The HMGB1/RAGE Pro-Inflammatory Axis in the Human Placenta: Modulating Effect of Low Molecular Weight Heparin

Abstract

We evaluated whether physiological and pre-eclamptic (PE) placentae, characterized by exacerbated inflammation, presented alterations in pro-inflammatory High Mobility Group Box 1 (HMGB1) and its Receptor of Advanced Glycation End products (RAGE) expression. Moreover, we investigated, in physiological placental tissue, the ability of Low Molecular Weight Heparin (LMWH) to modify HMGB1 structural conformation thus inhibiting RAGE binding and HMGB1/RAGE axis inflammatory activity. HMGB1, RAGE, IL-6 and TNFα (HMGB1/RAGE targets) mRNA expression were assessed by Real Time PCR. HMGB1, RAGE protein levels were assessed by western blot assay. Physiological term placental explants were treated by 0.5 U LMWH for 24 or 48 h. HMGB1 and RAGE expression and association were evaluated in LMWH explants by RAGE immunoprecipitation followed by HMGB1 immunoblot. HMGB1 spatial localization was evaluated by immuofluorescent staining (IF). HMGB1 expression was increased in PE relative to physiological placentae while RAGE was unvaried. 24 h LMWH treatment significantly up-regulated HMGB1 expression but inhibited HMGB1/RAGE complex formation in physiological explants. RAGE expression decreased in treated relative to untreated explants at 48 h. IF showed HMGB1 localization in both cytoplasm and nucleus of mesenchymal and endothelial cells but not in the trophoblast. IL-6 and TNFα gene expression were significantly increased at 24 h relative to controls, while they were significantly down-regulated in 48 h vs. 24 h LMWH explants. Our data depicted a new molecular mechanism through which LMWH exerts its anti-inflammatory effect on PE placentae, underlying the importance of HMGB1/RAGE axis in PE inflammatory response.

Keywords: HMGB1; heparin; placenta; pre-eclampsia; receptor for advanced glycation end products (RAGE).

Figure 1

HMGB1 and RAGE expression levels in normal (N = 19) versus PE (N = 18) placentae. (A) HMGB1 mRNA (left panel) and protein (right panel) expression levels as assessed by Real Time PCR and western blot analysis; (B) RAGE protein expression assessed by western blot. Statistical significance (*) has been considered as p < 0.05.

Figure 2

HMGB1 expression in LMWH-treated vs. untreated physiological placental villous explants. (A) HMGB1 mRNA expression levels in placental villous explants treated for 24 h (ctrl n = 8 and 0.5 U LMWH n = 10) and 48 h (ctrl n = 11 and 0.5 U LMWH n = 10) by 0.5 U of LMWH or plain culture medium, assessed by Real Time PCR; (B) Representative western blot for HMGB1 protein expression in LMWH-treated vs. untreated placental villous explants for 24 h (ctrl n = 4 and 0.5 U LMWH n = 4) and 48 h (ctrl n = 10 and 0.5 U LMWH n = 10). Statistical significance (*) has been considered as p < 0.05.

Figure 3

RAGE expression in LMWH-treated vs. untreated physiological placental villous explants. Representative western blot for RAGE protein expression level in physiological term placental villous explants treated for 24 h (ctrl n = 7 and 0.5 U LMWH n = 7) and 48 h (ctrl n = 10 and 0.5 U LMWH n = 10) by 0.5 U of LMWH.

Figure 4

RAGE/HMGB1 binding in LMWH-treated vs. untreated placental villous explants. HMGB1 protein levels were evaluated by western blot after immunoprecipitation with RAGE antibody of protein lysates from placental villous explants treated by 0.5 U of LMWH for 24 h and 48 h. (A) Representative WB for HMGB1 protein levels after RAGE IP at 24 h (ctrl n = 16 and 0.5 U LMWH n = 15) and 48 h (ctrl n = 5 and 0.5 U LMWH n = 6); (B) Representative WB for HMGB1 protein levels in RAGE IP supernatant at 24 h (ctrl n = 8 and 0.5 U LMWH n = 10) and 48 h (ctrl n = 5 and 0.5 U LMWH n = 6). Statistical significance (*) has been considered as p < 0.05.

Figure 5

HMGB1 spatial localization in untreated or 0.5 U LMWH treated physiological villous explants assessed by immunofluorescent staining. (A–C) HMGB1 spatial localization in untreated (n = 6) physiological villous explants assessed by immunofluorescent staining; (B–D) HMGB1 spatial localization in physiological villous explants treated with 0.5 U of LMWH (n = 6); (E) Absence of positive immunoreactivity for HMGB1 in section stained with control IgG. Cell nuclei are showed in blue by DAPI signal. TR, trophoblast cells; M, mesenchyme; V, vessel. Original magnifications, 60×.

Figure 6

IL-6 and TNFα mRNA expression in LMWH-treated explants vs. controls assessed by Real Time PCR. (A) IL-6 gene expression in villous explants treated by 0.5 U of LMWH for 24 h (ctrl n = 8 and 0.5 U LMWH n = 10) and 48 h (ctrl n = 11 and 0.5 U LMWH n = 10). (B) TNFα gene expression levels in villous explants treated by 0.5 U of LMWH for 24 h (ctrl n = 8 and 0.5 U LMWH n = 10) and 48 h (ctrl n = 11 and 0.5 U LMWH n = 10). Statistical significance (*#) has been considered as p < 0.05.

Figure 6

IL-6 and TNFα mRNA expression in LMWH-treated explants vs. controls assessed by Real Time PCR. (A) IL-6 gene expression in villous explants treated by 0.5 U of LMWH for 24 h (ctrl n = 8 and 0.5 U LMWH n = 10) and 48 h (ctrl n = 11 and 0.5 U LMWH n = 10). (B) TNFα gene expression levels in villous explants treated by 0.5 U of LMWH for 24 h (ctrl n = 8 and 0.5 U LMWH n = 10) and 48 h (ctrl n = 11 and 0.5 U LMWH n = 10). Statistical significance (*#) has been considered as p < 0.05.