Artigo Acesso aberto Revisado por pares

Exocytosis of Endothelial Lysosome-Related Organelles Hair-Triggers a Patchy Loss of Glycocalyx at the Onset of Sepsis

2015; Elsevier BV; Volume: 186; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2015.10.001

ISSN

1525-2191

Autores

Joseph Zullo, Jie Fan, Tala Azar, Wanyi Yen, Min Zeng, Jun Chen, Brian B. Ratliff, Jun Song, John M. Tarbell, Michael S. Goligorsky, Bingmei M. Fu,

Tópico(s)

Traumatic Brain Injury and Neurovascular Disturbances

Resumo

Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality. Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality. Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. It afflicts >700,000 individuals annually in the United States alone, has mortality rates of 30%, and is the 11th leading cause of death. One of the key molecular causes of Gram-negative septicemia is endotoxin that consists of lipopolysaccharides (LPSs) bound with high affinity to LPS-binding glycoprotein. Complex LPS-binding glycoprotein is recognized by cognate receptor Toll-like receptor 4 and co-receptor CD14 on monocytes/macrophages and endothelial cells.1Aird W.C. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome.Blood. 2003; 101: 3765-3777Crossref PubMed Scopus (901) Google Scholar Considering the systemic nature of septicemia, vascular endothelium represents the first line of exposure to bacterial endotoxins.2Morrison D.C. Ulevitch R.J. The effects of bacterial endotoxins on host mediation systems. A review.Am J Pathol. 1978; 93: 526-618PubMed Google Scholar It responds to endotoxins with a complex system of danger signals, which are chronologically sequenced and spatially propagated.3Ratliff B.B. Rabadi M.M. Vasko R. Yasuda K. Goligorsky M.S. Messengers without borders: mediators of systemic inflammatory response in AKI.J Am Soc Nephrol. 2013; 24: 529-536Crossref PubMed Scopus (73) Google Scholar Functionally, these waves of danger signaling tend to secure proper organismal responses, both proinflammatory and anti-inflammatory. Among the earliest responses of activated endothelial cells to endotoxin are exocytosis of Weibel-Palade bodies (WPBs) and secretory lysosomes.4Kuo M.C. Patschan D. Patschan S. Cohen-Gould L. Park H.C. Ni J. Addabbo F. Goligorsky M.S. Ischemia-induced exocytosis of Weibel-Palade bodies mobilizes stem cells.J Am Soc Nephrol. 2008; 19: 2321-2330Crossref PubMed Scopus (47) Google Scholar WPBs are rod-shaped members of lysosome-related organelles (0.2 μm by 2 to 3 μm in size) characteristic to endothelial cells and containing an array of proteins, peptides, and cytokines, which can be released emergently on demand. The endothelial lysosomes contain acid/secretory sphingomyelinase, glycohydrolases, cathepsins, fucosidase, phosphatases, heparan sulfate (HS) sulfatase, among others, but mechanisms of exocytosis of lysosome-related organelles have only been partially elucidated. Endothelial surface glycocalyx (ESG) represents a superficial layer that consists of glycoproteins, proteoglycans, and glycosaminoglycans. Because of its unique location, this structure provides a passive barrier to water and solute transport, the interaction between circulating cells and the endothelial cells that form the inner wall of blood vessels, serves as a sensor of mechanical forces, such as shear stress and pressure, and represents a shielding instrument for cell surface receptors to prevent their hyperactivation.5Reitsma S. Slaaf D.W. Vink H. van Zandvoort M.A. oude Egbrink M.G. The endothelial glycocalyx: composition, functions, and visualization.Pflügers Arch. 2007; 454: 345-359Crossref PubMed Scopus (1223) Google Scholar, 6Becker B.F. Chappell D. Jacob M. Endothelial glycocalyx and coronary vascular permeability: the fringe benefit.Basic Res Cardiol. 2010; 105: 687-701Crossref PubMed Scopus (184) Google Scholar This structure is, however, quite vulnerable and tends to disintegrate after application of various stressors, such as endotoxins, ischemia/hypoxia/reperfusion, oxidative stress, among others. Damage to and modification of the ESG are observed in many diseases, including diabetes, ischemia, myocardial edema, chronic infectious diseases, atherosclerosis, and tumor metastasis.5Reitsma S. Slaaf D.W. Vink H. van Zandvoort M.A. oude Egbrink M.G. The endothelial glycocalyx: composition, functions, and visualization.Pflügers Arch. 2007; 454: 345-359Crossref PubMed Scopus (1223) Google Scholar, 6Becker B.F. Chappell D. Jacob M. Endothelial glycocalyx and coronary vascular permeability: the fringe benefit.Basic Res Cardiol. 2010; 105: 687-701Crossref PubMed Scopus (184) Google Scholar, 7Cai B. Fan J. Zeng M. Zhang L. Fu B.M. Adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel wall increases microvascular permeability via degradation of endothelial surface glycocalyx.J Appl Physiol (1985). 2012; 113: 1141-1153Crossref PubMed Scopus (35) Google Scholar, 8Chappell D. Heindl B. Jacob M. Annecke T. Chen C. Rehm M. Conzen P. Becker B.F. Sevoflurane reduces leukocyte and platelet adhesion after ischemia-reperfusion by protecting the endothelial glycocalyx.Anesthesiology. 2011; 115: 483-491Crossref PubMed Scopus (102) Google Scholar, 9Fu B.M. Tarbell J.M. Mechano-sensing and transduction by endothelial surface glycocalyx: composition, structure, and function.Wiley Interdiscip Rev Syst Biol Med. 2013; 5: 381-390Crossref PubMed Scopus (120) Google Scholar, 10van den Berg B.M. Vink H. Spaan J.A. The endothelial glycocalyx protects against myocardial edema.Circ Res. 2003; 92: 592-594Crossref PubMed Scopus (384) Google Scholar, 11VanTeeffelen J.W. Brands J. Vink H. Agonist-induced impairment of glycocalyx exclusion properties: contribution to coronary effects of adenosine.Cardiovasc Res. 2010; 87: 311-319Crossref PubMed Scopus (25) Google Scholar The disintegration of this structure predisposes to leukocyte adhesion, emigration, and tissue infiltration by polymorphonuclear cells, monocyte/macrophages, and lymphocytes. It also leads to hyperactivation of plasma membrane receptors by unhindered availability of ligands and further activation of danger signaling by endothelial cells. Until now, the ESG remained poorly studied because of difficulties in preserving its structure on tissue fixation, limited number of experimental tools for its visualization, and requirements for an ultra-high resolution microscope. Therefore, there is no confidently chartered time course of damage and disintegration of ESG. Yet, the precise chronology of the loss of ESG is of critical significance, because it determines whether and to what extent the loss of glycocalyx is involved in the early pathogenic steps of systemic inflammatory response or only in its maintenance or in both. We hypothesized that exocytosis of WPBs and lysosomes, being an early response of endothelial cells to endotoxin, results in the focal disintegration of ESG. This singular event hair-triggers an avalanche of secondary pathologic processes, such as attraction of leukocytes and platelets, thrombosis, increased vascular permeability. The proof of this hypothesis lies in the demonstration of two key facts, namely, that exocytosis of WPBs and lysosomes does indeed trigger the initial loss of glycocalyx and that maneuvers directed toward preservation of glycocalyx improve the course of sepsis and overall survival. The recent discovery and implementation of the stochastic optical reconstruction microscopy (STORM) offered us a unique opportunity to detect the integrity of glycocalyx at the molecular resolution. Data presented herein support the validity of this hypothesis. Immortalized human umbilical vein endothelial cells (HUVECs; ATCC, Manassas, VA) and bEnd3 mouse brain microvascular endothelial cells (ATCC) were used in these studies. Cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin, and 50 μg/mL streptomycin, and 2 mmol/L l-glutamine under conditions of 37°C and 5% CO2. Cells were seeded onto 50 μg/mL fibronectin-coated 35-mm glass-bottom culture dishes with 14-mm No.1.5 coverslips (Mat Tek Corp., Ashland, MA) at a density of 4 × 104 cells/mL and cultured until confluence. HUVECs were incubated with 50 to 75 nmol/L of LysoTracker Red (Invitrogen, Carlsbad, CA), in prewarmed (37°C) DMEM with 10% fetal bovine serum and 1% penicillin and streptomycin medium at 37°C for 30 minutes. After removing the medium, phenol red-free DMEM with 10% fetal bovine serum was added. Endothelial progenitor cells (EPCs) were isolated from developmental age embryonic day 7.5 to 7.8 murine embryos and generously provided by Dr. Antonis K. Hatzopoulos (Vanderbilt University, Nashville, TN).12Hatzopoulos A.K. Folkman J. Vasile E. Eiselen G.K. Rosenberg R.D. Isolation and characterization of endothelial progenitor cells from mouse embryos.Development. 1998; 125: 1457-1468PubMed Google Scholar EPCs were cultured in DMEM supplemented with 20% fetal bovine serum, 0.1 mmol/L β-mercaptoethanol, 1 mmol/L minimum essential amino acid at 37°C in 5% CO2. The animal study protocol was in accordance with the NIH Guide for the Care and Use of Laboratory Animals13Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar and approved by the Institutional Animal Care and Use Committee. For cecal ligation puncture (CLP) to induce sepsis, male mice (C57/BL6 age, >16 weeks) under isoflurane anesthesia had the cecal tip ligated with 4-0 silk suture. The cecum was punctured twice with a 21-guage needle, then gently squeezed. Wound was sutured, and mice were closely monitored by investigators with the use of a video camera. HA-hydrogels (Glycosan Biosystems, Salt lake City, UT) embedded with EPCs were implanted subcutaneously in the ears of sedated mice, as we reported previously.14Ghaly T. Rabadi M.M. Weber M. Rabadi S.M. Bank M. Grom J.M. Fallon J.T. Goligorsky M.S. Ratliff B.B. Hydrogel-embedded endothelial progenitor cells evade LPS and mitigate endotoxemia.Am J Physiol Renal Physiol. 2011; 301: F802-F812Crossref PubMed Scopus (23) Google Scholar Implantation of hydrogels occurred during the CLP and LPS in vivo procedures. A total of 1 million cells were delivered to each mouse. Ear implants were injected with 300 U/mL collagenase (Sigma-Aldrich, St. Louis, MO) and 100 U/mL hyaluronidase (Sigma-Aldrich) to permit mobilization of embedded cells immediately after the CLP procedure. The cell monolayers were quickly washed with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde/0.1% glutaraldehyde for 20 minutes, followed by 0.1% NaBH4 for approximately 7 minutes at room temperature. After washing three times with PBS, the cells were blocked with 2% goat serum for 30 minutes, followed by an overnight incubation at 4°C with monoclonal primary antibody for HS (dilution 1:100; 10E4 epitope; AMS Biotechnology, Abingdon, United Kingdom) and von Willebrand factor (vWF)-labeled WPBs (vWF/WPBs; dilution 1:100; Dako Denmark A/S, Glostrup, Denmark). After washing three times with PBS, the cells were incubated for 1 hour at room temperature with the fluorescence-conjugated secondary antibody. Alexa Fluor 488-conjugated secondary antibody to HS and 20 μmol/L fluorescein isothiocyanate-conjugated Triticum vulgaris lectin (fluorescein isothiocyanate–wheat germ agglutinin; Sigma-Aldrich) were used for confocal microscopy, and Alexa Fluor 647-labeled and ATTO 488-labeled secondary antibodies to HS and vWF/WPBs were used for STORM, respectively, followed by washes three times. Magic Red Cathepsin detection kit with the use of the fluorophore cresyl violet (Immunochemistry Technologies, Bloomington, IN) was used to detect cathepsin B in the media of cultured cells. In brief, cells were grown to 80% to 90% confluence in phenol-free complete media, before adding 5 μg/mL LPS. Media were collected and incubated with Magic Red Cathepsin detection kit at room temperature for 15 minutes. Fluorescence was detected at excitation/emission of 592/628 nm with the use of a Biotek synergyHT microplate reader and analyzed with the companion software (Gen5 version 1.11; Biotek, Winooski, VT). We imaged the ESG on the aorta of male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME). The detailed animal preparation was described in our previous study.15Yen W. Cai B. Zeng M. Tarbell J.M. Fu B.M. Quantification of the endothelial surface glycocalyx on rat and mouse blood vessels.Microvasc Res. 2012; 83: 337-346Crossref PubMed Scopus (65) Google Scholar After anesthesia, the mice were perfused with cold 1% bovine serum albumin-PBS to remove the blood and perfusion-fixed with 2% paraformaldehyde. Then the aorta was cut into approximately 1-cm long rings or prepared en face with a longitudinal cut. After washing with the PBS, the aortic rings were blocked for 30 minutes with 1% bovine serum albumin saponin solution. Then the aorta segments were incubated with mouse anti-HS antibody and anti-vWF/WPBs overnight. After a brief wash with PBS, the specimens were blocked for 30 minutes and incubated with the Alexa Fluor 488-labeled and Alexa Fluor 647-labeled goat anti-mouse IgG for 2 hours, respectively, to anti-HS and anti-vWF/WPBs, followed by washes three times. Longitudinal segments for en face preparations used fluorescein isothiocyanate–wheat germ agglutinin as an alternate to the anti-HS antibody. Samples were incubated with 20 μmol/L fluorescein isothiocyanate–wheat germ agglutinin for 30 minutes at 4°C, then washed three times with PBS immediately before imaging. For fluorescence imaging, cells were cultured in fibronectin-coated 60-mm glass-bottom cell culture dishes (MatTek Corp.) until confluence (2 to 3 days). To prepare EPC-conditioned medium (EPC-CM), EPCs were cultured in 60-mm cell culture dishes for 24 hours. After 24 hours, DMEM was replaced with serum- and phenol-free medium for an additional 24 hours of incubation. Thus, prepared CM was filtered with sterile cell strainer, divided into aliquots, and stored until use at −80°C. Individual HUVECs were selected and monitored by time-lapse videomicroscopy before and after application of 5 μg/mL LPS. For the LPS + NG-hydroxy-l-arginine (NOHA; Sigma-Aldrich) group, 100 μg/mL NOHA was added 20 minutes before applying 5 μg/mL LPS in the medium. For the EPC-CM10 and EPC-CM50 groups, 1:10 or 1:50 dilutions of EPC-CM were added 20 minutes before applying 5 μg/mL LPS in the medium. NOHA and EPC-CM were selected as treatment options on the basis of the previous demonstration of the nitric oxide donor's ability to curtail exocytosis of lysosome-related organelles16Matsushita K. Morrell C.N. Cambien B. Yang S. Yamakuchi M. Bao C. Hara M.R. Quick R.A. Cao W. O'Rourke B. Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor.Cell. 2003; 115: 139-150Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar and our previous demonstration of the therapeutic activity of EPCs against septic vascular damage.3Ratliff B.B. Rabadi M.M. Vasko R. Yasuda K. Goligorsky M.S. Messengers without borders: mediators of systemic inflammatory response in AKI.J Am Soc Nephrol. 2013; 24: 529-536Crossref PubMed Scopus (73) Google Scholar In another group of experiments, bEnd3 cells were incubated with 50 nmol/L LysoTracker Red for 15 minutes. After a wash with a fresh medium, 5 μg/mL LPS was added for 10, 30, and 60 minutes. Then the cells were fixed with 3.7% paraformaldehyde, immunolabeled with primary anti-HS and then Alexa Fluor 488-labeled secondary antibody and observed by confocal microscopy. Dihydroethidium fluorescence intensity changed after LPS treatment. HUVECs were cultured to confluence, labeled with dihydroethidium, and treated with LPS at concentrations that ranged from 0 to 10 μg/mL. The oxidation of dihydroethidium was evaluated by fluorescent plate reader with excitation/emission wavelengths set at 485 ± 20/620 ± 40 nm. The fluorescence intensity of various treatment groups at each time point was normalized against the unlabeled group for each time point. A Nikon Y-FL epifluorescence intravital microscope (Nikon Inc., Melville, NY) equipped with an intensified Cool SNAP HQ tube camera (Photometrics, Tucson, AZ) and 60× Nikon Plan Apo objective was used for microscopy and image acquisition. Intravital videomicroscopy was performed over a time period of 120 minutes with a time-lapse interval set to 1 minute. Fluorescence was recorded, and fluorescence intensity was quantified for lysosomal particles in the cells with the use of MetaVue software version 6 (Universal Imaging, Downingtown, PA). Lysosomal particle/point tracking analysis and measuring of lysosomal trajectories used the ImageJ software version 1.45m (NIH, Bethesda, MD; http://imagej.nih.gov/ij) with plugin Manual Tracking (Fabrice Cordelières, Institut Curie, Orsay, France). The lysosomes were selected from a large pool at random and with the only criterion that the candidate lysosome could be tracked unambiguously throughout the session. For example, two neighboring lysosomes in close proximity within the same plane could not be differentiated from each other. If this event occurred during the tracking, both lysosomes were excluded from analysis. Automation was attempted, using computerized particle tracking; however, results were inconsistent and subpar compared with manual tracking. Each lysosome was recorded in intervals of 2 minutes with the use of time-lapse video microscopy. All images were converted into negatives for better visualization. All data were expressed as means ± SEM. Differences in distances between groups at every time point were analyzed by two-way repeated measures analysis of variance, with time and groups as variables, and followed by the Holm-Sidak test for multiple comparisons. Comparison of trajectories among group differences tested by one-way analysis of variance, followed by the Holm-Sidak multiple comparison test. P < 0.05 was regarded as statistically significant. We used a Zeiss LSM 710/510 confocal microscope (Carl Zeiss, Jena, Germany) with 63×/NA1.4 or 40×/NA1.3 objective lens to image the ESG of endothelial monolayers in vitro and mouse aorta ex vivo that were fixed and mounted on a coverslip. With the use of a motorized substage on the confocal microscope, z-stack images were generated of the cell monolayers and vessels. As described by Yen et al,15Yen W. Cai B. Zeng M. Tarbell J.M. Fu B.M. Quantification of the endothelial surface glycocalyx on rat and mouse blood vessels.Microvasc Res. 2012; 83: 337-346Crossref PubMed Scopus (65) Google Scholar stacked confocal images were reconstructed and analyzed by ImageJ to quantify changes in ESG, lysosome, and vWF/WPBs distribution in cell monolayers and vessels. The algorithm created space-filling ellipses and circles to highlight areas depleted of glycocalyx. The automatized process used ImageJ. Briefly, a 63× confocal image was first converted to monochromatic, made into binary, and then processed with the use of a watershed transformation. The results allowed for large cell populations to be analyzed consistently, effectively, and without bias. All functions pertaining to the algorithm are inherent to ImageJ. We used a three-dimensional multicolor ultra-high resolution Nikon-STORM system to image the ESG and vWF/WPBs of bEnd3 monolayers. The system consisted of a Nikon TiE Inverted Microscope stand with internal encoded Z-drive with 25-nm resolution and Nikon Perfect Focus System. The high magnification and high numerical aperture objective 100×/NA1.494 (oil immersion) enables super resolution images by offering high transmission and superior chromatic aberration correction throughout a wide wavelength range, from near-ultraviolet to near-infrared. The secondary antibodies conjugated with the photo switchable dyes, Alexa Fluor 647 and ATTO 488, were used to identify HS of the ESG and vWF, respectively. Kaplan-Meyer survival curves were generated with male C57BL/6 mice subjected to CLP over a 72-hour observation period. Double-blind randomization and administration of treatment was performed for healthy controls, NOHA, EPC-CM, CLP, CLP + EPC-CM, and CLP + NOHA. Mice in each treatment group (n = 10 each) were monitored at hourly intervals and provided water and food ad lib. Studies were sufficiently powered to detect significant changes in fluorescence imaging. A calculated biological replica of n = 8 was sufficient to detect differences at α = 0.05, powered to 0.9 (PASS software version 12.0.10; NCSS version 9.0.15; NCSS Statistical Software, Kaysville, UT). Hypothesized means and SDs in ESG fluorescence intensity and ESG focal degradation sizes were based on preliminary data, and results were aggregated from the literature. An analysis of variance with an appropriate Bonferroni post hoc test was performed with NCSS (NCSS9 software; NCSS LLC), considering P ≤ 0.05 to be statistically significant. A log-rank test was performed for analysis of survival curves (NCSS9 software; NCSS LLC), considering P ≤ 0.05 as statically significant. Under basal conditions, endothelial cells labeled with lysotracker displayed Brownian movement of lysosomes. Application of LPS resulted in agitated lysosomal movement already after several minutes. Figure 1 shows the traces of several lysosomes (different colors indicate different lysosomes) under control and various treatments. Figure 2A shows representative traces of lysosomes over the course of 60 minutes under different conditions. Movement was traced in intervals of 2 minutes. The total length that a lysosome travels is defined as the mean square of the distance, which is the summation of the square of the distance in each step a lysosome travels during a specific time period (each step takes 2 minutes). The mean square of the distance is a commonly held mathematical relation, accounting for time, random motion, and vector distance. The purpose of squaring the distance is to avoid canceling out a distance that a particle travels in the opposite direction during an observed time period. The total length of the LPS group showed a significant increase compared with the control group, starting from 10 minutes (P < 0.05) (Figure 2B). The vector distance (square of the distance from the start point of the track to the end point) of the LPS group was significantly increased compared with control, indicating directional movement (Figure 2C). Pretreatment of endothelial cells with the medium conditioned by EPCs, at dilutions 1:10 and 1:50, significantly prevented LPS-induced lysosomal motility. This effect could be mimicked by the pretreatment with a donor of NO, NOHA (Figure 2, A–C).Figure 2Trace, total length, and vector length of lysosomal exocytosis in iHUVECs and DHE fluorescence intensity. A: Representative and normalized XY-position traces of labeled lysosomes tracking over 60 minutes. The trajectories of peripheral lysosomes show significantly longer tracks in LPS, LPS + NOHA, and EPC-CM10 and EPC-CM50 groups. B and C: Individual organelles were randomly selected from a time-lapse series obtained before and after application of LPS, NOHA, and EPC-CM. In control, movements were non-vectorial. After LPS, lysosomal movement became more dynamic and directional. When nitric oxide donor NOHA or EPC-CM was used for pretreatment before LPS, lysosomal movements were reduced. D: DHE fluorescence intensity after LPS treatment of cultured HUVECs. Data are expressed as means ± SEM. n = 4 to 5 per group and 30 lysosomal particles per cell (B and C); n = 12 for 10 and 2.5 μg/mL groups (D); n = 6 for other tested groups (D). *P < 0.05 versus total trajectory (B and C); †P < 0.05 versus control (B and C); and ‡P < 0.05 versus LPS + NOHA and EPC-CM10 and EPC-CM50 (B and C), differences tested by one-way analysis of variance followed by the Holm-Sidak multiple comparison test. *P < 0.05, **P < 0.01 versus all lower LPS treatment group(s) at indicated times within each time point (D); †P < 0.05 versus no LPS (D). CM, conditioned medium; DHE, dihydroethidium; EPC, endothelial progenitor cell; iHUVEC, immortalized human umbilical vein endothelial cell; LPS, lipopolysaccharide; NOHA, NG-hydroxy-l-arginine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The motility of the peripherally located lysosomes differed across the treatments, as illustrated by the representative traces (Figure 2A). The trajectories of peripheral lysosomes showed significantly longer distances during the same period of time (the time from one point to another is 2 minutes) in LPS-treated cells than those of the LPS + EPC-CM10 (and to a lesser degree EPC-CM50) or LPS + NOHA groups. Remarkably, these differences were observed already after 10 minutes (P < 0.05) (Figure 2, B and C). The observed changes in motility were preceded by the surge in production of superoxide radicals, as detected with the use of dihydroethidium fluorescence. Application of LPS resulted in an upstroke of fluorescence intensity already within 5 minutes (Figure 2D). Having established the rapidity of LPS-induced exocytosis of peripheral lysosomal pool and demonstrating the ability of EPC-CM or NOHA to halt it, we next addressed a critical question: How early after application of LPS is the loss of ESG detected? STORM microscopy is characterized by its super-resolution to discern detailed structures and distributions at approximately 20 nm horizontal and approximately 50 nm vertical resolution. Results obtained with STORM are summarized in Figure 3. It provides representative images of ESG in control and 10-minute LPS-treated cells costained with antibodies to vWF. For the control group (Figure 3A), ESG was richly represented by fluorescently labeled anti-HS antibody. However, after 10 minutes of LPS treatment, vWF became externalized and was surrounded by the halo areas of lost HS-decorated ESG. Such a nano-scaled picture for the ESG and exocytotic vWF/WPBs was first observed because of the super-resolution STORM. Figure 3B demonstrates the relative intensity of anti-HS and vWF/WPBs after LPS treatment compared with the control. It indicates that after 10 minutes of LPS treatment, HS decreases to approximately one-third, whereas vWF increases to greater than twofold of their control values, respectively. With the use of confocal microscopy, similar results were obtained. Figure 4A illustrates (two-dimensional intensity distribution) the loss of ESG at the sites where lysosomal markers are detected in nonpermeabilized cells. The same was true for vWF and HS staining when analyzed by the confocal microscopy (Figure 4B). The relative intensity of the HS decreased when the time of LPS treatment increased, whereas the relative intensity of lysosomes and vWF/WPBs increased (Figure 4, A and B). The longer the length of LPS treatment, the more was the ESG loss and detected exocytosis of lysosomes and vWF/WPBs. The enlarged view in Figure 4C shows string-like externalized vWF; no colocalization of HS and vWF markers was found. Increased levels of cathepsins in the media were found after 10 minut

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