Ablation catheter-induced mechanical deformation in myocardium: computer modeling and ex vivo experiments.

Cardiac catheter ablation requires an adequate contact between myocardium and catheter tip. Our aim was to quantify the relationship between the contact force (CF) and the resulting mechanical deformation induced by the catheter tip using an ex vivo model and computational modeling. The catheter tip was inserted perpendicularly into porcine heart samples. CF values ranged from 10 to 80 g. The computer model was built to simulate the same experimental conditions, and it considered a 3-parameter Mooney-Rivlin model based on hyper-elastic material. We found a strong correlation between the CF and insertion depth (ID) (R2 = 0.96, P < 0.001), from 0.7 ± 0.3 mm at 10 g to 6.9 ± 0.1 mm at 80 g. Since the surface deformation was asymmetrical, two transversal diameters (minor and major) were identified. Both diameters were strongly correlated with CF (R2 ≥ 0.95), from 4.0 ± 0.4 mm at 20 g to 10.3 ± 0.0 mm at 80 g (minor), and from 6.4 ± 0.7 mm at 20 g to 16.7 ± 0.1 mm at 80 g (major). An optimal fit between computer and experimental results was achieved, with a prediction error of 0.74 and 0.86 mm for insertion depth and mean surface diameter, respectively.

Evaluation of erythrocyte membrane oxidation due to their exposure to shear flow generated by extracorporeal blood pump.

Several similarities have been found between shear stress-induced erythrocyte damage and physiological aging of erythrocytes in terms of elevated mechanical fragility, increased erythrocyte aggregation, and decreased membrane surface charge. Accordingly, we hypothesized that blood pump circulation, which generates shear stress, would accelerate erythrocyte aging, manifesting as oxidation. Therefore, the purpose of this study was to investigate the effect of blood pump circulation on erythrocyte oxidation. Fresh porcine blood was acquired from a slaughterhouse and anticoagulated with sodium citrate. About 500 mL of anticoagulated whole blood was circulated for 180 min in an in vitro test circuit comprising a BP-80 blood pump with a pump speed and a pump pressure head of 100-120 mmHg. A blood sample was taken at the start of the circulation and 180 min afterward. The hemolysis level and oxidation amount of the erythrocyte membrane were analyzed and compared between samples. Hemolysis increased with the prolongation of shear exposure inside the pump circuit. After 180 min of blood pumping in circuit, the oxidation level of the erythrocyte membrane showed an increase of 0.1 nmol/mg protein. Moreover, the membrane oxidation levels of sheared erythrocytes were greater than those of control erythrocytes. These results suggest that blood pump circulation accelerates erythrocyte aging and give us a greater understanding of the effects of blood pump perfusion.

Tocotrienols Prevent the Decline of Learning Ability in High-Fat, High-Sucrose Diet-Fed C57BL/6 Mice.

Obesity has been increasing worldwide and is well-known as a risk factor for cognitive decline. It has been reported that oxidative stress in the brain is deeply involved in cognitive dysfunction in rodent models. While there are many studies on oxidation in the liver and adipose tissue of obese mice, the relationship between obesity-induced cognitive dysfunction and brain oxidation has not been elucidated. Here, we show that obesity induced by a high-fat, high-sucrose diet (HFSD) alters cognitive function in C57BL/6 male mice, and it may involve the acceleration of brain oxidation. Tocotrienols (T3s), which are members of the vitamin E family, can prevent HFSD-induced cognitive changes. To elucidate these mechanisms, respiratory metabolism, locomotor activity, temperature around brown adipose tissue, and protein profiles in the cerebrum cortex were measured. Contrary to our expectation, respiratory metabolism was decreased, and temperature around brown adipose tissue was increased in the feeding of HFSD. The proteins that regulate redox balance did not significantly change, but 12 proteins, which were changed by HFSD feeding and not changed by T3s-treated HFSD compared to control mice, were identified. Our results indicated that HFSD-induced obesity decreases mouse learning ability and that T3s prevent its change. Additionally, feeding of HFSD significantly increased brain oxidation. However, further study is needed to elucidate the mechanisms of change in oxidative stress in the brain by obesity.

The shear rate promotes pinocytosis of extracellular dextran in platelets.

BACKGROUND: Several conventional studies focused on platelet pinocytosis for possible utilization as drug delivery systems. Although platelet pinocytosis is important in such utilization, the impact of the shear rate on pinocytosis is unclear. OBJECTIVE: Our objective was to investigate the relationship between shear rate and platelet pinocytosis in vitro. In addition, this study addressed the change in platelet aggregation reactivity with adenosine diphosphate (ADP) stimulation after pinocytosis. METHOD: Porcine platelet-rich plasma was mixed with fluorescein isothiocyanate (FITC)-conjugated dextran and incubated for 15 min under shear conditions of 0, 500, and 1500 s-1. After incubation, confocal microscopic scanning and three-dimensional rendering were performed to confirm the internalization of FITC-dextran into platelets. The amount of FITC-dextran accumulated via platelet pinocytosis was compared using flow cytometry at each shear rate. In addition, light transmission aggregometry by ADP stimulation was applied to platelets after pinocytosis. RESULTS: The amount of intracellular FITC-dextran increased with higher shear rates. Platelets with increased amounts of intracellular FITC-dextran did not show changes in the aggregation reactivity to ADP. CONCLUSIONS: A higher shear rate promotes platelet pinocytosis, but enhanced pinocytosis does not affect aggregation sensitivity, which is stimulated by ADP.

Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

BACKGROUND: Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. METHODS AND RESULTS: Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. CONCLUSION: Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

Mechanoresponses mediated by the TRP11 channel in cilia of Chlamydomonas reinhardtii.

Cilia are organelles involved in motility and sensory transduction, but how these two functions coexist has not been elucidated in depth. Here, the involvement of the ciliary transient receptor potential (TRP) channel TRP11 in mechanoresponses is studied in Chlamydomonas reinhardtii using a TRP11-knockout mutant. The mutant has defects in the conversion of the bending mode of the cilium from forward to reverse when tapped with a glass rod, the detachment of cilia when shear is applied, the increase in ciliary beat frequency upon application of mechanical agitation by vortex mixing, and the initiation of gliding while both cilia are attached in opposite directions to a glass surface. These observations indicate that TRP11 can perceive mechanical stimuli with distinct intensities and durations and induce various types of ciliary responses.

Novel blood typing method by discrimination of hemagglutination and rouleaux using an erythrocyte aggregometer.

BACKGROUND: In pretransfusion blood typing, pretreatments such as centrifugation and suspension of red blood cells (RBCs) and mixing them with sufficient amounts of reagents are required, but these steps are time-consuming and costly. OBJECTIVE: Aiming to develop a new blood typing method that requires no dilution and only a small amount of reagent, we attempted to determine blood type using syllectometry, an easy-to-use and rapid optical method for measuring the RBC aggregation that occurs when blood flow is abruptly stopped in a flow channel. METHODS: Samples of whole blood from 20 healthy participants were mixed with antibody reagents for blood typing at mixing ratios of 2.5% to 10% and measured with a syllectometry device. RESULTS: Amplitude (AMP), one of the aggregation parameters, showed significant differences between agglutination and non-agglutination samples at mixing ratios from 2.5% to 10%. Although there were significant individual differences in aggregation parameters, calculation of AMP relative to that of blood before reagent mixing reduced the individual differences and enabled determination of blood type in all participants. CONCLUSIONS: This new method enables blood typing with a small amount of reagent, without the time-consuming and labor-intensive pretreatments such as centrifugation and suspension of RBCs.

Determination of the erythrocyte sedimentation rate using the hematocrit-corrected aggregation index and mean corpuscular volume.

BACKGROUND: Determination of the erythrocyte sedimentation rate (ESR) by measurement of erythrocyte aggregation is an alternative to the Westergren method and can be performed rapidly. However, its principle is opaque and the ESR values obtained can deviate from Westergren method values (WG ESR) due to hematocrit. Furthermore, WG ESR is affected by particle size, but no studies have examined the effect of individual mean corpuscular volumes (MCVs). METHODS: Simultaneous measurement of the erythrocyte aggregation index (AI) over a 5-s interval and determination of the complete blood count in 80 µL blood from 203 patients were performed (hematocrit, 21.4%-52.3%; MCV, 62.7-114.1 fL). ESR values were calculated with the hematocrit-corrected AI (HAI) for comparison with WG ESR. We improved the calculation formula by using MCV. RESULTS: The sedimentation velocity of a single erythrocyte in the samples agreed well with an exponential function of HAI. ESR values calculated using HAI showed excellent correlation with WG ESR (r = 0.899, p < 0.001; Bland-Altman analysis: bias 2.76, limits of agreement (LOA) -24.5 to 30.0), but the difference between the calculated ESR and WG ESR increased with decreasing MCV. Calculation of ESR considering both HAI and MCV eliminated the MCV-dependent deviation and improved the correlation with WG ESR (r = 0.920, p < 0.001, bias -2.17, LOA -24.6 to 20.3). CONCLUSION: Calculation using HAI and MCV can rapidly provide ESR values that are highly correlated with WG ESR in clinical specimens over a wide range of hematocrit and MCV values.

C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes.

Podoplanin (PDPN) is intensely expressed on the podocyte membrane in an evolutionally conserved manner. CLEC-2, the endogenous ligand of PDPN, is highly expressed in platelets and also exists in a soluble form in plasma. Normally, podocytes are sequestered from CLEC-2, but when the glomerular barrier is injured, podocytes gain access to CLEC-2. We tested the effects of CLEC-2 in podocytes in vitro and in vivo. Cultured podocytes treated with Fc-CLEC-2 demonstrated that CLEC-2 induced the dephosphorylation of ezrin, radixin, and moesin (ERM) proteins. Podocytes treated with Fc-CLEC-2 also showed the dissociation of F-actin filaments from PDPN, F-actin degradation, detachment, and round morphology. Next, we perfused normal mouse kidney in vivo with FLAG-CLEC-2. CLEC-2 induced dephosphorylation of ERM and widening of the foot processes of podocytes. Platelets were detected by immunostaining for CD41 in the urine of mice with podocyte injury, indicating that podocytes can encounter platelets when glomeruli are injured. Collectively, these observations suggest that when platelets leak through the injured glomeruli, CLEC-2 from the platelets acts on PDPN in podocytes and induces morphological change and detachment, which may further aggravate podocyte injury. Thus, PDPN on podocytes may work as a leaked-platelet sensor.

A rapid and accurate method for estimating the erythrocyte sedimentation rate using a hematocrit-corrected optical aggregation index.

Although both the erythrocyte sedimentation rate (ESR) and optically measured erythrocyte aggregation parameters are affected by the hematocrit, this interaction is not considered by the method used to estimate ESR that considers aggregation parameters. In this study, we investigated the relationship between the ESR obtained by the Westergren method and that obtained with an aggregation parameter, namely, the aggregation index (AI) of multiple hematocrit values and fibrinogen-spiked samples with an analysis time of 5-60 s, and attempted to develop a rapid and accurate ESR estimation method. The AIs obtained from 5- and 10-s optical measurements with a fixed hematocrit were highly correlated with the erythrocyte sedimentation velocity. Furthermore, the rate of the AI increase with an increasing hematocrit was not significantly affected by the fibrinogen concentration at these measurement times. On the basis of these results, we defined the hematocrit-corrected aggregation index (HAI). The exponential function of the HAI obtained from the 5-s measurement agreed well with the sedimentation velocity calculated to eliminate the effect of hindered settling, and the HAI and hematocrit could be used to calculate the time constant of the sedimentation curve with a linear regression equation. The ESR value at 1 h was calculated based on the modified Stokes' law and the HAI obtained from the 5-s measurement and showed an excellent correlation (R = 0.966) with the ESR value obtained by the Westergren method over a wide range of hematocrit and fibrinogen concentrations.

Catheter contact area strongly correlates with lesion area in radiofrequency cardiac ablation: an ex vivo porcine heart study.

PURPOSE: Our previous study confirmed that not only force but also the catheter contact angle substantially impacted the contact area and its morphology. Therefore, in this study, we aimed to further investigate the relationship between the catheter contact area and the dimensions of the ablation lesion area as a function of catheter contact angle and force in radiofrequency catheter ablation. METHODS: The radiofrequency catheter ablation test was performed for 5 contact angles and 8 contact forces at a fixed ablation time of 30 s. The initial impedance was 92.5 ± 2.5 Ω, the temperature during ablation was 30 °C, and the power was 30 W. The irrigation rate during ablation was set to 17 mL/min. Each experiment was repeated 6 times. RESULTS: The catheter contact area showed a strong correlation with the ablation lesion area (r = 0.8507). When the contact area was increased, the lesion area also increased linearly in a monotonic manner. The relationships between catheter contact force and ablation lesion area and between catheter contact force and ablation lesion depth are logarithmic functions in which increased contact force was associated with increased lesion area and depth. The catheter contact angle is also an important determinant of the lesion area. The lesion area progressively increased when the contact angle was decreased. In contrast, the lesion depth progressively increased when the contact angle was increased. CONCLUSIONS: The catheter contact area was strongly correlated with the ablation lesion area. Additionally, catheter contact force and contact angle significantly impacted the dimensions of the lesion in radiofrequency catheter ablation procedures.

Discrete responses of erythrocytes, platelets, and von Willebrand factor to shear.

Despite decades of technological advancements in blood-contacting medical devices, complications related to shear flow-induced blood trauma are still frequently observed in clinic. Blood trauma includes haemolysis, platelet activation, and degradation of High Molecular Weight von Willebrand Factor (HMW vWF) multimers, all of which are dependent on the exposure time and magnitude of shear stress. Specifically, accumulating evidence supports that when blood is exposed to shear stresses above a certain threshold, blood trauma ensues; however, it remains unclear how various constituents of blood are affected by discrete shears experimentally. The aim of this study was to expose blood to discrete shear stresses and evaluate blood trauma indices that reflect red cell, platelet, and vWF structure. Citrated human whole blood (n = 6) was collected and its haematocrit was adjusted to 30 ± 2% by adding either phosphate buffered saline (PBS) or polyvinylpyrrolidone (PVP). Viscosity of whole blood was adjusted to 3.0, 12.5, 22.5 and 37.5 mPa·s to yield stresses of 3, 6, 9, 12, 50, 90 and 150 Pa in a custom-developed shearing system. Blood samples were exposed to shear for 0, 300, 600 and 900 s. Haemolysis was measured using spectrophotometry, platelet activation using flow cytometry, and HMW vWF multimer degradation was quantified with gel electrophoresis and immunoblotting. For tolerance to 300, 600 and 900 s of exposure time, the critical threshold of haemolysis was reached after blood was exposed to 90 Pa for 600 s (P < 0.05), platelet activation and HMW vWF multimer degradation were 50 Pa for 600 s and 12 Pa for 300 s respectively (P < 0.05). Our experimental results provide simultaneous comparison of blood trauma indices and thus also the relation between shear duration and magnitude required to induce damage to red cells, platelets, and vWF. Our results also demonstrate that near-physiological shear stress (<12 Pa) is needed in order to completely avoid any form of blood trauma. Therefore, there is an urgent need to design low shear-flow medical devices in order to avoid blood trauma in this blood-contacting medical device field.

Erythrocyte morphological symmetry analysis to detect sublethal trauma in shear flow.

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. Their viscoelasticity is intrinsically linked to physical properties of the cytosol, cytoskeleton, and membrane-all of which are highly sensitive to supraphysiological shear exposure. Given the need to minimise blood trauma within artificial organs, we observed RBC in supraphysiological shear through direct visualisation to gain understanding of processes leading to blood damage. Using a custom-built counter-rotating shear generator fit to a microscope, healthy red blood cells (RBC) were directly visualised during exposure to different levels of shear (10-60 Pa). To investigate RBC morphology in shear flow, we developed an image analysis method to quantify (a)symmetry of deforming ellipsoidal cells-following RBC identification and centroid detection, cell radius was determined for each angle around the circumference of the cell, and the resultant bimodal distribution (and thus RBC) was symmetrically compared. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased. These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow.

Relationship of Catheter Contact Angle and Contact Force with Contact Area on the Surface of Heart Muscle Tissue in Cardiac Catheter Ablation.

PURPOSE: The aims of this study were to develop an experimental procedure for setting the catheter angle with respect to the surface of the heart muscle and the catheter contact force and to investigate the catheter contact area on the heart muscle as a function of catheter contact angle and force. METHODS: Visualization tests were performed for 5 contact angles (0°, 30°, 45°, 60°, and 90°) and 8 contact forces (2, 4, 6, 10, 15, 20, 30, and 40 gf). Each experiment was repeated 6 times with 2 different commercially available catheter tips. RESULTS: The morphology of the contact area was classified into rectangular, circular, ellipsoidal, and semi-ellipsoidal. The correlation between contact force and contact area was a logarithmic function; increasing contact force was associated with increased contact area. At the same contact force, the correlation between contact angle and contact area was inverse; decreasing contact angle was associated with a corresponding increase in contact area. CONCLUSION: Both the catheter contact angle and contact force substantially impact the contact area and morphology in catheter ablation procedures.

Shear-dependent platelet aggregation size.

Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s-1 (200-300 µm2 ) was significantly larger compared to those sheared at >6000 s-1 (50-100 µm2 ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.

Podoplanin is indispensable for cell motility and platelet-induced epithelial-to-mesenchymal transition-related gene expression in esophagus squamous carcinoma TE11A cells.

BACKGROUND: The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood. METHODS: Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed. RESULTS: PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-ß-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-ß-induced EMT induction. CONCLUSIONS: These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.

Usefulness of next-generation DNA sequencing for the diagnosis of urinary tract infection.

Acute urinary tract infection (UTI) is a highly common clinical condition. Although bacterial culture is the gold standard diagnostic test, false negative results may be possible, leading to the pathogen being unidentified. In recent years, bacterial DNA sequencing analysis has garnered much attention, but clinical studies are rare in Japan. In this study, we assessed the usefulness of next-generation DNA sequencing (NGS) analysis for acute UTI patients. We thus performed an observational, retrospective case series study. Urine and blood samples were collected from ten acute UTI patients, of whom four had also been diagnosed with urosepsis. Seven variable regions of bacterial 16S rRNA genes were amplified by PCR and then sequenced by IonPGM. The identified bacterial species were compared with those identified using the culture tests and the clinical parameters were analyzed. As a result, the NGS method effectively identified predominant culture-positive bacteria in urine samples. The urine NGS also detected several culture-negative species, which have been reported to be potentially pathogenic. Out of four urosepsis cases, three were pathogen-positive in blood NGS results, while two were pathogen-negative in blood culture. In one sepsis case, although blood culture was negative for Escherichia coli, this species was detected by blood NGS. For non-sepsis cases, however, blood NGS, as well as blood culture, was less effective in detecting bacterial signals. In conclusion, NGS is potentially useful for identifying pathogenic bacteria in urine from acute UTI patients but is less applicable in patients who do not meet clinical criteria for sepsis.

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.

Papaverine identified as an inhibitor of high mobility group box 1/receptor for advanced glycation end-products interaction suppresses high mobility group box 1-mediated inflammatory responses.

The interaction of high mobility group box 1 (HMGB1), which is secreted from immune and dying cells during cellular infection and injury, and receptor for advanced glycation end-products (RAGE) appears to be critical for acute and chronic inflammatory disorders. Here we designed a unique cyclic ß-hairpin peptide (Pepb2), which mimics the predicted RAGE-binding domain of HMGB1. Pepb2 competitively inhibited HMGB1/RAGE interaction. We then identified papaverine as a Pepb2 mimetic by in silico 3D-structural similarity screening from the DrugBank library. Papaverine was found to directly inhibit HMGB1/RAGE interaction. It also suppressed the HMGB1-mediated production of pro-inflammatory cytokines, IL-6 and TNF-α, in mouse macrophage-like RAW264.7 cells and bone marrow-derived macrophages. In addition, papaverine attenuated mortality in cecal ligation puncture-induced sepsis model mice. Taken together, these findings indicate that papaverine could become a useful therapeutic against HMGB1/RAGE-mediated sepsis and other inflammatory diseases.

Shear-stress mediated nitric oxide production within red blood cells: A dose-response.

BACKGROUND: Red blood cells (RBC) are exposed to varying shear stress while traversing the circulatory system; this shear initiates RBC-derived nitric oxide (NO) production. OBJECTIVE: The current study investigated the effect of varying shear stress dose on RBC-derived NO production. METHODS: Separated RBC were prepared with the molecular probe, diamino-fluoreoscein diacetate, for fluorometric detection of NO. Prepared RBC were exposed to discrete magnitudes of shear stress (1-100 Pa), and intracellular and extracellular fluorescence was quantified via fluorescence microscopy at baseline (0 min) and discrete time-points (1-30 min). RESULTS: Intracellular RBC-derived NO fluorescence was significantly increased (p < 0.05) following shear stress exposure when compared to baseline at: i) 1 min-100 Pa; ii) 5 min-1, 5 Pa; iii) 15 min-1, 5, 35 Pa; iv) 30 min-35 Pa. Extracellular RBC-derived NO fluorescence was significantly increased (p < 0.05) following shear stress exposure when compared to baseline at: i) 5 min - 100 Pa; ii) 15 min-100 Pa; iii) 30 min-40, 100 Pa. CONCLUSIONS: These data indicate that: i) a dose-response exists for the RBC-derived production of NO via shear stress; and ii) exposure to supra-physiological shear stress allows for the leakage of RBC intracellular contents (e.g., RBC-derived NO).