<b>The long-term effect of DTT on capillary-like tube formation</b>.

<div><p> <b>A</b> Porcine aortic endothelial cells were seeded on growth factor-reduced Matrigel and stimulated with the vehicle alone for 1 day (I) or 1 mM DTT (II–V) and observed at (II) 1, (III) 7, (IV) 14, (V) 21, and (VI) 28 days.</p> <p> <b>B</b> Histograms showing the quantitative analysis of the mean total tube length per 40× field.</p> <p>Results are the mean (±SEM, <i>bars</i>) of three different fields.</p> <p>*, <i>p</i><0.01;**, <i>p</i><0.001 <i>versus</i> control.</p></div

<b>The effect of DTT and vitamin C on NO-release and eNOS activation in endothelial cells</b>.

<div><p>(<b>A</b>): Porcine aortic endothelial cells (PAEC) were grown to confluence in 24-well plates and stimulated with increasing concentrations of DTT and vitamin C for 1 hour and NO measured in the supernatants.</p> <p>(<b>B</b>): Total NO release from PAEC following 1 hour incubation with 500 µM DTT and vitamin C in the absence or presence of the NOS inhibitor, L-NNA (500 µM).</p> <p>NO was measured directly using a Sievers NOA 280 chemiluminescene analyzer and the results were corrected for background levels of NO present in the medium.</p> <p>Results are the mean (±SEM, <i>bars</i>) of two experiments (n = 12), *<i>p</i><0.01 <i>versus</i> control.</p> <p>(<b>C</b>): Western blots showing the effect of increasing concentrations of DTT, vitamin C or phorbol myristate acetate (PMA) on the phosphorylation of eNOS (phospho-eNOS) at serine-1177 in PAEC after 15 min.</p> <p>(<b>D</b>): Western blot showing the presence of S-nitrosylated proteins of PAEC pre-treated with 30 µM SNAP (lanes 1–3) or vehicle (lane 4) for 1 hour and then incubated with 500 µmol/L DTT (lane 2) or 500 µM vitamin C (lane 3) or vehicle (lanes 1 and 4) for 1 hour.</p></div

<b>The effect of DTT on </b><b><i>in vitro</i></b><b> tube formation</b>.

<div><p> <b>A</b> Endothelial cells grown on growth factor-reduced Matrigel were stimulated with (I) the vehicle alone (control) and with (II) 10, (III) 100, (IV) 200, (V) 500, and (VI) 1000 µM DTT.</p> <p>Cells were observed 24 hours after stimulation and results recorded digitally.</p> <p> <b>B</b> Quantitative analysis of the results representing the mean total tube length per 40× field.</p> <p>The results are the mean (±SEM, <i>bars</i>) of three different fields.</p> <p>*, <i>p</i><0.01 <i>versus</i> 200 µM DTT.</p></div

<b>DTT-induced capillary-like tube formation is dependent on guanylyl cyclase and PKG activity</b>.

<div><p>Porcine aortic endothelial cells (PAEC) plated on growth factor-reduced Matrigel were pre-incubated for 30 minutes with the NOS inhibitors, L-NNA (500 µM) and L-NIO (10 µM), guanylyl cyclase inhibitor, ODQ (10 µM), or PKG inhibitor, KT-5823 (1 µM), at 37°C in medium containing 0.2% BSA prior to stimulation with 500 μM DTT or vitamin C for 24 hours.</p> <p> <b>A</b> Vehicle alone (I), DTT (II), DTT and L-NNA (III), DTT and L-NIO (IV), <b>B</b> Histogram showing the mean total tube length per 40× field of PAEC following stimulation with DTT in the presence of L-NNA and L-NIO following 24 hour incubation.</p> <p> <b>C</b> Vehicle alone (I), DTT (II), DTT and ODQ (III), DTT and KT-5823 (IV), <b>D</b> Histogram showing the mean total tube length per 40× field of PAEC following stimulation with DTT in the presence of ODQ or KT-5823 following 24 hour incubation.</p> <p> <b>E</b> Histogram showing the mean total tube length per 40× field of PAEC following incubation with vitamin C in the presence of L-NNA, L-NIO, ODQ or KT-5823.</p> <p>The results are the mean (±SEM <i>bars</i>) of three different fields.</p></div

<b>The effect of vitamin C on capillary-like tube formation</b>.

<div><p> <b>A</b> Porcine aortic endothelial cells seeded on growth factor-reduced Matrigel were stimulated with either the vehicle alone (I), 10 µM CuSO₄ (II), 200 µM vitamin C (III), 200 µM vitamin C and 10 µM CuSO₄ (IV), 500 µmol/L mmol/L vitamin C (V) and 500 µM vitamin C and 10 µM CuSO₄ (VI), for 24 hours.</p> <p> <b>B</b> Histogram showing the mean total tube length per 40× field.</p> <p>The results are the mean (±SEM, <i>bars</i>) of three different fields of three independent experiments.</p></div

Characterization of the chimeric immune system.

<p>Blood, bone marrow and splenic digests were analysed by flow cytometry to define the leukocyte populations (n = 26 mice). Treatment groups were matched by degree of peripheral blood chimerism prior to IgG injection. hMono = human monocytes. There was no significant difference in degree of chimerism or human granulocyte reconstitution between the experimental groups.</p

Anti-PR3 antibodies induce infiltration of kidneys with leukocytes of murine and human origin.

<p>Kidney sections were incubated with anti-mCD45 (red) and anti-hCD45 (green) antibodies and images were captured by fluorescence microscopy (T = tubule). Occasional (<5%) glomeruli of anti-PR3 treated mice displayed intense extracapillary leukocyte infiltration (A) in the shape of crescents (arrows). Most glomeruli in animals treated with anti-PR3 antibodies (n = 18) had evidence of intraglomerular (B,G) and peri-glomerular (C,G) leukocyte infiltration. These were comprised mostly of mCD45+ cells, although some hCD45 leukocytes were also present (arrowheads). In addition, there was a significant increase in peri-vascular leukocyte (mCD45+ and hCD45+) infiltration in anti-PR3 treated mice (D,G [per arteriolar section (art.sec.)]). Sections were also stained for deposition of IgG [red] (E,G) and C3 [green] (F,G). IgG was detectable within periglomerular cells, but there was minimal deposition within the glomeruli. Mouse C3 was weakly deposited in glomeruli but was no different between control group (n = 8) and anti-PR3 group (n = 18). Note mouse C3 can be detected normally binding avidly to tubular basement membranes. (Marker = 10 µm) (*<i>P</i><0.05, **<i>P</i><0.01. median ± IQ ± max/min values). (H) Kidney sections from anti-PR3 and control treated animals were incubated with anti-PR3 positive ANCA IgG. In the peritubular capillaries of chimera mice that received anti-PR3 hIgG occasional leukocytes detected by anti-hPR3 hIgG could be detected. No positively stained human neutrophils were seen in glomeruli.</p

Anti-PR3 antibodies cause kidney disease.

<p>(A–C) PAS stained images of glomeruli from chimera mice 6 days after injection with anti-PR3 (n = 18, A, 400×; C, 600×) or control IgG (n = 8, B, 600×). Note extra-capillary proliferation and peri-glomerular inflammation (arrowhead) (A), and mesangiolysis (C, arrow) in anti-PR3 treated mice. (D–F) H & E stained sections of kidney from chimera mice treated with anti-PR3 (D, 40×) or disease control (E, 40×) IgG. There are regions of tubulointerstitial injury, with red cell cast formation (arrow). (F) Demonstrates intense peri-glomerular inflammation in an animal treated with anti-PR3 IgG (arrowhead, 400×). By comparison mice treated with disease control IgG showed minimal glomerular or tubulointerstitial changes. (G) Fractions of glomeruli affected in anti-PR3 (n = 18) and control IgG (n = 8) treated animals (Error bars depict SEM; ***<i>p</i> = 0.001) (H). Degree of tubulointerstitial disease in mice treated with anti-PR3 antibodies and control antibodies (*<i>P</i><0.05, median ± IQ ± max/min values). (Bars = 50 µm).</p

Characterization of chimerism in NOD-<i>scid</i>-<i>IL2Rγ^−/−</i> mice.

<p>(A–D) Flow cytometric analysis of leukocytes from tail bleeds six weeks after administration of HSCs (n = 26 mice). (A) Plots showing mouse leukocytes labelled with anti-mouse CD45 antibodies. Compared with control wild-type mouse blood, chimeras have populations of mCD45 negative leukocytes that show SSC characteristics of granulocytes (High), monocytes (Int) and lymphocytes (low). (B) Chimera blood leukocytes express human CD45 and many of these express CD11b. hCD45+,CD11b+ leukocytes predominantly express hCD15 and hCD66b compared with hCD45+,CD11b− leukocytes shown in histograms. (C) A proportion of hCD45+ leukocytes express CD19. (D) Some hCD45 leukocytes are CD14^high and some are CD16+,CD14^low. (E) In chimera bone marrow there are CD11b+ leukocytes which do not express mCD45 and among hCD45+ leukocytes a proportion express CD14 and a proportion express CD66b. (F) In chimera spleen there are CD11b+ leukocytes which express hCD45 and among hCD45+ leukocytes many express both CD14 and CD16. (G) Bone marrow spreads from wild type or chimera mice, labelled with anti-hMPO or anti-hPR3 IgG antibodies (red) purified from patients with vasculitis. Note that chimera bone marrow demonstrates anti-hMPO or anti-hPR3 antibody positive leukocytes with characteristic human neutrophil nuclear morphology. Wild type mouse bone marrow shows no cells positive for these antigens indicating that the anti-human antibodies do not cross react with mouse neutrophils.</p

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

<div><p>Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH₂ and SLIGKV-NH_2, respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.</p> </div