Biophysical Properties and Motility of Human Mature Dendritic Cells Deteriorated by Vascular Endothelial Growth Factor through Cytoskeleton Remodeling.

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role in the initiation, regulation, and maintenance of the immune responses. Vascular endothelial growth factor (VEGF) is one of the important cytokines in the tumor microenvironment (TME) and can inhibit the differentiation and functional maturation of DCs. To elucidate the potential mechanisms of DC dysfunction induced by VEGF, the effects of VEGF on the biophysical characteristics and motility of human mature DCs (mDCs) were investigated. The results showed that VEGF had a negative influence on the biophysical properties, including electrophoretic mobility, osmotic fragility, viscoelasticity, and transmigration. Further cytoskeleton structure analysis by confocal microscope and gene expression profile analyses by gene microarray and real-time PCR indicated that the abnormal remodeling of F-actin cytoskeleton may be the main reason for the deterioration of biophysical properties, motility, and stimulatory capability of VEGF-treated mDCs. This is significant for understanding the biological behavior of DCs and the immune escape mechanism of tumors. Simultaneously, the therapeutic efficacies may be improved by blocking the signaling pathway of VEGF in an appropriate manner before the deployment of DC-based vaccinations against tumors.

The biomechanical alterations in the CD14+ monocytes of patients with living donor renal transplantation.

Living-donor renal transplantation is an ideal treatment for patients with end stage renal disease because it affords earlier transplantation and better graft for long term survival. CD14+ monocytes are the predominant inflammatory cells in renal allograft intimal arteritis. The biomechanical alterations in CD14+ monocytes would affect the function of graft. The aim of the present study was to explore the changes in the biorheological properties of CD14+ monocytes before and after the living donor renal transplantation. We found that the viscoelastic properties of CD14+ monocytes were greatly decreased after renal transplantation. Confocal microscopy showed that the F-actin content was increased when the oral immunosuppressive agents started. We also found that two cytoskeletal regulatory proteins, cofilin1 and profilin1, changed. Our results suggest that the immunosuppressive agents could significantly change the biorheological characteristics of the CD14+ mononuclear cells and the biomechanical changes may greatly affects their function, which would play a critical role to gain longer immune-tolerance stage.

Investigations of the functional states of dendritic cells under different conditioned microenvironments by Fourier transformed infrared spectroscopy.

BACKGROUND: Dendritic cells are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. The dendritic cell-based vaccination against cancer has been clinically achieved promising successes. But there are still many challenges in its clinical application, especially for how to identify the functional states. METHODS: The CD14+ monocytes were isolated from human peripheral blood after plastic adherence and purified to approximately 98% with cocktail immunomagnetic beads. The immature dendritic cells and mature dendritic cells were induced by traditional protocols. The resulting dendritic cells were cocultured with normal cells and cancer cells. The functional state of dendritic cells including immature dendritic cells (imDCs) and mature dendritic cells (mDCs) under different conditioned microenvironments were investigated by Fourier transformed infrared spectroscopy (FTIR) and molecular biological methods. RESULTS: The results of Fourier transformed infrared spectroscopy showed that the gene transcription activity and energy states of dendritic cells were specifically suppressed by tumor cells (P < 0.05 or 0.01). The expression levels of NF-kappa B (NF-κB) in dendritic cells were also specifically inhibited by tumor-derived factors (P < 0.05 or 0.01). Moreover, the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were closely correlated with the expression levels of NF-κB (R2:0.69 and R2:0.81, respectively). CONCLUSION: Our results confirmed that the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were positively correlated with the expression levels of NF-κB, suggesting that Fourier transformed infrared spectroscopy technology could be clinically applied to identify the functional states of dendritic cell when performing dendritic cell-based vaccination. It's significant for the simplification and standardization of dendritic cell-based vaccination clinical preparation protocols.

Transforming growth factor-ß1 deteriorates microrheological characteristics and motility of mature dendritic cells in concentration-dependent fashion.

Dendritic cells (DCs) are potent and specialized antigen-presenting cells that play a crucial role in initiating and amplifying both the innate and adaptive immune responses. Tumor cells can escape from immune attack by secreting suppressive cytokines which solely or cooperatively impair the immune function and microrheological properties of DCs. However, the underlying mechanisms are not fully defined. Transforming growth factor-ß1 (TGF-ß1) has been identified as a major cytokine in the tumor microenvironment. To determine the effects of TGF-ß1 on mature DCs (mDCs) from microrheological viewpoint, cells were treated with different concentrations of TGF-ß1. The results showed that the impaired microrheological parameters, including osmotic fragility, electrophoretic mobility, deformability, membrane fluidity, F-actin organization and so on, as well as motilities of mDCs relied heavily on TGF-ß1 concentration. Moreover, these changes were correlated with the expression levels of fascin1, cofilin1, phosphorylated cofilin1 and profilin, this could be one of the crucial aspects of immune escape mechanisms of tumors, hinting that the signal pathway of TGF-ß1 should be blocked in appropriate way before performing DCs-based immunotherapy against cancer. It is clinically important to understand the biological behavior of DCs and immune escape mechanism of tumor as well as how to improve efficiency of the anti-tumor therapy based on DCs.

The effects of non-ionic contrast medium on the hemorheology in vitro and in vivo.

Contrast media are the commonly used agents in radiology. However, because of their characteristics of high osmolality, high viscosity, and chemical toxicity, the administrations of contrast media have been shown to cause adverse effects especially on hemorheology in short time course. The present study is to find the effects of a non-ionic contrast medium, iopromide, on the hemorheology in long time course both in vitro and in vivo. For in vitro treatment, human peripheral blood samples were incubated with contrast medium at 37°C for 0.5, 1 and 2 h. For in vivo study, about 15 ml of contrast medium was injected into rabbits and blood samples were collected at 0.5, 2, 6, and 24 h after the bolus injection. Hemorheological parameters were examined. Results showed that hematocrit adjusted whole blood viscosity increased significantly at 1 h after in vitro treatment of contrast medium, while it decreased at 0.5 h and remained low till 6 h after bolus injection. Ektacytometer showed that erythrocyte deformability decreased to the lowest level at 2 h in vitro and it dropped at 0.5 h and resumed to normal after 2 h in vivo. Erythrocyte small deformation indices were reduced by contrast medium in both in vitro and in vivo studies. Erythrocyte orientation index was also reduced in in vivo study. Erythrocyte electrophoresis rates at all time points decreased but osmotic fragility did not change in both studies. These impaired hemorheological parameters may disturb the microcirculation and cause adverse effects in patients with kidney diseases.

The effects of long term aerobic exercise on the hemorheology in rats fed with high-fat diet.

Hypercholesterolemia is one of the cardiovascular risk factors sensitive to preventive and control interventions. Here we created a hypercholesterolemia model to investigate the effect of the long term aerobic exercise (swimming) on the hemorheology of rats fed with high-fat diet. We found that the rats fed with high-fat diet developed hypercholesterolmia and hepatic steatosis and their hemorheological and coagulative properties were all impaired as compared to those of the rats fed with standard diet. But after exercise, the total cholesterol and triglyceride in the plasma were significantly decreased and the severity of hepatic steatosis were reduced. Exercise greatly improved the erythrocytes' hemorheological properties, including deformability, electrophoretic mobility and osmotic fragility. Exercise also markedly lowered the activated partial thromboplastin time (APTT) but had moderate effects on other coagulative parameters. The high oxidative stress level, as indicated by plasma MDA concentration, in rats with high-fat diet was significantly attenuated to the normal level after exercise. The present study suggests that long term aerobic exercise could remarkably improve the abnormal hemorheological property and the oxidative stress in rats with hypercholesterolemia.

Effects of Gekko sulfated polysaccharide-protein complex on the defective biorheological characters of dendritic cells under tumor microenvironment.

We previously isolated a sulfated polysaccharide-protein complex from Gekko swinhonis Guenther, a traditional Chinese medicine, and have demonstrated its direct anti-cancer effect on human hepatocellular carcinoma cell line SMMC-7721. Here we investigated the effects of Gekko sulfated polysaccharide-protein complex (GSPP) on the defective biorheological characters of dendritic cells (DCs) under SMMC-7721 microenvironment. Our findings have shown that the biorheological properties of DCs were severely impaired by SMMC-7721 microenvironment, including decreased cell deformability, migration, and electrophoresis mobility, increased osmotic fragilities, and changed organizations of cytoskeletal proteins. We also found decreased secretion of interleukin (IL)-12 and increased secretion of IL-10 in DCs. However, supernatant collected from nonmalignant liver cells had no effect on these parameters. SMMC-7721 cells were treated with GSPP and the supernatant was used to culture DCs. We found that the defective biorheological parameters of DCs, except for osmotic fragility, were partially or completely improved. The secretion of IL-12 did not change as compared with that of DCs in SMMC-7721 microenvironment, but the secretion of IL-10 was resumed to the control level. Our results indicate that GSPP could partially restore the defective biorheological characteristics of DCs via modifying the tumor microenvironment and decreasing the secretion of IL-10 of DCs.

Knockdown of hTERT alters biophysical properties of K562 cells resulting in decreased migration rate in vitro.

It has been shown that 90% of tumors, including hematological malignant tumors and leukemia, have much higher levels of telomerase expression than normal cells. To investigate the effect of telomerase on leukemia cells, we transfected K562, a human erythroleukemia cell line with an antisense-hTERT (human telomerase reverse transcriptase) cDNA vector, and examined the biological and biophysical properties of the stably transfected cells (referred to as KAT). Un-transfected cells (K562) and cells transfected with the empty vector (referred to as KC) were used as controls. Cell growth curve and (3)H-TdR test showed that the growth rate and DNA synthesis of KAT decreased compared with those of K562 and KC cells. Apoptosis and cell cycle distribution in KAT cells under normal culture condition were similar to those of K562 and KC cells, but changed after serum deprivation. KAT cells had significantly different biophysical characteristics from K562 and KC in terms of cell electrophoresis, membrane fluidity, membrane fluidity, and viscoelasticity. Furthermore, the transendothelial migration rate of KAT was much lower than those of K562 and KC cells. Confocal microscopy showed that KAT cells had higher F-actin content, suggesting the reorganization of cytoskeleton. Flow cytometry analysis revealed a lowered intracellular calcium concentration and CD71 expression, explaining the high F-actin content in KAT cells. In conclusion, we found that the knockdown of hTERT in K562 cells changed their cytoskeleton and biophysical features, and reduced the cell migration.

[The capping protein on the slow-growing end of actin filament: erythrocyte tropomodulin].

Erythrocyte tropomodulin (E-Tmod) is first isolated from human erythrocyte membrane as a TM-binding protein. Its N-terminus contains two TM-binding sites and one TM-dependent actin capping domain and C-terminus contains 5 leucine-rich repeats and a TM-independent actin capping domain. As the unique capping protein at the slow-growing end of F-actin, E-Tmod binds to N-terminus of TM and actin and decreases the TM-coated F-actin depolymerization. E-Tmod encoding gene is highly conserved and E-Tmod is distributed widely in erythrocytes and cardiomyocytes, etc. E-Tmod plays a pivotal role in organizing F-actin and cytoskeleton and maintaining the mechanical properties of the cells.

Hemorheological changes in patients with living-donor renal transplantation.

Living-donor renal transplantation is the preferred treatment for patients with end stage renal disease since it affords earlier transplantation and better graft for long term survival. The aim of the present study was to explore the hemorheological changes in patients undergone living-donor renal transplantation. We investigated the dynamic changes in the hemorheological properties of blood taken from the patients before renal transplantation and at 1 week, 2 week, 3 week and >1 month after the operation. As compared with pre-operation, the whole blood viscosity at different shear rates decreased significantly; the erythrocyte aggregation index decreased; the erythrocyte deformation index (DI) and integrated deformation index (IDI) had a remarkable improvement; the erythrocyte electrophoresis rate was raised. We also found that the osmotic fragility of RBCs at 145 mOsm/kg was greatly decreased after renal transplantation. Importantly, the ratio of erythrocyte membrane protein 4.1 a to 4.1 b decreased, which may explain the changes in DI, IDI, and osmotic fragility. Our results suggest that these hemorheological changes may greatly improve the organ microcirculation, which would play a critical role in reduce the ischemia-reperfusion injury in the graft.

Biorheological changes of dendritic cells at the different differentiation stages.

The differentiation, maturation and functioning of Dendritic cells (DCs) are dynamic processes. This study investigated the changes of DCs' migration ability and biorheological properties during their differentiation. Transmigration assay showed that, DCs' migration rate was improved significantly as they differentiate (p < 0.05); NSC (Rac1 blocker) treatment could significantly decrease their migration rates (p < 0.05). Confocal images showed that, F-actin uniformly distributed in monocytes; with DC's differentiation, F-actin began to remodel and gather at the site of dendrites; the images presented surface ruffles and uneven sawtooth-like cytoskeletal structures. Fluorescence polarization analysis showed that, membrane fluidity was increased significantly with DC's differentiation (p < 0.05). CD62L was upregulated significantly (p < 0.05) on the third and ninth days. CD2 was upregulated significantly (p < 0.05) until the seventh day. DC's electrophoretic mobility was increased continuously, especially increased significantly from the third day to the fifth day and the final stage (p < 0.05). These results indicate that there are significant changes in the biorheological properties of DCs during their differentiation.

Biomechanical alterations of dendritic cells by co-culturing with K562 CML cells and their potential role in immune escape.

Dendritic cells (DCs), which are potent antigen presenting cells (APCs), are utilized to deliver the signals essential for the initiation of immune responses. In this study, we used an interdisciplinary approach to characterize the effect of K562 cells, a human chronic myeloid leukemia (CML) cell line, on the biomechanical characteristics and immune functions of DCs. When co-cultured with K562 cells, the biomechanical and immunological characteristics of immature DCs (imDCs) and mature DCs (mDCs) were severely impaired compared with controls. The changes include increased membrane viscoelasticity, reorganized cytoskeleton (F-actin), suppressed capability of antigen uptake, transendothelium migration, and activation of naïve T cells. In exploring the mechanisms of these changes, we identified several genes and proteins by microarray analysis and 2D gel electrophoresis. Changes were found in the cytoskeleton-related genes and proteins (such as cofilin1 and profilin1) and matrix-related genes and proteins (such as TIMP1 and MMP9). These findings provide a molecular basis for the biomechanical and immunological changes of DCs in response to K562 and may help to elucidate the mechanism for tumor immune escape.

Acute dichlorvos poisoning induces hemorheological abnormalities in rabbits via oxidative stress.

Dichlorvos is an important insecticide used largely. Some studies have demonstrated that organophosphate pesticide has effects on erythrocyte membrane structures, which is critical to erythrocyte function and hemorheology. The aim of the present study was to explore the effect of oxidative stress on hemorheological changes during dichlorvos poisoning in rabbits. Data indicated that after dichlorvos exposure the hematocrit adjusted viscosity at high shear rate increased and erythrocyte membrane fluidity decreased. Data obtained from plasma showed that lipid peroxidative substance-malonaldehyde was elevated and superoxide dismutase was reduced. In summary, oxidative stress does occur in dichlorvos poisoning and may lead to hemorheological alterations. The changes of hemorheology may be responsible for the pathophysiology of the dichlorvos poisoning.

Effects of Gekko sulfated polysaccharide-protein complex on human hepatoma SMMC-7721 cells: inhibition of proliferation and migration.

AIM OF THE STUDY: Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Here we investigated the structural characterization and anti-cancer effects of sulfated polysaccharide-protein complex (GSPP) isolated from Gekko swinhonis Guenther. MATERIALS AND METHODS: The structure of GSPP was characterized by high performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, beta-elimination reaction, and NMR spectroscopy. SMMC-7721 cells were used to assess the influence of GSPP on hepatocellular carcinoma. Cell proliferation and survival was determined by trypan blue exclusion assay. Cell migration was performed by wound-healing and transwell assay. The secretion of IL-8 was detected by an enzyme-linked immunosorbent assay kit. Flow cytometry was used to analyze intracellular calcium concentration, as well as cell cycle distribution and apoptosis. Confocal microscopy was used to assess the localization and configuration of actin filaments. RESULTS: GSPP was chemically characterized as a sulfated polysaccharide-protein complex with O-glycopeptide linkages. Our results showed that GSPP inhibited the proliferation of SMMC-7721 cells and blocked cells in the S phase. No direct toxicity against cells was observed. Furthermore, GSPP inhibited the migration of SMMC-7721 cells with the reduction of intracellular calcium. Actin filaments were polymerized and accumulated in the cytoplasm of the treated cells, whereas the secretion of IL-8 was not significantly changed after GSPP exposure. CONCLUSION: We describe an identified sulfated polysaccharide-protein complex, and demonstrate its direct effect on hepatocellular carcinoma cell migration via calcium-mediated regulation of the actin cytoskeleton reorganization.

Ryanodine receptor 1 mediates Ca2+ transport and influences the biomechanical properties in RBCs.

Ryanodine receptors (RyRs) are a family of Ca2+ channel proteins that mediate the massive release of Ca2+ from the endoplasmic reticulum into the cytoplasma. In the present study, we manipulated the incorporation of RyR1 into RBC membrane and investigated its influences on the intracellular Ca2+ ([Ca2+](in)) level and the biomechanical properties in RBCs. The incorporation of RyR1 into RBC membranes was demonstrated by both immunofluorescent staining and the change of [Ca2+](in) of RBCs. In the presence of RyR1, [Ca2+](in) showed biphasic changes, i.e., it increased with the extracellular Ca2+ ([Ca2+](ex)) up to 5muM and then decreased with the further increase of [Ca2+](ex). However, [Ca2+](in) remained constant in the absence of the RyR1. The results of biomechanical measurements on RBCs, including deformability, osmotic fragility, and membrane microviscosity, reflected similar biphasic changes of [Ca2+](in) mediated by RyR1 with the increases of [Ca2+](ex). Therefore, it is believed that RyR1 can incorporate into RBC membrane in vitro, and mediate Ca2+ influx, and then regulate RBC biomechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca2+ release channel.

Hepatocellular carcinoma cells deteriorate the biophysical properties of dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells and induce antigen-specific immune responses in the organism. The dysfunction of DCs has been implicated in tumor-bearing host. In order to elucidate the effects of tumor microenvironment on the functions of DCs from interdisciplinary aspects, we characterized the biophysical properties of DCs co-cultured with hepatocellular carcinoma cells (HCC). The results showed that the biophysical characteristics of immature and mature DCs were severely impaired by HCC compared with those under normal conditions, including the increased osmotic fragilities, decreased cell membrane fluidities, increased membrane viscoelastic properties, dysfunction and increased expression of cytoskeleton protein F-actin, as well as the deteriorated transendothelium migration. The impaired biophysical properties of DCs may be one of many aspects of the immune escape mechanisms of tumors. These results are clinically and instructionally significant with regard to how to enhance efficiency of the anti-tumor therapy based on DCs.

Improving abnormal hemorheological parameters in ApoE-/- mice by Ilex kudingcha total saponins.

The aim of this study was to test the effects of Ilex kudingcha total saponins on hemorheology of ApoE-/- mice suffering from hypercholesterolemia induced by high-cholesterol diet. The mice were randomly divided into six groups: the control group, the high-cholesterol diet group, 50 mg/kg atorvastatin treatment group, 75, 150 and 300 mg/kg Ilex kudingcha saponins treatment groups, and all the drug treatment groups were fed with a high-cholesterol diet. After administration with saponins (150 mg/kg or more) and atorvastatin (50 mg/kg) for six weeks, the plasma total cholesterol (TC), whole blood viscosity (WBV), plasma viscosity (PV), and erythrocyte aggregation index (EAI) had a remarkable decrease compared with that of the high-cholesterol diet group, but the hematocrit (Hct) and erythrocyte deformation index (DI) had no significant changes. In addition, it is found that the improving effects of saponins on reducing plasma fibrinogen (Fg) levels and prolonging the blood coagulation times including activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). In conclusion, the Ilex kudingcha total saponins may have a significant therapy application of hypercholesterolemia and atherosclerosis by considering its actions on hemorheological characteristics.

Effects of myakuryu on hemorheological characteristics and mesenteric microcirculation of rats fed with a high-fat diet.

There is evidence that hyperlipidemia can induce hemorheological and microcirculatory disturbances. Myakuryu, a Chinese traditional medicine is efficacious in promoting lipid metabolism and protecting oxidative stress, but whether this drug can ameliorate rheologic disturbances caused by hyperlipidemia is still unknown. The present study was conducted to investigate the effects of myakuryu on hemorheological and microcirculatory disturbances induced by hyperlipidemia. Wistar rats were divided into a group on control diet (n=8) and a group on high-fat diet (HFD, n=44). Eight weeks later, plasma triglyceride (TG) and total cholesterol (TC) were determined. Sixteen animals with the highest levels of hyperlipidemia from the HFD group were randomly divided into two sub-groups: the untreated hyperlipidemia group (n=8) and the group treated with myakuryu (n=8). At the end of the sixteenth week, rheological and microcirculatory parameters were measured. Chemical analysis showed that myakuryu treatment caused significant reductions of plasma TG and TC levels (P<0.01), and the cholesterol/phospholipid ratio in the erythrocyte membrane (P<0.05). Rheological and microcirculatory measurements showed that myakuryu treatment led to a significant decrease in the erythrocyte aggregation index, plasma viscosity and blood viscosity at shear rates of 50, 100 and 150 s(-1) and in adherent leukocytes in mesenteric venules. There was a significant increase in erythrocyte deformation, electrophoretic mobility, membrane fluidity and F-actin content in the erythrocyte membrane as well as in red cell velocity in mesenteric venules. Our findings suggest that myakuryu treatment can improve blood flow and reduce adherent leukocytes in the venules of rats fed with HFD by ameliorating blood viscosity, erythrocyte deformability and aggregation, and other hemorheological characteristics.

Effects of cardiotonic pill on RBC rheologic abnormalities in HFD-induced mice and LPL deficient mice.

The lipoprotein lipase deficient (LPL-/-) mice and high fat-diet (HFD) induced hypertriglyceridemic mice were used to investigate the effects of cardiotonic pill (CP) on RBC rheologic abnormalities. Mice were randomly divided into the following five groups: the control group; the untreated HFD group; the untreated LPL-/- group; the treated HFD group; and the treated LPL-/- group, and the treated HFD and LPL-/- mice were administered with CP twice a day (400 mg/kg/day) orally for four weeks. Then, plasma triglyceride (TG), RBC deformation index (DI), orientation index (DI)or and RBC electrophoretic time (EPT) were measured. Compared with the untreated HFD mice, TG level and EPT reduced and DI and (DI)or increased markedly in the treated HFD mice (P<0.05). However, compared with the untreated LPL-/- mice, these parameters in the treated LPL-/- mice had no statistically significant changes (P>0.05). Our data show that CP can lower plasma TG level and ameliorate RBC rheologic abnormalities in the HFD-induced hypertriglyceridemic mice, but it losses its capacity in the LPL deficient animals. The results indicate that LPL may be one of the important targets for CP regulating lipometabolism and rheologic abnormalities.

Synergistic effect of cytokines EPO, IL-3 and SCF on the proliferation, differentiation and apoptosis of erythroid progenitor cells.

Erythroblasts were obtained from murine spleen. After cultured for 12 hr, the cells were divided into four groups with the use of the following cytokines in culture: EPO, EPO+SCF, EPO+IL-3, and EPO+IL-3+SCF. Cell proliferation assay was done. Apoptosis rates were obtained by using a flow cytometer. Mitochondrial membrane potential (MMP) was assessed in flow cytometry (FCM) by labeling with rhodamine 123. Mitochondrial enzyme activity (MEA) was evaluated with MTT colorimetric assay. The cells were labeled with Fluo-3/Am Ester and Ca(2+) concentration was measured. The expression of Bax mRNA and Bcl-2 mRNA was analyzed by RT-PCR. At same time, the expression of Bax and Bcl-2 was analyzed by western blotting. Our results showed that IL-3 and SCF have synergistic effects with EPO on the proliferation, differentiation and apoptosis of erythroid progenitors.