Inflammation Research

, Volume 67, Issue 3, pp 265–275 | Cite as

Angiotensin 1–7, but not the thrombin-cleaved osteopontin C-terminal fragment, attenuates osteopontin-mediated macrophage-induced endothelial-cell inflammation

  • Rachel Hamias
  • Assaf Rudich
  • George Greenberg
  • Gabriel Szendro
  • Talya Wolak
Original Research Paper
  • 59 Downloads

Abstract

Objective and design

Evaluating the pro-/anti-inflammatory activity of the C-terminal cleavage product of osteopontin in comparison to angiotensin 1–7.

Material and subjects

Human coronary endothelial cells (hcEC) treated with conditioned media from human U937 macrophages.

Treatment

Macrophages were (pre)treated with C-terminal, full-length or N-terminal osteopontin (OPN-C, OPN-FL, OPN-N, respectively), angiotensin II, angiotensin 1–7 or TNF-α. OPN-C modulatory capacity was compared to that of Ang1–7 in inhibiting subsequent Ag II, OPN-FL or OPN-N-induced macrophage-mediated endothelial inflammation.

Methods

Protein expression of NFκB, IκB, vCAM-1 and iCAM-1 was assessed using western blot. Promotor activation by NFκB was also assessed by dual-luciferase reporter assay.

Results

Conditioned media of macrophages treated with OPN-C induced hcECs’ NfκB activation to a lower degree than OPN-FL or OPN-N. Priming of macrophages with angiotensin 1–7 attenuated the endothelial pro-inflammatory effect induced by subsequent exposure of the macrophages to angiotensin II, OPN-FL or OPN-N. This was evidenced by both NfκB activation and vCAM and iCAM expression. In contrast, priming macrophages with OPN-C did not significantly attenuate the subsequent response to the pro-inflammatory cytokines.

Conclusions

OPN-C induces lower macrophage-induced endothelial inflammation compared to OPN-FL or OPN-N, but unlike angiotensin 1–7, fails to prevent endothelial inflammation induced by subsequent pro-inflammatory macrophage stimulation.

Keywords

Osteopontin OPN-N terminal OPN-C terminal NFκB IκB 

Notes

Acknowledgements

This study was supported by grant from the Israel Ministry of Health (Grant no. 8736511; 3-9937 for TW and AR).

Supplementary material

11_2017_1120_MOESM1_ESM.tif (96 kb)
Supplementary Figure 1. Ag II, OPN-FL and OPN-N expression in conditioned media. Western blot of AgII, OPN-FL and OPN-N MQ condition media: before PBS wash versus the conditioned media that was applied onto the endothelial cells; after PBS wash and incubated overnight for additional 16 h with fresh medium (TIF 96 KB)
11_2017_1120_MOESM2_ESM.tif (159 kb)
Supplementary Figure 2. NfκB and IκB expression induced by inflammatory stimulation. (A) Macrophages from U937 cells were treated with vehicle, angiotensin II (Ag II), angiotensin 1-7 (Ag 1-7), TNF-α, OPN-FL or vehicle OPN-FL, OPN-N, OPN overnight, then washed and incubated overnight with fresh medium. This medium was transferred onto human coronary endothelial cells for another 16 h. (B) Representative blots of cytoplasmatic NfκB, IκB and nuclear NfκB after stimulation with vehicle, Ag 1-7, Ag II, TNF-α or OPN-FL. Quantification of signals from western blot: column bars (mean±SEM) treated with vehicle, Ag 1-7, Ag II, TNF-α or OPN-FL: for cytoplasmatic NfκB: p<0.001; nuclear NfκB: p=0.0017. One-way Anova with Kruskal–Wallis test. All data are the mean change of n=3 per treatment (TIF 159 KB)
11_2017_1120_MOESM3_ESM.tif (159 kb)
Supplementary Figure 3. The effect of pretreatment with Ag 1-7 and OPN-C on OPN-rFL induced NfκB and IκB endothelial expression. (A) Representative blots of cytoplasmatic NfkB and IkB and nuclear NfkB of control and after pretreatment with vehicle, OPN-C or Ag 1-7 before stimulation with OPN-rFL. (B) Quantification of western blot signals (mean±SEM) bars column treated with control, pretreatment with vehicle, OPN-C, or Ag 1-7 from cytoplasmatic NfκB:p<0.001; IκB: p=0.0014; nuclear NfκB: p<0.001. The effect of pre treatment with Ag 1-7 and OPN-C on OPN-rFL induced vCAM and iCAM endothelial expression. (C) Representative blots of whole cell lysates of vCAM and iCAM of control and after pretreatment with vehicle, OPN-C, or Ag 1-7 before stimulation with OPN-rFL. (D) Quantification of signals from vCAM western blot: column bars (mean±SEM) of control, vehicle, OPN-C, or Ag 1-7: p=0.0014. Quantification of signals iCAM from western blot: column bars (mean±SEM) of control, vehicle, OPN-C or Ag 1-7: p=0.014. One-way Anova with Kruskal–Wallis test. All data are the mean change of n=3 per treatment (TIF 159 KB)

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  • Rachel Hamias
    • 1
  • Assaf Rudich
    • 2
    • 3
  • George Greenberg
    • 4
  • Gabriel Szendro
    • 4
  • Talya Wolak
    • 5
  1. 1.Clinical Biochemistry and Pharmacology, Faculty of Health SciencesBen-Gurion University of the Negev and Soroka University Medical CenterBeer ShevaIsrael
  2. 2.Department of Clinical Biochemistry and Pharmacology, Faculty of Health SciencesBen-Gurion University of the Negev and Soroka Medical CenterBeer ShevaIsrael
  3. 3.National Institute of BiotechnologyNegevIsrael
  4. 4.Department of Vascular SurgerySoroka University Medical CenterBeer-ShevaIsrael
  5. 5.Hypertension UnitSoroka University Medical Center and Ben-Gurion University of the NegevBeer-ShevaIsrael

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