Research ArticleHEALTH AND MEDICINE

The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability

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Science Advances  24 Apr 2015:
Vol. 1, no. 3, e1500175
DOI: 10.1126/sciadv.1500175
  • Fig. 1 N-α-PGP activates endothelial cell signaling through CXCR2.

    (A to C) HUVECs were serum-starved for 2 hours before stimulation with N-α-PGP (0.5 mg/ml) for indicated times, and activation of Rac1 (A) and phosphorylation of PAK (pPAK) and ERK (pERK) (B) and VE-cadherin (pVE-cad) (C) were determined by Western blot. Shown are representative Western blots together with quantification. Bar graphs show means ± SEM (n = 3). *P < 0.05 relative to time 0 by one-way analysis of variance (ANOVA) with Tukey post-test. (D) HUVECs were untreated or treated with N-α-PGP (0.5 mg/ml) (30 min) alone or after pretreatment with 200 nM SB225002, and Rac1 activity and phosphorylation of ERK, PAK, and VE-cadherin were determined by Western blot. Shown are representative Western blots together with quantification. Bar graphs show means ± SEM (n = 3). *P < 0.05 relative to time 0, #P < 0.05 relative to PGP by one-way ANOVA with Tukey post-test.

  • Fig. 2 N-α-PGP induces endothelial permeability through CXCR2.

    (A) HUVECs were grown on 3.0-μm Transwell membranes and left untreated or stimulated with N-α-PGP (0.5 mg/ml), PGG (0.5 mg/ml), or VEGF (50 ng/ml) for 30 min. (B) HUVECs were cultured as before, and some cells were pretreated with 200 nM SB225002 for 30 min before stimulation with N-α-PGP (0.5 mg/ml). HRP leak from the upper chamber to the lower chamber was measured as described in Materials and Methods. (C) As a separate readout of permeability, HUVECs were grown on biotinylated collagen and stimulated with N-α-PGP (0.5 mg/ml) for 0 to 60 min or with thrombin (0.2 U/ml) for 10 min, and monolayer leak was determined by staining with streptavidin–Alexa 488. (D) HUVECs were treated with N-α-PGP (0.5 mg/ml) with or without 200 nM SB225002 for 30 min with streptavidin–Alexa 488 staining. *P < 0.05 versus control; #P < 0.05 versus N-α-PGP. Data are means ± SEM [n = 6 for (A) and (B), and n = 3 for (C) and (D)].

  • Fig. 3 N-α-PGP induces skin and pulmonary microvascular permeability.

    (A to C) Mice (n = 4 to 5 per group) were injected via the tail vein with Evans blue and then received abdominal subcutaneous injection of PBS alone, N-α-PGP (250 μg), PGG (250 μg), or VEGF (50 ng). Evans blue leak to the skin tissue was visually assessed (A) and quantified (B), and VE-cadherin phosphorylation after treatment was determined by Western blot (representative image) (C). *P < 0.05 versus PBS control by one-way ANOVA with Tukey post-test. (D to F) Mice (n = 4 to 5 per group) were intraperitoneally injected with saline alone or containing N-α-PGP (250 μg), PGG (250 μg), LPS (75 μg) once a day for 4 days, and then the total IgM in BAL fluid was measured by immunoassay (D) and total BAL cell (E), and VE-cadherin phosphorylation in lung lysate was measured by Western blot (F). *P < 0.05 versus saline or PGG, #P < 0.05 versus PGG by one-way ANOVA with Tukey’s multiple comparison post-test. All values represent means ± SEM.

  • Fig. 4 RTR attenuates LPS-induced pulmonary microvascular permeability.

    Mice were injected via the tail vein with 50 μl of PBS alone or containing 50 μg of RTR and then intraperitoneally administered with 75 μg of LPS (in 100 μl of PBS) once a day. (A) After 4 days of treatment, mice were sacrificed for serum measurements of PGP (n = 6) or injected via the tail vein with Evans blue. (B) Evans blue leak to the lung was quantified and normalized to protein; data show fold change relative to saline (n = 7 to 11). (C) IgM levels were measured in the BAL (n = 4 to 6). (D) VE-cadherin phosphorylation in lung homogenates was assessed by Western blot. Representative image. *P < 0.05 versus saline control, #P < 0.05 relative to LPS for (A) to (C) by one-way ANOVA with Tukey post-test; #P < 0.05 relative to LPS by t test for (D). All values represent means ± SEM.

  • Fig. 5 ARDS plasma induces endothelial activation, which is attenuated by RTR.

    (A) Plasma was collected from patients with ARDS and normal control, and plasma PGP and N-α-PGP levels were measured via electrospray ionization–liquid chromatography–tandem mass spectrometry. *P < 0.05 by t test between ARDS and normal for all PGP peptides (n = 6). PMVECs were treated with N-α-PGP, and permeability was assessed (see fig. S4F). (B) Maximal changes in relative cell index as a function of N-α-PGP concentrations. Inset compares the effects of N-α-PGP and PGP (both at 1 ng/ml). (C) After 2 hours of serum starvation, HUVECs were treated with pooled ARDS (from three patients) or non-lung disease intensive care unit (ICU) patient (n = 3) plasma at two different time points, and VE-cadherin phosphorylation was measured by Western blot. Representative image. HUVECs were treated as above for 15 min with plasma collected from individual ARDS with or without RTR (30 μg/ml), and changes in VE-cadherin phosphorylation were assessed. (D) Representative Western blot. (E) Quantitation. *P < 0.05 relative to control, #P < 0.05 relative to ARDS by one-way ANOVA with Tukey post-test (n = 3). Measurements of cellular impedance were made in HUVECs over 75 min with ARDS plasma versus ARDS plasma + RTR. (F) Representative traces from one patient with four intraexperimental replicates. Arrow denotes addition of plasma. (G) Percent attenuation by RTR on ARDS plasma time-dependent permeability changes. *P < 0.05 by two-way repeated-measures ANOVA with Bonferroni post-test. All values represent means ± SEM (n = 3). AUC, area under the curve.

  • Table 1 Demographics for ARDS and non-lung disease ICU patients.

    Values represent means ± SD. All ARDS subjects were secondary to documented gram-negative sepsis and intubated because of respiratory failure. Non-lung disease control subjects were intubated for various reasons unrelated to respiratory failure (one for airway protection secondary to gastrointestinal bleed, two for metabolic acidosis secondary to diabetic ketoacidosis, two for overdose, and one for airway protection status post–cerebrovascular accident). ns, no significant difference.

    GroupAgeSexRacePaO2/FIO2APACHE II score30-Day mortality
    ARDS (n = 6)59.5 (6.8)50% male, 50% female66% white, 34% black149.5 (60)25.5 (2.6)66%
    Non-lung disease (n = 6)46.1 (9.7)66% male, 34% female66% white, 34% black469.2 (80)11.0 (4.9)0%
    P (between groups)<0.05nsns<0.05<0.05<0.05

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/3/e1500175/DC1

    Fig. S1. Multiple endothelial cells demonstrate ERK phosphorylation with IL-8 stimulation.

    Fig. S2. PGG stimulation does not activate HUVECs.

    Fig. S3. Blockade of ERK mitigates N-α-PGP–mediated VE-cadherin activation in endothelial cells.

    Fig. S4. Effects of N-α-PGP on PMVEC permeability.

    Fig. S5. N-α-PGP does not induce proinflammatory signaling in endothelial cells.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Multiple endothelial cells demonstrate ERK phosphorylation with IL-8 stimulation.
    • Fig. S2. PGG stimulation does not activate HUVECs.
    • Fig. S3. Blockade of ERK mitigates N-α-PGP–mediated VE-cadherin activation in endothelial cells.
    • Fig. S4. Effects of N-α-PGP on PMVEC permeability.
    • Fig. S5. N-α-PGP does not induce proinflammatory signaling in endothelial cells.

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