Nitric oxide influences red blood cell velocity independently of changes in the vascular tone

Nitric oxide (NO) plays a key role in regulation of vascular tone and blood flow. In the microcirculation blood flow is strongly dependent on red blood cells (RBC) deformability. In vitro NO increases RBC deformability. This study hypothesized that NO increases RBC velocity in vivo not only by regulating vascular tone, but also by modifying RBC deformability. The effects of NO on RBC velocity were analysed by intra-vital microscopy in the microcirculation of the chorioallantoic membrane (CAM) of the avian embryo at day 7 post-fertilization, when all vessels lack smooth muscle cells and vascular tone is not affected by NO. It was found that inhibition of enzymatic NO synthesis and NO scavenging decreased intracellular NO levels and avian RBC deformability in vitro. Injection of a NO synthase-inhibitor or a NO scavenger into the microcirculation of the CAM decreased capillary RBC velocity and deformation, while the diameter of the vessels remained constant. The results indicate that scavenging of NO and inhibition of NO synthesis decrease RBC velocity not only by regulating vascular tone but also by decreasing RBC deformability.     

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

Background

Red blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold.

Methods/Findings

Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects.

Conclusion/Significance

This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.     

Nicotinic acid inhibits angiogenesis likely through cytoskeleton remodeling

Angiogenesis is a physiological procedure during which the new blood vessels develop from the pre-existing vessels. Uncontrolled angiogenesis is related to various diseases including cancers. Clinical inhibition of undesired angiogenesis is still under investigation. We utilized nicotinic acid, a family member of the B-vitamin niacin (vitamin B3) that has been used in the prevention and treatment of atherosclerosis or other lipid-metabolic disorders, to treat human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM), and investigated its influence on angiogenesis in vitro and in vivo. We found that nicotinic acid could obviously inhibit HUVEC proliferation induced by vascular endothelial growth factor. Both the in vitro and in vivo assays showed that nicotinic acid could significantly inhibit the process of angiogenesis. To further investigate the mechanism underlying the effect of nicotinic acid on angiogenesis, we found that it might function via regulating the cytoskeleton arrangements, especially the rearranging the structures of F-actin and paxillin. In summary, we discovered that nicotinic acid could obviously inhibit the process of angiogenesis by changing the angiogenesis factor expression levels and inducing the cytoskeleton rearrangement of endothelial cells.     

Effects of nitric oxide on red blood cell deformability

In addition to its known action on vascular smooth muscle, nitric oxide (NO) has been suggested to have cardiovascular effects via regulation of red blood cell (RBC) deformability. The present study was designed to further explore this possibility. Human RBCs in autologous plasma were incubated for 1 h with NO synthase (NOS) inhibitors [N(omega)-nitro-l-arginine methyl ester (l-NAME) and S-methylisothiourea], NO donors [sodium nitroprusside (SNP) and diethylenetriamine (DETA)-NONOate], an NO precursor (l-arginine), soluble guanylate cyclase inhibitors (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and methylene blue), and a potassium channel blocker [triethylammonium (TEA)]. After incubation, RBC deformability at various shear stresses was determined by ektacytometry. Both NOS inhibitors significantly reduced RBC deformability above a threshold concentration, whereas the NO donors increased deformability at optimal concentrations. NO donors, as well as the NO precursor l-arginine and the potassium blocker TEA, were able to reverse the effects of NOS inhibitors. Guanylate cyclase inhibition reduced RBC deformation, with both SNP and DETA-NONOate able to reverse this effect. These results thus indicate the importance of NO as a determinant of RBC mechanical behavior and suggest its regulatory role for normal RBC deformability.     

Protection of erythrocytes from sub-hemolytic mechanical damage by nitric oxide mediated inhibition of potassium leakage

The effects of mechanical stress on red blood cell (RBC) deformability were evaluated by subjecting cells to a uniform fluid shear stress of 120 Pa for 15-120 seconds at 37 degrees C. This level of stress induced significant impairment of RBC deformability as assessed by ektacytometry, with the degree of impairment independent of extracellular calcium concentration. Inhibition of RBC nitric oxide (NO) synthesis by a competitive inhibitor of NO synthases (N-omega-nitro-L-arginine methyl ester, L-NAME) had no effect on deformability after exposure to mechanical stress. The NO donor sodium nitroprusside (SNP) prevented the deterioration of RBC deformability in a dose-dependent manner with 10(-4) M being the most effective concentration. A similar protective effect by the non-selective potassium channel blocker, tetraethylammonium chloride (TEA) suggests that the effect of NO might be mediated by the inhibition of potassium leakage from RBC. These results suggest that NO may prevent mechanical deterioration of RBC exposed to high shear stresses. While RBC are not exposed to such high levels of shear stresses for prolonged periods under normal circulatory conditions, comparable levels of mechanical stress can be encountered under certain situations (i.e., artificial organs, extracorporeal circulation) and may result in subhemolytic damage and hemorheological alterations.     

The role of red blood cell adenylyl cyclase activation in changes of erythrocyte membrane microrheological properties

The ability of red blood cells (RBCs) for the reversible change of their shape under passing through capillaries in microcirculation mainly depends on membrane elasticity of these cells. Phosphorylation of some membrane proteins can result in the changes of microrheological red blood cell properties. Here we show a significant increase in RBC deformability (RBCD) after incubation with isoproterenol (10⁻⁶ M). Red blood cell aggregation (RBCA) decreased under these conditions only slightly. When forskolin (10 μM), an adenylyl cyclase (AC) stimulator, was added to the RBC suspension, RBCD increased significantly (p < 0.05). Some more changes of deformability were found after incubation of RBC with stable penetrating analog of cyclic adenosine phosphate (cAMP), dibutyryl-cAMP, (dB-cAMP, 50 μM) and after phosphodiesterase (PDE) activity inhibition with Vinpocetine, Rolipram, or IBMX. It was found that Gs-proteins inhibitor Clonidine and specific Gi-protein stimulator Mastaparan 7 increased both RBCD and RBCA. On the whole, the data clearly show that the RBC aggregation and deformation changes are related with activation of the different intracellular signaling pathways. We suppose that RBCD increase was mainly associated with activation of the adenylyl-cyclase-cAMP system.     

Moderate Exercise Promotes Human RBC-NOS Activity,NO Production and Deformability through Akt Kinase Pathway

Background

Nitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs.

Methods/Findings

Fifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer¹¹⁷⁷ was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr⁴⁹⁵ were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability.

Conclusion/Significance

This human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.     

Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress

Abstract

The role of peroxiredoxin-2 (PRDX2) in preventing hydrogen peroxide-induced oxidative stress in the red blood cell was investigated by comparing blood from PRDX2 knockout mice with superoxide dismutase-1 (SOD1) knockout and control mice. Loss of PRDX2 increased basal levels of methemoglobin and heme degradation (a marker for oxidative stress), and reduced red blood cell deformability. In vitro incubation under normoxic conditions, both with and without inhibition of catalase, resulted in a lag phase during which negligible heme degradation occurred followed by a more rapid rate of heme degradation in the absence of PRDX2. The appreciable basal increase in heme degradation for PRDX2 knockout mice, together with the lag during in vitro incubation, implies that PRDX2 neutralizes hydrogen peroxide generated in vivo under the transient hypoxic conditions experienced as the cells pass through the microcirculation.     

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22 ± 2 °C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10^?7–10^?4 M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10^?3 M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.     

Rapid Rather than Gradual Weight Reduction Impairs Hemorheological Parameters of Taekwondo Athletes through Reduction in RBC-NOS Activation

Purpose

Rapid weight reduction is part of the pre-competition routine and has been shown to negatively affect psychological and physiological performance of Taekwondo (TKD) athletes. This is caused by a reduction of the body water and an electrolyte imbalance. So far, it is unknown whether weight reduction also affects hemorheological properties and hemorheology-influencing nitric oxide (NO) signaling, important for oxygen supply to the muscles and organs.

Methods

For this purpose, ten male TKD athletes reduced their body weight by 5% within four days (rapid weight reduction, RWR). After a recovery phase, athletes reduced body weight by 5% within four weeks (gradual weight reduction, GWR). Each intervention was preceded by two baseline measurements and followed by a simulated competition. Basal blood parameters (red blood cell (RBC) count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean cellular hemoglobin and mean cellular hemoglobin concentration), RBC-NO synthase activation, RBC nitrite as marker for NO synthesis, RBC deformability and aggregation parameters were determined on a total of eight investigation days.

Results

Basal blood parameters were not affected by the two interventions. In contrast to GWR, RWR decreased activation of RBC-NO synthase, RBC nitrite, respective NO concentration and RBC deformability. Additionally, RWR increased RBC aggregation and disaggregation threshold.

Conclusion

The results point out that a rapid weight reduction negatively affects hemorheological parameters and NO signaling in RBC which might limit performance capacity. Thus, GWR should be preferred to achieve the desired weight prior to a competition to avoid these negative effects.     

Vasomotor control in mice overexpressing human endothelial nitric oxide synthase

Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.     

Erythrocyte deformability is a nitric oxide-mediated factor in decreased capillary density during sepsis

Erythrocyte deformability has been recognized as a determinant of microvascular perfusion. Because nitric oxide (NO) is implicated in the modulation of red blood cell (RBC) deformability and NO levels increase during sepsis, we tested the hypothesis that a NO-mediated decrease in RBC deformability contributes to decreased functional capillary density (CD) in remote organs. With the use of a peritonitis model of sepsis in the rat [cecal ligation and perforation (CLP)] and aminoguanidine (AG) to prevent increases in NO, we measured CD in skeletal muscle (intravital microscopy), mean erythrocyte membrane deformability (; micropipette aspiration), systemic NO production [plasma nitrite/nitrate (NO(x)) chemiluminescence], and NO accumulation in RBC [NO bound to hemoglobin (HbNO) detected by electron paramagnetic resonance spectroscopy]. In untreated CLP animals relative to sham, NO(x) increased 254% (P < 0.05), stopped flow capillaries increased 149% (P < 0.05), and decreased 12.7% (P < 0.05), with a subpopulation (5%) of RBC with deformabilities below the normal range. AG prevented increases in NO(x), accumulation of HbNO, and decreases in both and functional CD. We found no evidence of leukocyte plugging postcapillary venules. Our findings suggest that decreased functional CD during sepsis resulted from a NO-mediated decrease in erythrocyte deformability.     

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.Download video file.^{(53M, mov)}     

Estimation of the microcirculation state in cerebrovascular disorders using laser doppler flowmetry data and hemorheological parameters

The microcirculation state was assessed in the group of patients with ischemic stroke (n = 30) and the control group of healthy individuals (n = 27) using laser Doppler flowmetry and the wavelet analysis of the amplitude-frequency range of microvascular blood flow oscillations combined with absorption spectroscopy. The hemorheological parameters (blood and plasma viscosity, the degree of red blood cell aggregability and deformability) were assessed in both groups, as were their correlations with the microcirculation parameters. Decreased tissue perfusion (by 25%) and specific oxygen consumption (by 21%) were revealed in a cerebrovascular accident. Changes in the tone-forming regulatory mechanisms of microcirculation of vasodilating nature (decreased microvascular tone, activation of the secretory function of endothelium) may be regarded as a compensatory reaction aimed at maintaining the blood supply of organs and tissues in stroke. The blood viscosity increase in patients due to the plasma viscosity increase and increased red blood cell aggregability and their decreased deformability cause the blood flow to slow down and the wall shear stress to increase, which activates the endothelial secretory function and vasodilation of microvessels. Correlation between the rheological parameters and the passive (respiratory and cardiac) rhythm amplitudes was observed in the control group. In patients, the hemorheological parameters were correlated with the characteristics of the active factors of microvascular blood flow modulation (endothelial, neurogenic, and myogenic), which confirms the role of changed blood properties and regulatory tone-forming mechanisms in the maintenance of tissue perfusion in cerbrovascular accidents.     

Nitrite infusion increases cerebral blood flow and decreases mean arterial blood pressure in rats: A role for red cell NO

It has been proposed that the reduction of nitrite by red cells producing NO plays a role in the regulation of vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of red cell NO. The relative magnitude of the effects on MAP and CBF as well as the time dependent changes during nitrite infusion are used to distinguish between the effects on the peripheral circulation and the effects on the cerebral circulation undergoing cerebral autoregulation. The nitrite infusion was found to reverse the 96% increase in MAP and the 13% decrease in CBF produced by L-NAME inhibition of e-NOS. At the same time there was a 20-fold increase in oxygen stable red cell NO. Correlations of the red cell NO for individual rats support a role for red cell nitrite reduction in regulating vascular tone in both the peripheral and the cerebral circulation. Furthermore, data obtained prior to treatment is consistent with a contribution of red cell reduced nitrite in regulating vascular tone even under normal conditions.     

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells

Aim

To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.

Method

RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.

Results

The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine¹¹⁷⁷ phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N⁵-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.

Conclusion

This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

In vivo characterization of tumor and tumor vascular network using multi‐modal imaging approach

We present a multi‐modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)