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.1177 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.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-NOSSerin 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.This human  Nitric oxide (NO), a highly reactive and short-lived diffusible molecule regulates central physiological mechanisms, e.g. vascular tone, macrophage-mediated neurotoxicity, anti-apoptotic activity In vitro investigations have shown that pharmacological stimuli positively influence RBC-NOS activation 1177 (RBC-NOSSer1177)-residue that resulted in increased NO production. It was further shown that the phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway is involved in this process in vitro examinations have shown that the application of continuous shear stress also activates RBC-NOS and NO production Importantly, it is known that physical exercise results in increased peripheral shear stress in the vascular bed in vivo investigation In contrast, our group demonstrated in a human in vitro, but also possibly in vivo, shear stress induced e.g. by moderate exercise does not lead to protein catabolism and therefore improves RBCs rheological characteristics. However, the underlying in vivo mechanisms of these regulations are far from being resolved. Therefore, we hypothesized in the present study that acute moderate physical exercise stimulates and improves RBC-NOS activity, RBC NO production and deformability in humans. Additionally, we proved the questions whether NOS-regulating upstream located signaling pathways, specifically Akt kinase signaling in vivo.It can be stated that The protocols used in this study were approved by the ethics committee of the German Sports University Cologne. These protocols align with the Declaration of Helsinki and all participants gave written informed consent to participate in this study.Fifteen male subjects participated in this study. Basal anthropometric parameters of the subjects were as follows (mean ± SD): age [years]: 27±3.6, height [cm]: 182±4.0, weight [kg]: 79±8.9. All subjects abstained from alcohol consumption for 24 hours prior to and during the training intervention and were non-smokers.–1 and the speed increased every 5 min by 0.5 m*sec–1 until subjective exhaustion. Every interval was followed by a 30 sec break to obtain 20 µl blood from the earlobe for lactate analysis. Capillary blood lactate concentrations were measured with EBIO plus . A calculation matrix developed at the German Sport University was used to determine the individual 4 mmol lactate threshold, the 70% of this threshold and the corresponding velocity applied during AMRT. The average running speed during AMRT was 2.39±0.41 m*sec–1.During the acute moderate running test (AMRT) the participants exercised at a velocity corresponding to 70% of their individual 4 mmol lactate threshold for 1 hour. This velocity was determined for each subject in a preliminary incremental running step test (IRT) which was performed 21 days prior AMRT. This three week interruption phase between the tests was chosen to avoid post-exercise plasma expansion, intensified haemolysis or disturbances in erythropoiesis 2), partial pressure of oxygen that is dissolved in arterial blood (pO2), and arterial haemoglobin oxygen saturation (sO2) Pre AMRT and Post AMRT  in order to obtain pH, partial pressure of arterial carbon dioxide  connected to a 5 mL syringe. 4 mL blood was slowly drawn from the vein and immediately anticoagulated using either Heparin vacutainers .in vitro approach was carried out to investigate the influence of wortmannin on RBC-NOS serine1177 phosphorylation (RBC-NOSSer1177), RBC NO production and RBC deformability. Wortmannin has been shown to inhibit PI3 kinase, thereby reducing Akt signaling. This reduction decreases phosphorylation of the eNOSSer1177An additional 1177 and RBC deformability depending on PI3 kinase inhibition. Parameters were determined before (0 min) and after (30 min) wortmannin incubation. Simultaneous sample incubation with phosphate buffered saline  (1x), without calcium and magnesium, PAA, Paschin, Austria) adjusted to physiological pH 7.4 served as control.Post AMRT additional blood was taken to also investigate phosphorylation of RBC-NOSSer1177, eNOSThr495 , cGMP  or phosphoAktSer473  for 1 hour. The control area was incubated in the absence of the first antibody. After repeated washing the sections were incubated with a secondary goat-anti-rabbit antibody  for 1 hour. A streptavidin-horseradish-peroxidase complex  was applied as a detection system (dilution 1∶150) for 30 min. The staining was developed using 3,3-diaminobenzidine-tetrahydrochloride solution  in 0.1 M TBS.Immunostaining of RBCs as described hereinafter has been applied in various studies Following the protocol of Kleinbongard et al. 6 RBCs were used for the extraction. RBCs were repeatedly washed  at 800×g for 10 min at 4°C using supplied washing buffer. RBCs were then incubated with supplied extraction buffer for 10 min at 4°C by gentle shaking to release cytosolic proteins. After centrifugation at 1,000×g for 10 min at 4°C supernatant with cytosolic proteins were stored on ice until further processing. Subsequently, a second extraction buffer was added to the remaining pellet, incubated for 30 min at 4°C and the sample was centrifuged at 20,000×g for 10 min and 4°C. The supernatant with the solubilized membrane proteins was stored on ice. A third extraction buffer was added to the remaining pellet, incubated for 10 min at 4°C on a shaker and the sample was then centrifuged at 20,000×g for 10 min and 4°C. This supernatant is aimed to contain nucleic proteins and was discarded because mature RBCs are anucleate cells Additional western blot analyses were performed to confirm the results obtained in the immunohistochemical procedure. For this purpose heparinized venous blood was taken Pre and Post AMRT. RBCs were separated at 800×g for 10 min at 4°C. RBC protein fractions were extracted using the ProteoExtract Subcellular Proteome Extraction Kit . The mild procedure yields proteins in their native state. 5×101177, phosphorylated eNOSThr495 , cGMP  or phosphorylated AktSer473 . The membrane was then incubated with a secondary goat-anti-rabbit peroxidise conjugated antibody . After development of immunoreactive bands by means of an enhanced chemiluminescence kit , relative band intensities were quantified using software “Image J”. The difference in intensity between Pre and Post exercise condition was calculated.Protein concentration of all four fractions was determined using the DC-Protein Assay Kit . A preliminary test revealed that the relevant proteins were present in the cytosolic fraction that therefore was used in subsequent western blot analysis. A total of 150 µg protein (fraction 1) was loaded fivefold into the lanes of a 4–12% Bis-Tris gel  next to the Precision Plus Protein™ Standard . Proteins were separated according to their charge and mass in a 1 x MOPS running buffer  and transferred to a polyvinylidene fluoride membrane (0.45 µm pore size) and probed with antibodies against total eNOS , phosphorylated eNOSSer2O3), which is formed by autoxidation of NO in air As described by Kojima et al. −1, viscosity 32 mPa*s at 37°C; Mechatronics, Netherlands) for deformability measurements. All measurements were performed at physiological pH 7.4 and 37°C. Deformability was determined at various physiological and supra-physiological fluid shear stresses by laser diffraction analysis using the laser-assisted optical rotational red cell analyzer . The LORRCA has been described in detail elsewhere –1, where L and W represent length and width of the diffraction pattern, respectively. EI values were calculated for nine shear rates, in particular 0.3, 0.57, 1.08, 2.04, 3.87, 7.34, 13.92, 26.38 and 50 Pa. EI values were plotted as a function and according to Baskurt et al. Immediately after sampling, venous heparinized blood was mixed  with an isotonic viscous medium (0.14 mM Polyvinylpyrrolidone (PVP), osmolality 300 mOsmol*LAdditionally, Post AMRT heparinized blood samples were also incubated with either PBS or wortmannin (c = 10 µM) for 30 min at 37°C to inhibit PI3 kinase. RBC deformability was measured after 0 and 30 min using the LORRCA as described previously.P<0.05.Statistical analyses of the data were performed by using statistics software packages . Data were analyzed by paired Student’s t-test to determine the significance of any differences between the Pre and Post situation. For comparison of wortmannin data one-way analysis of variances (ANOVA) was applied. Descriptive statistics of the data is presented as mean ± standard error of means (S.E.M.) unless described otherwise. Statistical differences were considered to be significant for values of Immunohistochemical straining was performed Pre and Post AMRT to investigate whether moderate exercise influences RBC-NOS activation pathway.473AktSer. To determine the impact of the intervention on Akt kinase activation, phosphorylation of Akt kinase on its Ser473 residue was examined before and after the test. The statistical analysis of phosphoAktSer473 revealed that the value significantly increased from 1.16±0.29 au Pre exercise to 1.61±0.39 au Post exercise  vs. Post: 10.22±1.78 au, P>0.05, 1177 showed significantly increased levels Post exercise compared to Pre exercise  and Post situation  is a prominent target of NO in endothelial cells P<0.05, ex-vivo results revealed that moderate exercise activates both Akt kinase and RBC-NOS. We thus performed in vitro experiments to verify the observed in vivo results. The conducted in vitro approach investigated RBC-NOS activation via the PI3 kinase/Akt kinase pathway. For this purpose phosphorylation of RBC-NOS at Ser1177 was investigated after PI3 kinase inhibition using wortmannin P<0.01, The Western Blot procedure served as an additional method, beside immunohistochemical approaches, to investigate whether moderate exercise influences RBC-NOS activation pathway and was performed Pre and Post AMRT. For the determination of relative intensity all obtained Pre-values were set to “1”. The corresponding Post-values were then calculated as the quotient of the Pre-values.473 , RBC-NOSSer1177 , and cGMP , were significantly increased compared to Pre-AMRT (P>0.05) and RBC-NOSThr495  remained unchanged  and directly after the AMRT intervention (Post). Due to an increased level of RBC-NOS phosphorylation at the SerThe addition of the indirect RBC-NOS inhibitor wortmannin Post AMRT caused a down-regulation in NO production in human RBCs .P<0.05, P<0.05, RBC deformability is crucial for the passage of RBCs through the microvasculature bed in vitro wortmannin-treated samples significantly decreased from 0.61±0.002 (0 min) to 0.59±0.006 . EI max of control samples was determined to be 0.61±0.003 (0 min incubation) and 0.61±0.003 after the 30 min incubation . SS ½ of control samples was 2.36±0.09 (0 min) and 2.40±0.09 after the 30 min incubation (P>0.05; Maximal RBC deformability of  P>0.05; . In wort P>0.05; .NO generation and –mediated signaling are highly important for the regulation of the peripheral vascular bed in vitro that defined shear stresses regulate and manipulate the activation of RBC-NOS with subsequent NO production in vivo by moderate physical exercise on RBC regulation, specifically on RBC-NOS activation, NO generation, and subsequently on RBCs rheological characteristics.Although it has been shown in vivo effect of reinforced shear stress, caused by moderate exercise, on RBC-NOS protein, its activations sites, and –mediated NO signaling to outline that moderate physical exercise exerts a positive influence on the regulation of this biologically important enzyme.In the present study, we therefore studied the 1177, indicating increased enzyme activity 495 residue, indicating inhibition of NOS enzyme activity The phosphorylation of RBC-NOSSer1177 phosphorylation and thus eNOS activation depends on the phosphorylation of Akt kinase at its Ser473 through PI3 kinase in vivo phosphorylation of RBCs Akt kinase at Ser473 significantly increased after AMRT, indicating a direct molecular interaction between phosphorylation of Akt- and RBC-NOS activation. These data complement the study of Mihov and colleagues who showed, using an in vitro assay, that pharmacological stimulations increase Akt kinase activity through PI3 kinase activation in RBCs Next, signaling pathways that contribute to the activation of the RBC-NOS under exercise conditions were aimed to be identified. In endothelial cells eNOSSer2y purinergic receptors, thereby resulting in synthesis of endothelial NO NO, as an unstable radical, reacts rapidly within the cell The function of cGMP in RBCs is only speculative, but it is conceivable that RBC -cGMP also influences vascular tonus possibly by activating potassium channels that control membrane potentials in response to altered intracellular calcium Our data on human RBC-NOS modulation are the first to add important and molecular knowledge to the question whether exercise-induced increased shear stress promotes the activation of Akt kinase and RBC-NOS in RBCs thereby initiating beneficial alterations of RBC rheological properties.in vivo results on RBC-NOS-related signaling, we conducted additional in vitro experiments by applying the PI3 kinase/Akt kinase inhibitor wortmannin 1177 phosphorylation. In line with these observations, NO production and RBC deformability were significantly decreased indicating a causal relation between RBC-NOS activation and RBC deformability that depends on PI3/Akt kinase signaling.To verify and support our obtained human 1177. Activated RBC-NOS generates NO in RBCs that is mandatory to beneficially promote RBC rheological functions, e.g. deformability.The novel key findings of this study are that moderate physical exercise is a sufficient stimulator for the activation of RBC-NOS directly resulting in improved RBC rheological properties. The molecular mechanisms are highly similar to those observed in endothelial cells. Exercise-induced shear stress results in activation of the PI3 kinase/Akt kinase pathway that in turn activates RBC-NOS by phosphorylation at SerTherefore, the presented data gain important insights into the molecular pathways underlying RBC deformability. This knowledge is of great interest, because nowadays physical exercise programs are fundamental for the treatment of patients suffering from cardiac and cardiovascular diseases who may benefit from improved rheological properties. Further elucidations are required to investigate the effect of chronic exercise interventions on the discussed molecular RBC-NOS activation pathways and resulting biological functionality of RBC-NO- produced NO.


Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants.Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins.The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity. Protein modifications play important roles in biological process, and can serve as diagnostic indicators of pathological events . HoweverPresented here is a quantitative mass spectrometric immunoassay for cystatin C variants. Cystatin C is a serine proteinase inhibitor belonging to the type 2 cystatin gene family ,8. It isIn the past we have utilized homologous proteins from other animal species that are recognized by the same antibody that binds the human antigen, so that upon spiking of the IRS into the analytical sample, the human protein and its animal homologue are retrieved by the single antibody. The two proteins register in the same region of the mass spectrum, but at a slightly different m/z value ,19,20. HThe first step in the assay optimization was determination of the ratios of the immobilized cysC and BL antibodies in the affinity pipettes. The general idea is that there should be more CysC antibody so that it can accurately quantify the varying concentrations of CysC in the samples; and less BL antibody because the amount of BL spiked in the samples can be easily controlled. After several empirical iterations, which involved immobilizing both CysC and BL antibodies in various mass ratios onto the microcolumns, it was determined that the optimal mass ratio of the two antibodies was 8.5:1 (CysC:BL). The optimal concentration and volume of BL spiked into the samples were determined next, by starting first with fixed spike volumes containing increasing BL concentrations , and then using fixed concentrations of BL but increasing the spiking volumes . From these experiments, the optimal BL concentration and volume were determined to be 10 mg/L and 5 μL, respectively. At this concentration, the BL present in the analytical sample saturates the anti-BL antibody, thus producing constant signals in the resulting mass spectra (this is an important prerequisite for the IRS). Next, various volumes  and dilutions  of human serum or plasma were utilized to determine the optimal sample volume into the analytical samples. It is very important not to saturate the anti-CysC antibody so that increased concentrations of cysC can be readily quantified with the assay. It was determined that 5 μL of serum or plasma, diluted 2-fold into assay buffer, provided the best results. At normal concentrations of cystatin C in plasma (~1 mg/L), these volume and concentration translate into less than 200 fmoles of cystatin C in the analytical samples.2 = 0.998) and standard error of estimate (SEE = 0.0708). It was found that that this range of the standard curve was sufficient to determine the concentration of cystatin C in all of the examined human plasma and serum samples. From the ratios of the CysC/BL peak heights and using a standard curve that was generated in parallel with the analytical samples analyses, the concentrations of cystatin C and its variants in the samples were determined. Operationally, we first determined the CysC/BL peak heights ratios for each cysC form in the sample, summed them up, determined the total cystatin C concentration using the standard curve, and then determined the concentration of the individual forms based on their percentage of the total cystatin C concentration.The standard curve was optimized and determined in parallel with the sample volume optimization. After several iterations, a 6-point standard curve was selected, spanning the range from 0.0312 to 1.0 mg/L cystatin C. An example of the standard curve, along with representative mass spectra, is shown in Figure A typical mass spectrum resulting from the cystatin C mass spectrometric immunoassay of a human plasma sample is shown in Figure The intra-assay precision (within-run) was determined by analyzing three plasma samples, in triplicates, each with a single standard curve. The inter-assay precision (run-to-run) was determined by analyzing one plasma sample three times, on different days, with separate standard curves each time. The results are shown in Table The fully-developed, characterized, and validated cystatin C mass spectrometric immunoassay was then used to analyze a set of human plasma samples in a high throughput mode. Forty-four sodium heparin plasma samples were utilized to prepare 88 analytical samples  and placed in columns 2-12 of a 96-well microplate. The standard curve samples were placed in column 1, along with a control sample with a known cystatin C concentration. The 96 cystatin C assays were executed in parallel using the Multimek 96 channel pipettor. Following mass spectrometry analysis and spectra processing, a standard curve was constructed from the data in column 1, and the performance of the assay verified with the control sample. The concentrations of cystatin C and its variants were then individually determined, averaged  and are presented in Table a-priori for detection of specific peptides, which eliminates the ability to detect novel truncations and mutations. On the other hand, the assay and methodology presented here utilize a simplistic two-step approach to native and intact protein analysis - in many ways similar to that in well established enzymatic immunoassays. However, it is the last step of the assay - the mass spectrometric detection - that enables differentiation of the multiple protein variants. Enzymatic immunoassay are oblivious to protein modifications, unless an antibody is designed that specifically recognizes a single protein variant (a Herculean task by itself). The mass spectrometric immunoassays, on the other hand, are designed with detection of protein modifications in mind. With the added quantitative feature, these assays are poised to change the way we look at protein modifications and their role in diseases - one protein at a time.The assay presented in this works utilizes mass spectrometry to fully quantify cystatin C variants. Similar MS assays can also be designed for other proteins to enable full quantification of their post-translationally modified forms. There are other affinity-based MS methods for protein quantification. Among the earliest is SELDI, which utilizes MALDI target-bound antibodies for protein retrieval . In a siRabbit anti-human polyclonal antibody to cystatin C (cysC) was obtained from DAKO . Rabbit anti-human polyclonal antibody to beta-lactoglobulin (BL) was obtained from GeneTex . Recombinant human cystatin C was purchased from HyTest . Beta-lactoglobulin from bovine milk (Cat. No. L8005), 1,1' Carbonyldiimidazole (115533), TWEEN 20 (P7949), TRIS (T-6128), Dithiothreitol  and α-cyano -4-hydroxycinnamic acid (476870) were obtained from Sigma-Aldrich . Affinity pipettes fitted with porous microcolumns were obtained from Intrinsic Bioprobes . Phosphate buffered saline was obtained from Thermo Scientific . Sterile water (Cat. No. AB02120), acetone (AB00636), MES (AB01235), acetonitrile (AB00120) and trifluoracetic acid (AB02010) were purchased from American Bionalytical . Methyl-1 pyrrolidone-2 was obtained from EMD Chemicals . N-octylglucoside was obtained from Roche Applied Science . Cystatin ELISA kit was obtained from Biovendor . Sequencing grade modified trypsin was obtained from Promega .Autoflex II MALDI-TOF (linear spectra) and Autoflex II TOF/TOF MALDI-TOF (reflectron spectra) mass spectrometers . ELISA readouts were obtained on Cary 50 spectrophotometer equiped with a microplate reader accesory . The cystatin C ELISA was carried out according to the kit manufacturer instructions Derivatization of the affinity pipettes with antibodies and high-throughput mass spectrometric immunoassays were performed on a Multimek 96 automated 96-channel pipettor . Manual mass spectrometric immunoassays were performed with the help of an 8-channel Finnpipette Novus multichannel pipetter . Mass spectrometry was performed on an Forty-four Na-heparin human plasma samples and eighteen human serum samples were obtained from ProMedDX . The samples were collected at certified blood donor and medical centers and designated as normal based on their non-reactivity for common blood infectious agents and the donor information itself. The samples were labeled only with a barcode and supplied with an accompanying specification sheet containing information only about the gender and age, ensuring proper privacy protection.Ninety-six affinity pipettes were mounted on the head of the Multimek 96 pipettor and initially rinsed with 200 mM HCl , followed by a water rinse (5 cycles) and acetone rinse (5 cycles). To activate the surface of the microcolumns contained within, the pipettes were immersed into a tray containing 100 mg/mL 1,1' Carbonyldiimidazole (in methyl-1 pyrrolidone-2), and 500 cycles of 100 μL aspirations and dispenses through each affinity pipette were performed. Two rinses with methyl-1 pyrrolidone-2  and a final rinse with acetone  followed. The affinity pipettes were then immediately immersed into a microwell plate containing the antibodies solutions  and 800 cycles of aspirations and dispenses of 50 μL volumes were performed to bind the antibodies to the activated microcolumns surfaces. Two rinses with 60 mM HCl followed , ending with two final rinses with assay buffer . The antibody-derivatized pipettes were stored at 4°C until used.For the generation of the standard curve, a solution containing 1.0 mg/L recombinant cystatin C was prepared (Standard #1) and serially diluted with assay buffer to 0.5 mg/L (Standard #2), 0.25 mg/L (Standard #3), 0.125 mg/L (Standard #4), 0.0625 mg/L (Standard #5), and 0.0312 mg/L (Standard #6). Five microliters of each of these standards were added to microtubes containing 140 μL assay buffer and 5 μL of 10 mg/L beta-lactoglobulin. For the analytical samples, the standard solutions were substituted with human plasma or serum sample (diluted 2-fold into assay buffer).The antibody-derivatized affinity pipettes were mounted onto the head of the Multimek pipettor and initially rinsed with assay buffer . Next, the pipettes were immersed into a microplate containing the samples and 100 aspirations and dispense cycles were performed (100 μL volumes each) allowing for affinity capture of cystatin C and beta lactoglobulin. The pipettes were then rinsed with assay buffer (100 cycles), and twice with water (10 cycles each). In preparation of elution, the affinity pipettes containing the retrieved protein were rinsed with 1 mM N-octylglucoside  in order to homogenize the subsequent MALDI matrix draw and elution, by completely wetting the porous microcolumns inside the pipettes. For elution of the captured proteins, 6 μL aliquots of MALDI matrix (25 g/L α-cyano-4-hydroxycinnamic acid in aqueous solution containing 33% (v/v) acetonitrile, and 0.4% (v/v) trifluoroacetic acid) were aspired into the affinity pipettes, and after a 10 second delay , the eluates from all 96 affinity pipettes containing the targeted proteins were dispensed directly onto a 96-well formatted MALDI target. Following air-drying and visual inspection of the sample spots, linear mass spectra were acquired with a delayed extraction mode using a 1.7 kV draw out pulse, 200 ns delay, and a full accelerating potential of 20 kV. Five mass spectra were acquired from each sample spot, each spectrum consisting of three-hundred laser shots. The mass spectra were internally calibrated with the singly and doubly charged beta-lactoglobulin signals, and further processed (baseline subtracted and smoothed) with Flex Analysis software . The peak heights for the cystatin C and beta-lactoglobulin signals were measured and entered into an Excel spreadsheet. The ratios of the cysC/BL peak heights were calculated, and the average ratio for each sample determined. A standard curve was generated by plotting the CysC/BL ratios against the concentration of the human cystatin C standards, and the data was fitted with a linear trendline using Sigma Plot . This standard curve was then utilized to determine the absolute concentration of cystatin C and its variants in the analytical samples.Tryptic digestion was performed directly on the cystatin C eluates deposited onto the MALDI target after the mass spectrometric immunoassay of the human serum samples . A 10 μLBL: beta-lactoglobulin; CysC: cystatin C; IRS: internal reference standard; MS: mass spectrometry.The authors declare that they have no competing interests.OT performed the experiments and analyzed the data. DN designed the experiments and drafted the manuscript. All authors read and approved the final manuscript.OT's permanent address is at the Institute of Chemistry, Faculty of Natural Sciences, Sts. Cyril and Methodius University, Skopje, Republic of Macedonia.


The migration of endothelial cells has been regarded as a potential target for the treatment of angiogenesis-related diseases. Previously, we demonstrated that norisoboldine (NOR), an alkaloid compound isolated from Radix Linderae, can significantly suppress synovial angiogenesis by selectively inhibiting endothelial cell migration. In this study, we evaluated the importance of various pathways in VEGF-induced endothelial cell migration using specific inhibitor. VEGF-induced endothelial cell migration and sprouting were significantly inhibited by H-89 (an inhibitor of protein kinase A (PKA)) but not by inhibitors of other pathways. NOR markedly suppressed VEGF-induced intracytoplasmic cAMP production and PKA activation and thereby down-regulated the activation of downstream components of the PKA pathway, including enzymes  and the transcription factor NF-κB. Moreover, the transcription activation potential of NF-κB, which is related to IκBα phosphorylation and the disruption of the p65/IκBα complex, was reduced by NOR. Meanwhile, NOR selectively inhibited the expression of p-p65 (ser276) but not p-p65 (ser536) or PKAc, indicating that PKAc participates in the regulation of NF-κB by NOR. Co-immunoprecipitation and immunofluorescence assays confirmed that NOR inhibited the formation of the PKAc/p65 complex and thereby decreased p65 (ser276) phosphorylation to prevent p65 binding to DNA. Docking models indicated that the affinity of NOR for PKA was higher than that of the original PKA ligand. Moreover, the fact that H-89 improved Notch1 activation, but DAPT (an inhibitor of Notch) failed to affect PKA activation, suggested that PKA may act on upstream of Notch1. In conclusion, the inhibitory effects of NOR on endothelial cell migration can be attributed to its modulation of the PKA pathway, especially on the processes of p65/IκBα complex disruption and PKAc/p65 complex formation. These results suggest that NOR inhibit VEGF-induced endothelial cell migration via a cAMP-PKA-NF-κB/Notch1 signaling pathway. Angiogenesis is defined as the generation of new vascular growth by sprouting from pre-existing vessels 2) in the endothelial cell membrane. VEGFR2, a receptor tyrosine kinase, can activate a series of downstream signaling pathways, including the p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Jun-amino-terminal kinase/stress-activated protein (JNK/SAPK), AKT, protein kinase C (PKC) and protein kinase A (PKA) pathways Numerous endogenous factors are involved in the regulation of angiogenesis. VEGF, the most well-described pro-angiogenic factor, plays a crucial role in both physiological and pathological angiogenesis Lindera aggregata used in traditional Chinese medicine. Previously, we reported a beneficial effect of NOR on joint destruction in adjuvant-induced arthritis (AIA) rats and demonstrated that the inhibition of synovial angiogenesis contributed, at least partially, to the effects of NOR Norisoboldine (NOR) is the primary isoquinoline alkaloid constituent of Radix Linderae, which is the dry roots of In the present study, we investigated which pathway plays the key role in the regulation of VEGF-induced endothelial cell migration. Next, we clarified the effect of NOR on this pathway and explored the interplay between this pathway and the Notch1 pathway using specific inhibitors.1H- and 13C-nuclear magnetic resonance, and its purity was tested by high-performance liquid chromatography 165 was obtained from Peprotech . The other chemicals and reagents used were of analytical grade.NOR (>98%) was prepared by Prof. Guixin Chou . Its strEthical approval for this research was obtained from the Human Research Ethics Committee of Nanjing Maternal and Child Health Hospital, Nanjing Medical University. Written informed consent was obtained from the mothers. All experiments were conducted in accordance with the ethical regulations and current laws of China.2 flasks that were pre-coated with 0.2% (w/v) gelatin . The culture medium consisted of M199 supplemented with 20% (v/v) NBCS, 2 mM L-glutamine, 5 U/ml penicillin G, 5 µg/ml streptomycin sulfate and 7.5 µg/ml endothelial cell growth supplement (Sigma). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed after 24 h and every two days thereafter until confluence was achieved. All of the experiments in this study were performed using HUVECs within passages 3–5.Umbilical cords were obtained from Nanjing Maternal and Child Health Hospital of Nanjing Medical University . Endothelial cells were isolated from the cords as described previously 5 cells/ml and cultured in a humidified 5% CO2-containing atmosphere at 37°C for 12 h. They were treated with different concentrations of NOR  for 24 h. Aliquots of MTT  were added to each well, and the plates were incubated for an additional 4 h at 37°C. The supernatants were carefully removed, and DMSO (150 µl/well) was added into each well to dissolve the formazan crystals. The absorbance at 570 nm (A570 nm) was read using a Model 1500 Multiskan spectrum microplate reader .Cell viability was evaluated using MTT assays. HUVECs were plated into 96-well plates at a density of 2×105 cells/ml. They were treated with the indicated concentrations of NOR for 24 h. After treatment with NOR, the cell supernatants were collected, and the levels of LDH were measured using ELISA kits according to the manufacturer’s instructions.For LDH assays, HUVECs were seeded into 96-well plates at a density of 2×102 and then incubated in 5 ml EGM-2 for an additional 12–16 h. The beads were then re-suspended at a concentration of 200 cell-coated beads/ml in 2.5 mg/ml fibrinogen (Sigma) and 0.15 units/ml aprotinin (Sigma) and seeded in a 24-well plate (0.5 ml/well), and then 0.625 units of thrombin (Sigma) were added to the wells. Capillary-like sprouts from the beads appeared on day 2–3. Lumen formation began on approximately day 5–6. Over the course of 10–20 days, extensive networks were formed. The indicated drug treatments were added on day 3, and five random ×200 fields were photographed for each well on day 14. The number and length of sprouts were measured, averaged and compared.This experiment was conducted as described previously 5 cells/ml. Total RNA (0.5 mg) was isolated using Trizol reagent . Reverse transcription was performed using an oligo (dT)18 primer and M-MLV reverse transcriptase (Invitrogen) at 37°C for 50 min. The primer sequences were chosen as follows: hairy/enhancer-of-split related with YRPW motif (HEY) 1 primers, (sense) 5′-CGA GGT GGA GAA GGA GAG TG-3′ and (antisense) 5′-CTG GGT ACC AGC CTT CTC AG-3′; HEY2 primers, (sense) 5′-TTC AAG GCA GCT CGG TAA CT-3′ and (antisense) 5′-GGG CAT TTT ACT TCC CCA AT-3′; and β-actin primers, (sense) 5′-ACA TCT GCT GGA AGG TGG AC-3′ and (antisense) 5′-GGT ACC ACC ATG TAC CCA GG-3′. The thermal cycling programs were as follows: To amplify HEY1 and β-actin, the reactions were subjected to 32 cycles of 94°C for 1 min, at 55°C for 45 s and at 72°C for 1 min and to amplify HEY2, the reactions were subjected to 40 cycles of 94°C for 1 min, at 55°C for 1 min and at 72°C for 1 min. The amplified products were electrophoresed on a 1% agarose gel and visualized by GoldView™  and ultraviolet irradiation RNA was extracted from cells and converted to cDNA as follows: HUVECs in the logarithmic growth phase were placed into six-well flat-bottom tissue culture plates at a density of 2×10The levels of PKAc, p-PKA, p-src, p-VASP, p-eNOS, p65, p-p65 (ser276), p-p65 (ser536), IκBα, p-IκBα and Cleaved Notch1 protein were detected by western blot analysis as described previously HUVECs were treated with NOR or curcumin in the presence of VEGF for 24 h. Next, cells were lysed with coimmunoprecipitation lysis buffer (SunShineBio). Cell lysates (containing 800 mg protein) were incubated with 0.2–2 µg of the indicated antibodies overnight at 4°C and then with 20 µl Protein A/G Plus-Agarose (Beyotime) for another 4 h at 4°C HUVECs were treated with NOR or curcumin in the presence of VEGF for 24 h. Immunofluorescence assays were performed as described previously 5′-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3′; 3′-TCT CTA ACG GAC TGC AGT CTC TCG ATC-5′), respectively. Nuclear extracts from HUVECs were prepared using a Nucleoprotein Extraction Kit . The nuclear extracts mixed with probe were incubated with 1 µg/µl of poly (deoxyinosinic-deoxycytidylic) acid in binding buffer for 30 min at 4°C, resolved on a 6.5% polyacrylamide gel at 100 V for 1 h, and then transferred to a nylon membrane. For supershift assays, the nuclear extracts from VEGF-treated cells were incubated with p65 or CREB antibodies for 30 minutes at 37°C before the complexes were analyzed by EMSA. The immunoreactive bands on nylon membranes were visualized by film exposure with enhanced chemiluminescence detection reagents The DNA-binding activities of NF-κB and CREB were confirmed with biotin-labeled oligonucleotide probes  and  for 10 h. The wound area was estimated using a light microscope  equipped with a digital camera, and three measurements of wound area were made at randomly chosen points. The extent of wound closure was presented as the percentage by which the original scratch area had decreased at each measured time point HUVECs were cultured in a six-well plate at a density of 2×105 cells/ml. The cells were treated with different concentrations of NOR or curcumin in the presence of VEGF for 24 h. After treatment, cell lysates were collected, and the levels of cAMP and activity of PKA were measured using ELISA kits according to the manufacturer’s instructions.HUVECs were seeded into 96-well plates at a density of 2×10http://www.rcsb.org/pdb/home/home.do) and docked NOR with PKA in AutoDock4.2. The original ligand in the complex structures was used as a reference compound to judge the affinity of NOR for the PKA target. The binding site was defined as a 40×40×40 Å cube centered on the occupied space of the original ligand with a spacing of 0.375 Å between the grid points.To further understand how NOR affects the PKA pathway, a preliminary in silico study was performed to identify potential targets. AutoDock4.2 software is commonly used for these analyses due to its automated docking capability. In this study, the 3D structure of NOR was generated and minimized in the MMFF94 force field by Discovery Studio, and the 2D structures of NOR were constructed and hydrogen atoms were added using ChemOffice software. We then used the protein-ligand complex crystal structures of PKA from the RCSB Protein Data Bank , RelB and c-Rel. In cytoplasm, they exist as homo- and hetero-dimers. The most common dimer is the p65–p50 complex To examine the disruption of the p65/IκBα complex, the effect of NOR on IκBα phosphorylation was first investigated. The results showed that NOR reduced the phosphorylation of IκBα but did not affect the protein expression of IκBα. Coimmunoprecipitation assay also revealed that NOR prevented the VEGF-induced disruption of the p65/IκBα complex, suggesting that NOR can inhibit NF-κB transcriptional activation by regulating the p65/IκBα complex. To evaluate the formation of the PKAc/p65 complex, the effect of NOR on the expression of p-p65 (ser276), p-p65 (ser536), p65 and PKAc was investigated. It was observed that NOR inhibited VEGF-induced p65 (ser276) phosphorylation. However, p-p65 (ser536), p65 and PKAc protein levels were not influenced by NOR, indicating that the regulation of NOR on p65 (ser276) is selective; this residue has also been reported to be selectively phosphorylated by PKAc A docking simulation using AutoDock4.2 suggested that a direct interaction between PKA and NOR-S and NOR-R occurs at the active site. Furthermore, the docking scores between PKA and NOR-S/NOR-R (5.06/4.96) calculated by the docking program were higher than that (4.61) of the original PKA ligand. Although the mechanism of interaction between NOR and PKA needs to be confirmed by a future X-ray crystallographic study, these results suggest that NOR inhibits PKA more strongly than its ligand. According to the docking program, the binding residues that interact with NOR-S and NOR-R were GLU127, ASP184, and LYS72 of PKA ; these rIn our previous studies, NOR was shown to promote VEGF-induced Notch1 activation and thereby showed an anti-migration activity In summary, the present study demonstrated the crucial role of the PKA pathway in VEGF-induced endothelial cell migration. The inhibitory effect of NOR was attributed to its modulation of the PKA pathway, key points of which were the disruption of p65/IκBα complex and the formation of the PKAc/p65 complex. These results suggest that NOR inhibits VEGF-induced endothelial cell migration via a cAMP-PKA-NF-κB/Notch1 signaling pathway.


These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca Chemotaxis, the directed migration of cells driven by a gradient of external factors, is critical for the initial phases of innate immunity in which neutrophils sense chemoattractant mediators and migrate from the circulation through the endothelium to combat invading pathogens at sites of infection in vitro, as they are terminally differentiated and have a short life span. Accordingly, studies of the functional roles of PAK in human neutrophils have been restricted to the use of gene transfection/knockdown strategies in leukemic cell lines. While PAK1- and PAK2-knockout mice have recently been established Most chemoattractants, including the bacteria-derived chemotactic peptide f-Met-Leu-Phe (fMLP), bind to G protein-coupled receptors (GPCRs) expressed on the neutrophil surface. GPCR-ligand binding activates heterotrimeric G proteins and triggers various intracellular signaling pathways. The Rho family of GTPases, including Rac, Cdc42 and RhoA, have been shown to play key roles in the spatial and temporal regulation of neutrophil cytoskeletal remodeling downstream of chemoattractant receptors during chemotaxis In this study, we characterized the roles of the PAK signaling in relation to PI3K and Rho GTPase systems during fMLP-driven cytoskeletal reorganization in primary human neutrophils. Our data suggest that PAK2 is activated and accumulates to the neutrophil leading edge in response to fMLP to support Rac/Cdc42-mediated actin dynamics in a localized manner. In addition, PAK inhibition altered the subcellular localization of active RhoA and induced aberrant formation of vinculin-rich complexes. PAK kinase activity played a critical role in chemotaxis of human neutrophils as PAK inhibition led to a loss of directionality, increased spreading and decreased migration speed, whereas Rac or PI3K inhibition resulted in impaired directionality or polarization, respectively. Taken together, these results suggest that PAKs establish a ‘frontness’ signal by negatively regulating the surface adhesion and Rho-dependent ‘backness’ signals in human neutrophils, thus providing a mechanism for the crosstalk between Rho-family GTPases in neutrophil cytoskeletal dynamics and cell migration.For immunohistochemistry and western blot experiments, anti-αPAK , γPAK , βPAK  and PAK4 (sc-28779) were purchased from Santa Cruz . Anti-PAK1/2/3 pThr423 (44–942G) was from Invitrogen . Anti-PAK2 pSer20 (2607) and myosin light chain2 pSer19 (3675) were from Cell Signaling . Anti-Rac1 (23A8) and Y27632 was from Millipore . Anti-active Rac-GTP (26903), active RhoA-GTP (26904) and active-Cdc42-GTP (26905) antibodies were from NewEast Biosciences . PF3758309 was prepared as previously described All human blood donors provided written informed consent in accordance with a protocol approved by the Oregon Health & Science University Institutional Review Board.2O for 30 sec, followed by the immediate addition of 10× PIPES buffer . After centrifugation at 400 g for 10 min, the pellet was resuspended in PMN buffer .Human neutrophils were purified as previously described After indicated treatments, human neutrophils were lysed in protein extraction reagent . Lysates were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 1 hr prior to incubation with primary antibodies overnight at 4°C. Following washing with TBS-T, membranes were incubated with the appropriate secondary antibodies. Subsequent immunoblotting was carried out as previously described 6/ ml) were incubated on fibronectin-coated surfaces at 37°C for 1 hr in the presence of inhibitors or vehicle (DMSO), followed by stimulation with fMLP. After fixation with 4% paraformaldehyde for 5 min, cells were permeabilized with 80% acetone for 3 min and blocked with blocking buffer  for 10 min. For phospho-PAK staining, cells were permeabilized with methanol. Neutrophils were stained with anti-PAK1/2/3 pThr423 (1∶100), anti-PAK2 pSer20 (1∶50), anti-Rac (1∶100), anti-active Rac-GTP (1∶100), anti-active RhoA-GTP (1∶200), anti-active Cdc42-GTP (1∶100) anti-myosin light chain2 pSer19 (1∶200), anti-vinculin (1∶100), or anti-actin (1∶100) in blocking buffer overnight at 4°C. Secondary antibodies conjugated with AlexaFluor 488 or AlexaFluor 546 (1∶500) and/or TRITC-phalloidin (1∶1000) in blocking buffer were added and incubated for 2 hr in dark. Coverslips were mounted onto glass slides and visualized with a Zeiss Axiovert fluorescent microscope. For TIRF microscopy, purified neutrophils were plated in 8-well chamber slide (Nunc) and treated with vehicle or inhibitors followed by stimulation with fMLP. Cells were fixed, permeabilized and stained for vinculin as described above. Vinculin immunofluorescence in a focal section of the neutrophil within ∼150 nm from the surface of coverslip was excited with a 488 nm-laser and detected via TIRF microscopy using a Nikon TE300 microscope equipped with a Nikon 60× oil immersion objective (NA = 1.49) and an electron multiplied CCD camera. For data presentation, fluorescent intensities of each image were adjusted based on signals detected in neutrophil samples treated without primary antibody. Image analyses were performed using Colocalization Finder of ImageJ, or a custom program in MATLAB .Purified human neutrophils (2×10Neutrophil chemotaxis was recorded as previously described 6/ ml) were loaded with fluo-4 AM  and plated on fibronectin-surface for 30 min at 37°C. After washing with Ca2+-free HBSS, cells were incubated for another 30 min at 37°C in PMN buffer and inverted onto an Insall chamber. Intracellular Ca2+ spikes in neutrophils were monitored for 15 min at 40×using a Zeiss Axiovert inverted fluorescent microscopy and analyzed using a custom MATLAB program.Human neutrophils  display a wide range of tissue distribution. PAK2 and PAK4 are ubiquitously expressed, while PAK1, PAK3, PAK5 and PAK6 expression are specific for tissues such as brain  and spleen (PAK1) We next examined whether the translocation of PAK2 to the leading edge is associated with kinase activation downstream of fMLP receptor signaling. In primary human neutrophils, PAK undergoes rapid phosphorylation at specific Ser and Thr residues in response to a number of stimuli Next, we investigated the mechanisms that regulate PAK activation in lamellipodia dynamics of activated neutrophils. To detect the activation of PAK, we performed immunofluorescence microscopy experiments using primary antibodies specific for phosphorylated PAK1/2 Thr423/402. Fluorescence microscopy revealed Threonine-phosphorylated PAK1/2 in neutrophils upon stimulation with fMLP, which localized to the actin-rich leading edge . To testIn neutrophils, Rac activation is in part regulated by PI3Ks which promote the localization of Rac-activating GEFs to the neutrophil leading edge Neutrophil morphological polarity is maintained through a balance of ‘frontness’ and ‘backness’ signals which have been shown to be mediated by the Rho GTPases Rac/Cdc42 GTPases and RhoA, respectively Active RhoA binds to and activates a number of downstream effectors including the Rho-associated kinase ROCK To stabilize the lamellipodium, small adhesion structures, known as focal complexes, are formed at the leading edge via Rac/Cdc42 activity which link integrins to surface and cytoskeletal proteins The above described PAK-mediated regulation of actomyosin contractility and surface adhesion led us to further examine the role of PAK in neutrophil chemotaxis. The determining components of efficient chemotaxis include directional sensing, morphological polarization and motility. Our data show that upon exposure to an fMLP gradient, neutrophils first uniformly spread and then immediately developed morphological polarity, in which the leading edge formed distinct lamellipodia to differentiate from the uropod . Within To better understand the roles of PI3K, Rho GTPases and PAK signaling pathways downstream of fMLP that regulate neutrophil chemotaxis, we characterized the directionality, polarity and motility of migrating neutrophils in the presence of pharmacological inhibitors. Neutrophil directionality was partially disrupted in the presence of the PI3K inhibitor, wortmannin . Althoug2+ transients via the activation of the Gβγ complex and phospholipase C, triggering Ca2+-dependent downstream pathways such as protein kinase C 2+ mobilization, neutrophils were loaded with the Ca2+-sensitive reporter dye fluo-4 AM and allowed to migrate under a fMLP gradient while intracellular Ca2+ release was measured for 15 min. As shown in 2+ release upon exposure to fMLP, followed by a gradually decreasing level of Ca2+ during chemotaxis. In contrast, neutrophils treated with the PAK inhibitor, PF3758309, displayed multiple peaks of Ca2+ release and failed to develop polarized morphology or migrate toward the fMLP gradient  The expression of the six PAK isoforms differs according to cell and tissue type, implying that PAK family isoforms may have specific and distinct functions in differing physiological contexts 2+-dependent myosin light chain kinase (MLCK), as the phosphorylation of MLCK by PAK has been shown to inhibit MLCK activity Antagonistic signals between Rac1/2, Cdc42 and RhoA stabilize the polarity of migrating cells by restricting actin polymerization to the leading edge and myosin-dependent contraction to the uropod In motile cells, adhesion strength is one of the determining factors for migratory ability. Focal complexes, consisting of integrins and adhesion complex proteins such as vinculin, stabilize the lamellipodium by supporting surface attachment, and contribute to efficient cell migration In summary, our data describe PAK as a key coordinator of cytoskeletal dynamics during cytoskeletal reorganization in human neutrophils and demonstrates that PAK mediates the crosstalk between the Rho GTPases to maintain the balance of front and back polarity signals. Future studies are required to characterize the functional roles of PAK in neutrophil inflammatory responses such as oxidative burst, degranulation, phagocytosis and neutrophil extracellular trap formation.Movie S1(MOV)Click here for additional data file.Movie S2(MOV)Click here for additional data file.