9 research outputs found
Regulatory Components of the Alternative Complement Pathway in Endothelial Cell Cytoplasm, Factor H and Factor I, Are Not Packaged in Weibel-Palade Bodies
<div><p>It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF —- but not factor H.</p></div
Changes in FH levels measured in plasma stored at 4°C for 26 days.
<p>Six individual units of plasma anticoagulated with citrate-phosphate-dextrose were stored at 4°C for 26 days. The FH levels were measured by fluorescent immunoassay on day 1 and after 14 and 26 days of storage at 4°C.</p><p>Changes in FH levels measured in plasma stored at 4°C for 26 days.</p
Colocalization coefficients of internal FH in HUVECs concurrently stained with VWF, β-actin and FI.
<p>Colocalization coefficients, Pearson’s (PCC) and Manders’ (M1 and M2), were measured in HUVEC images stained concurrently for FH and with VWF, β-actin, or FI, using the antibody pairs 2, 3 and 4 described under Internal HUVEC FH, VWF, β-actin and FI Detection in the Methods section. Data were analyzed in 2–4 images per experiment from 4–7 experiments with each antibody pair.</p><p><sup>a</sup>Indicates signal correlation in the two channels.</p><p>Colocalization coefficients of internal FH in HUVECs concurrently stained with VWF, β-actin and FI.</p
Intensity scatter plots of FH with FI indicate a high degree of colocalization in the HUVEC cytoplasm.
<p>HUVECs were formaldehyde-fixed, treated with Triton-X and stained concurrently for FH and FI. (A) FH was detected using 2 mouse monoclonal antibodies to human FH plus chicken anti-mouse AF IgG-647 (red). (B) FI was detected using polyclonal goat anti-human FI plus donkey anti-goat AF IgG-488 (green). (C) Shows the merged image detecting both FH (red) and FI (green) and the calculated values for the Pearson’s (PCC) and Manders’ (M1 and M2) correlation coefficients. (D) The intensity scatter plot of the merged image in (C) shows a single linear correlation indicative of a signal overlap. The population is skewed towards the y-axis on account of the higher green intensity values of FI detection.</p
Factor H is not present in HUVEC WPBs.
<p>Un-stimulated HUVECs were fixed with 1% p-formaldehyde and treated with 0.02% Triton-X to allow intracellular staining, and then stained with rabbit anti-human VWF antibody plus chicken anti-rabbit IgG-Alexa Fluor (AF) 488-secondary antibody. In panels A, B and C, VWF staining was followed by staining with two mouse anti-human FH monoclonal antibodies plus goat anti-mouse F(ab’)<sub>2</sub>-IgG-AF 647-secondary antibody. Cells were imaged at 100X and cell nuclei were detected with DAPI (blue). (A) FH detection, red-647 channel; (B) VWF detection, green-488 channel and (C) merged image with simultaneous VWF and FH detection. In panels D, E and F, VWF staining was followed by FH detection using goat anti-human FH antibody plus donkey anti-goat IgG-AF 647-secondary antibody and imaging at 60X. (D) FH detection, red-647 channel; (E) VWF detection, green-488 channel; and (F) merged image with simultaneous VWF and FH detection. Images shown are representative of VWF and FH staining in >10 separate HUVEC cultures.</p
Intensity scatter plots of FH with β-actin shows a colocalization in the HUVEC cytoplasm.
<p>HUVECs were formaldehyde-fixed, treated with Triton-X and stained concurrently for FH and <b>β</b>-actin. (A) FH was detected using 2 mouse monoclonal antibodies to human FH plus donkey anti-mouse AF IgG-488 (green). (B) <b>β</b>-actin was detected using polyclonal goat anti- <b>β</b>-actin plus chicken anti-goat antibody AF IgG-647 (red). (C) Shows the merged image detecting both FH (green) and <b>β</b>-actin (red) and the calculated values for the Pearson’s (PCC) and Manders’ (M1 and M2) correlation coefficients. (D) The intensity scatter plot of the merged image in (C) shows a single linear correlation indicative of a signal overlap.</p
Intensity scatter plots comparing FH with VWF do not show colocalization or a single correlation.
<p>HUVECs were treated and stained for FH (red) and VWF (green) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121994#pone.0121994.g001" target="_blank">Fig. 1AB</a>. Images are shown without the DAPI-stained nuclei. (A) FH detection with mouse anti-FH + goat anti-mouse AF-647 (red); (B) VWF detection with rabbit anti-VWF + chicken anti-rabbit AF-488 (green); and (C) Merged image of (A) and (B) detecting FH and VWF. The correlation coefficients, Pearson’s (PCC) and Manders’ (M1 and M2), for these 2 images are on the left bottom of the image. (D) The intensity scatter plot of the merged image in (C) showing 2 linear independent relationships of the data. The scattering of points along each of the axes indicates a low degree of signal overlap. The intensity delta is the distance from either outer color line to the plot’s center of density line and defines the area considered for colocalization. An intensity delta of 30 pixels was used for all of the colocalization measurements. The inset in (C) shows an enlargement of the plot’s origin.</p
FH release from HUVECs is not increased with histamine stimulation.
<p>Cell supernatants collected from 30 min to 7 hours after 100μM histamine stimulation or from unstimulated HUVECs in serum-free medium were quantified for <i>(</i>A) VWF levels and (B) FH levels by fluorescent immunoassays. Values shown (ng/ml) are means plus standard deviations from samples collected from 4–6 separate experiments. In (A) VWF levels were significantly different at each time point (P < 0.001 or 0.01) in samples from un-stimulated HUVECs compared to histamine-stimulated HUVECs.</p
VWF antigen levels increase in VWD patients after DDAVP administration without increases in FH levels.
<p>VWF and FH antigen levels were measured in citrated plasma samples from 6 VWD pediatric patients treated with DDAVP. VWF antigen levels were measured by standard clinical laboratory techniques and FH levels were determined by fluorescent immunoassays. For each patient, VWF and FH levels were measured in samples obtained prior to DDAVP administration, and in samples obtained 1 and 4 hours later. (A) Individual VWF levels in plasma samples. Values are in percentage of normal plasma VWF level. (B) The average VWF plasma levels of the 6 patients at the 3 different times, plus standard deviations. The mean response of VWF antigen to DDAVP was significantly greater 1-hour and 4-hours post-DDAVP compared with baseline (P = 0.01 and 0.007, respectively). (C) Individual FH levels of the same 6 patients before and after DDAVP administration. The values shown are means plus standard deviations from 3 FH immunoassays. There was either no change, or a decrease in plasma FH concentrations, at 1-hour and 4-hours post-DDAVP. (D) The average FH plasma level of the 6 patients at the 3 different time points. There was no statistically significant difference in the mean responses of FH to DDAVP among the 3 time points (P = 0.3 for baseline to 1-hour and P = 0.2 for baseline to 4-hours).</p