelet aging, platelet storage, hemocompatibility, biomaterials

DOI: 10.3233/CH-141876

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 159-170, 2014

Authors: Gerk, U. | Krüger, A. | Franke, R.P. | Jung, F.

Article Type: Research Article

Abstract: Radiographic contrast media can lead to drastic changes of the morphology of erythrocytes. The change of the erythrocyte morphology is associated with a decreased deformability possibly resulting from distinctions in the loss of constituents of the membrane cytoskeleton. However, it is unclear whether there is an intravascular hemolysis as a consequence of the disintegration of the erythrocyte membrane. The results of this study showed, that free haemoglobin increased from 16.8 ± 10.0 mg/dl to 21.6 ± 12.6 mg/dl after Iopromide application (p = 0.240), while it slightly decreased from 20.5 ± 10.3 mg/dl to 19.5 ± 12.2 mg/dl after Iodixanol …application (p = 0.547). The slight decrease of free haemoglobin after application of Iodixanol differed significantly compared to the increase of free haemoglobin after Iopromide application (p < 0.05). This different response is thought to give evidence to the assumption that the erythrocyte membrane integrity was compromised leading to the release of free haemoglobin as an indicator of hemolysis as well. Show more

Keywords: Radiographic contrast media, Iopromide, Iodixanol, hemolysis, free haemoglobin

DOI: 10.3233/CH-141879

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 171-174, 2014

Authors: Gori, Tommaso | Münzel, Thomas

Article Type: Research Article

Abstract: Coronary artery stenting is associated with endothelial dysfunction, probably caused by the mechanical injury as well as by the tissue reactions triggered by the metal, the polymer and the drug eluted by the stent. The clinical relevance, and the implications, of this dysfunction are still unclear. We revise the iterature on this topic and provide the first evidence regarding endothelial dysfunction after implantation of bioresorbable scaffolds.

Keywords: Endothelium/vascular type/nitric oxide, pathophysiology, coronary circulation

DOI: 10.3233/CH-141880

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 175-181, 2014

Authors: Teusch, V.I. | Wohlgemuth, W.A. | Piehler, A.P. | Jung, E.M.

Article Type: Research Article

Abstract: AIM: Aim of our pilot study was the application of a contrast-enhanced color-coded ultrasound perfusion analysis in patients with vascular malformations to quantify microcirculatory alterations. MATERIAL AND METHODS: 28 patients (16 female, 12 male, mean age 24.9 years) with high flow (n = 6) or slow-flow (n = 22) malformations were analyzed before intervention. An experienced examiner performed a color-coded Doppler sonography (CCDS) and a Power Doppler as well as a contrast-enhanced ultrasound after intravenous bolus injection of 1 – 2.4 ml of a second-generation ultrasound contrast medium (SonoVue® , Bracco, Milan). The contrast-enhanced examination was documented as a cine …sequence over 60 s. The quantitative analysis based on color-coded contrast-enhanced ultrasound (CEUS) images included percentage peak enhancement (%peak), time to peak (TTP), area under the curve (AUC), and mean transit time (MTT). RESULTS: No side effects occurred after intravenous contrast injection. The mean %peak in arteriovenous malformations was almost twice as high as in slow-flow-malformations. The area under the curve was 4 times higher in arteriovenous malformations compared to the mean value of other malformations. The mean transit time was 1.4 times higher in high-flow-malformations compared to slow-flow-malformations. There was no difference regarding the time to peak between the different malformation types. The comparison between all vascular malformation and surrounding tissue showed statistically significant differences for all analyzed data (%peak, TTP, AUC, MTT; p < 0.01). High-flow and slow-flow vascular malformations had statistically significant differences in %peak (p < 0.01), AUC analysis (p < 0.01), and MTT (p < 0.05). CONCLUSION: Color-coded perfusion analysis of CEUS seems to be a promising technique for the dynamic assessment of microvasculature in vascular malformations. Show more

Keywords: Contrast-enhanced ultrasound (CEUS), vascular malformations, color-coded perfusion analysis, microcirculation

DOI: 10.3233/CH-141878

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 183-193, 2014

Authors: Trottmann, M. | Marcon, J. | D'Anastasi, M. | Karl, A. | Stief, C.G. | Reiser, M. | Clevert, D.A.

Article Type: Research Article

Abstract: PURPOSE: Virtual touch tissue imaging quantification (VTIQ) is a newly developed technique for the sonographic quantification of tissue elasticity. It has been used in the assessment of breast lesions. The purpose of this study was to determine the diagnostic performance of VTIQ in indeterminate testicular lesions. METHODS: Twenty patients with known testicular pathology underwent conventional B-mode sonography with additional VTIQ of the testicular lesions using a Siemens Acuson S2000™ and S3000™ (Siemens Medical Solutions, Mountain View, CA, USA) system. Tissue mechanical properties were analysed in the VTIQ examination. The pathologic diagnosis was established after surgery or in the follow-up examination …for suspected benign lesions. RESULTS: Over 36 months, 22 focal testicular lesions (median lesion size, 18 mm; range, 4–36 mm in 20 patients (median age, 43 years; range, 22–81 years) were examined. Lesions were hyperechoic (n = 1), hypoechoic (n = 14), isoechoic (n = 1), of mixed echogenicity (n = 3) or anechoic (n = 3). Histological examination showed one benign lesion (6.25%) with a mean size of 7 mm and 15 malignant lesions (93.75%) with a mean size of 20 mm. Mean shear wave velocity for normal testicular tissue was 1.17 m/s. No shear wave velocity could be measured in cystic lesions. The rest of the benign lesions showed a mean shear wave velocity of 2.37 m/s. The value of the shear wave velocity in germ cell tumours showed a mean shear wave velocity of 1.94 m/s and for seminoma it showed a mean shear wave velocity of 2.42 m/s. CONCLUSIONS: VTIQ is a reliable new method for measuring qualitative and quantitative stiffness of testicular lesions and tissue. The qualitative shear-wave elastography features were highly reproducible and showed good diagnostic performance in unclear testicular lesions. The VTIQ technique is also useful in assessing small testicular nodules and pseudolesions. Show more

Keywords: Elastography, shear wave imaging, seminoma, germ cell tumour, testis

DOI: 10.3233/CH-141904

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 195-209, 2014

Authors: George, S. | Georgi, M. | Roggenbuck, D. | Conrad, K. | Küpper, J.-H.

Article Type: Research Article

Abstract: Many autoimmune diseases are characterized by autoantibodies directed against cell membrane proteins. We were intrigued to develop a strategy for targeting individual cell membrane proteins to various subcellular compartments as a prerequisite for their simultaneous immunofluorescence detection. We first employed GFP and RFP reporters that were equipped with defined intracellular localization signals. Expressing these protein reporters in HEp-2 cells we found by using fluorescence microscopy that protein localization in cytoplasm or at mitochondria can be clearly discriminated from localization at Golgi, ER or lysosomes. We then tested for muscle-specific kinase, a relevant cell membrane autoantigen in Myasthenia gravis, and NMDA …receptor which is relevant for autoimmune encephalitis, whether these autoantigens can be localized to the same intracellular compartments. To this end, we successfully targeted muscle-specific kinase to Golgi apparatus, mitochondria and cytoplasm. We found that its Golgi localization can be clearly distinguished from its natural cell membrane localization. The same we found for Golgi-localized NMDA receptor 1. Interestingly, cell membrane proteins kept at the Golgi system accumulated in higher amounts than their wild-type counterparts. The obtained results are the basis for the further development of multiplex assays for the immunofluorescence diagnostics of Myasthenia gravis and autoimmune encephalitis. Show more

Keywords: AKLIDES, autoimmune diseases, encephalitis, immunofluorescence, intracellular trafficking, multiplex diagnostics, Myasthenia gravis

DOI: 10.3233/CH-141897

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 211-228, 2014

Authors: Krüger, Anne | Mayer, Anke | Roch, Toralf | Schulz, Christian | Lendlein, Andreas | Jung, Friedrich

Article Type: Research Article

Abstract: Angiogenically stimulated alternative monocytes (aMO2) could be established as cellular release system accelerating the endothelialization of polymers rendering their surfaces hemocompatibility in a short-term study. However, for their clinical application it is essential that aMO2 do not switch back to the MO1 state sustaining their capability as cellular release system over an extended period of time. We explored whether aMO2 can maintain their differentiation state over 21 days in a mono- and in a co-culture with HUVEC. In comparison, the influence of recombinant VEGF-A165 on the endothelialization of biomaterials was assessed including endothelial cell (HUVEC) density, organisation of the …endothelial cytoskeleton, cytokine secretion profile and release of prostacyclin, thromboxane A2 and matrix metalloproteinases. In mono-culture aMO2 secreted high amounts of VEGF and other growth factors/cytokines. Co-cultured with HUVEC, aMO2 accelerated the formation of a confluent HUVEC monolayer. Furthermore, no pro-inflammatory cytokines were found, neither in aMO2-mono, nor in co-cultures with HUVEC indicating that the majority of the aMO2 remained stable in their aMO2 state during the 21 days of cultivation. In contrast, the addition of recombinant VEGF-A165 instead of the co-culture with aMO2 resulted in the formation of stress fibres, dissociated marginal filament bands, and a detachment of HUVEC. In addition, the profile of bioactive agents of HUVEC (e.g. prostacyclin, thromboxane A2, matrix metalloproteinases, IFN-γ and TNF-α) was influenced by the VEGF-A165 treatment inducing the detachment of HUVEC. In conclusion, in co-culture with HUVEC aMO2 remained stable in their type 2 state over 21 days confirming the suitability of aMO2 as biological release system for the endothelialization of biomaterial surfaces with constant release of angiogenic factors but without secretion of pro-inflammatory cytokines over three weeks. Therefore, this endothelialization approach seems to be appropriate to improve the hemocompatibility of cardiovascular implant materials in vitro, and proved to be superior to the use of recombinant VEGF-A165 . Show more

Keywords: Angiogenically stimulated alternative monocytes, endothelialization, biomaterials, hemocompatible surface

DOI: 10.3233/CH-141875

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 229-240, 2014

Authors: Manz, Patrick | Cadeddu, Ron-Patrick | Wilk, Matthias | Fritz, Birgit | Haas, Rainer | Wenzel, Folker

Article Type: Research Article

Abstract: INTRODUCTION: Softeners like phthalate esters are ubiquitous in the environment and have been detected in transfusion bags, though there is only a limited amount of studies on the effect of phthalates on blood cells. This study seeks to determine effects on cell migration of human promyelocytic leukemia cells (HL-60) incubated with di(2-ethylhexyl)phthalate (DEHP) at concentrations found in blood bags. MATERIAL AND METHODS: HL-60 cells were incubated with DEHP concentrations ranging from 0.1 μg/ml to 1000 μg/ml, diluted in DMSO, over 24 h, 48 h, and 72 h. Migration rate was analyzed along an SDF-1α gradient using Transwell migration inserts. RESULTS: …Of the applied concentrations 100 μg/ml, 250 μg/ml, 500 μg/ml, and 1000 μg/ml showed a significant decrease in migration rates relative to DMSO control at all measuring points (p < 0.05), with relative migration rates between 37.87 % for 100 μg/ml and 25.34 % for 1000 μg/ml relative to DMSO after 24 h of stimulation and 19.73 % for 100 μg/ml and 14.69 % for 1000 μg/ml respectively after 72 h of incubation. CONCLUSION: Our results indicate HL-60 to be a suitable in vitro model for examining effects of DEHP on the migration of blood and nucleated cells at concentrations found in blood bags. Show more

Keywords: Phthalates, DEHP, MEHP, HL-60 promyelocytes, vitality, migration, apoptosis

DOI: 10.3233/CH-141903

Citation: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 241-246, 2014