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Franke R.P.,University of Ulm | Scharnweber T.,Karlsruhe Institute of Technology | Fuhrmann R.,University of Ulm | Mrowietz C.,Institute for Heart and Circulation Research | And 3 more authors.
Clinical Hemorheology and Microcirculation | Year: 2014

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton-as it appeared in cells suspended in autologous plasma-was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media. © 2014-IOS Press and the authors.


Franke R.P.,University of Ulm | Scharnweber T.,Karlsruhe Institute of Technology | Fuhrmann R.,University of Ulm | Kruger A.,Helmholtz Center Geesthacht | And 3 more authors.
Clinical Hemorheology and Microcirculation | Year: 2013

A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to β-actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time. © 2013 - IOS Press and the authors. All rights reserved.


Franke R.P.,University of Ulm | Scharnweber T.,Karlsruhe Institute of Technology | Fuhrmann R.,University of Ulm | Mrowietz C.,Institute for Heart and Circulation Research | Jung F.,Helmholtz Center Geesthacht
Clinical Hemorheology and Microcirculation | Year: 2013

Red blood cells demonstrate a unique ability for repeated large deformation. Under the influence of a variety of agents, shapes other than the discocyte-e.g. stomatocytes or echinocytes - can be observed. Some radiographic agents induce shape changes from discocytic to echinocytic cells. Especially the echinocyte formation is associated with a rigidification of the cells bearing the risk of a hindered capillary passage of the echinocytes. The mechanisms leading to the formation of echinocytes are not well understood assuming that the membrane cytoskeleton is a key player. That is why this examination was focused on the participation of components of the membrane cytoskeleton in the formation of echinocytes and the protrusions accompanying the formation of echinocytes. Two radiographic contrast media approved for intra-arterial application were used to study echinocyte formation (Iodixanol320; Iopromide370). In the in vitro study serious changes in the membrane cytoskeleton were only found in those erythrocytes incubated in plasma supplemented with Iopromide370 (30%v/v). The shape of the spectrin net was completely altered; from the more homogeneous distribution - typical of cells in autologous plasma and also of cells in plasma supplemented with Iodixanol320-to a distribution of spectrin concentrated in the membrane-near regions with the appearance of spectrin-actin co-localization. Co-localized spectrin with actin was also found around the membranous roots of protrusions which resemble exocytotic processes. In central parts of the cells there was a pronounced dissociation of spectrin and actin; green coloured condensed spectrin bundles originating from the cell membrane reached up to the root of the protrusions. Separate from this there were also fine long actin fibres passing through the whole cell. The incubation of erythrocytes in plasma supplemented with Iopromide370 induced rounded bubble-like protrusions from the cell membrane containing almost completely long bundles of actin fibres. The examination confirmed earlier studies showing that some radiographic contrast media are able to induce echinocyte formation. Furthermore, subcellular mechanisms were revealed explaining the different effects of Iodixanol in comparison to Iopromide. © 2013 - IOS Press and the authors. All rights reserved.


Franke R.-P.,University of Ulm | Scharnweber T.,Karlsruhe Institute of Technology | Fuhrmann R.,University of Ulm | Wenzel F.,Cell Therapeutics | And 3 more authors.
PLoS ONE | Year: 2014

The membrane of red blood cells consists of a phospholipid bilayer with embedded membrane proteins and is associated on the cytoplasmatic side with a network of proteins, the membrane skeleton. Band3 has an important role as centre of the functional complexes e.g. gas exchange complex and as element of attachment for the membrane skeleton maintaining membrane stability and flexibility. Up to now it is unclear if band3 is involved in the morphology change of red blood cells after contact with radiographic contrast media. The study revealed for the first time that Iopromide induced markedly more severe alterations of the membrane skeleton compared to Iodixanol whose effects were similar to erythrocytes suspended in autologous plasma. A remarkable clustering of band3 was found associated with an accumulation of band3 in spicules and also a sequestration of band3 to the extracellular space. This was evidently accompanied by a gross reduction of functional band3 complexes combined with a dissociation of spectrin from band3 leading to a loss of homogeneity of the spectrin network. It could be demonstrated for the first time that RCM not only induced echinocyte formation but also exocytosis of particles at least coated with band3. © 2014 Franke et al.


PubMed | Karlsruhe Institute of Technology, Cell Therapeutics, Helmholtz Center Geesthacht, University of Ulm and Institute for Heart and Circulation Research
Type: Journal Article | Journal: Clinical hemorheology and microcirculation | Year: 2014

A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to -actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time.


Posch M.G.,Deutsches Herzzentrum Berlin | Posch M.G.,Charité - Medical University of Berlin | Waldmuller S.,Institute for Heart and Circulation Research | Muller M.,Institute for Heart and Circulation Research | And 13 more authors.
PLoS ONE | Year: 2011

Secundum-type atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD) and are associated with a familial risk. Mutations in transcription factors represent a genetic source for ASDII. Yet, little is known about the role of mutations in sarcomeric genes in ASDII etiology. To assess the role of sarcomeric genes in patients with inherited ASDII, we analyzed 13 sarcomeric genes (MYH7, MYBPC3, TNNT2, TCAP, TNNI3, MYH6, TPM1, MYL2, CSRP3, ACTC1, MYL3, TNNC1, and TTN kinase region) in 31 patients with familial ASDII using array-based resequencing. Genotyping of family relatives and control subjects as well as structural and homology analyses were used to evaluate the pathogenic impact of novel non-synonymous gene variants. Three novel missense mutations were found in the MYH6 gene encoding alpha-myosin heavy chain (R17H, C539R, and K543R). These mutations co-segregated with CHD in the families and were absent in 370 control alleles. Interestingly, all three MYH6 mutations are located in a highly conserved region of the alpha-myosin motor domain, which is involved in myosin-actin interaction. In addition, the cardiomyopathy related MYH6-A1004S and the MYBPC3-A833T mutations were also found in one and two unrelated subjects with ASDII, respectively. No mutations were found in the 11 other sarcomeric genes analyzed. The study indicates that sarcomeric gene mutations may represent a so far underestimated genetic source for familial recurrence of ASDII. In particular, perturbations in the MYH6 head domain seem to play a major role in the genetic origin of familial ASDII. © 2011 Posch et al.


Knaut M.,University Hospital | Matschke K.,University Hospital | Plotze K.,University Hospital | Steinmann C.,University Hospital | And 2 more authors.
Clinical Hemorheology and Microcirculation | Year: 2012

Heart failure patients are clinically characterized by extreme cardiomegaly, breathlessness, fluid retention and an early onset of fatigue. Studies have shown generalized restricted blood flow in those patients. Furthermore animal experiments proved an impaired blood flow and a diminished oxygen supply of the skeletal muscle in animals with chronic heart failure. Patients with chronic heart failure are limited to the extent of their ability to regulate their arterial pressure, especially in physical activity. It is however unclear in what way restriction of blood flow in the main arteries correlates with those in capillaries and to what extent. In this study it was examined the depth of capillary circulatory restriction as well as the disregulation of oxygen partial pressure in skeletal muscle in rest and stress conditions, in patients with terminal heart failure. © 2012 - IOS Press and the authors. All rights reserved.

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