Dual phenotypic characteristics of P-selectin in a mouse model of hemorrhagic shock and hepatectomy

Abstract

Background: Membrane-bound P-selectin induces endothelial adhesion of leucocytes and amplifies organ inflammations during major trauma, while soluble P-selectin (sP-sel) mediates survival rescue properties. This study characterized the differential effects of P-selectin in a "2-hit" model of hemorrhagic shock (HS) and partial hepatectomy (PH).

Materials and methods: HS was induced by withdrawing blood (0.3 mL) directly from the mouse femoral arteries. 70% or 50% of liver volumes were resected after inducing HS. Time of survival in P-selectin deficient (Selp -/-) mice treated with and without intraperitoneal injections of recombinant P-sel IgG-Fc fusion proteins (rP-sel-Fc, 1.5 mg/kg) were recorded for up to 72h after injury. In addition, liver regeneration at 72h after HS and 50% PH was assessed in wild-type and Selp -/- mice.

Results: Compared to wild-types, Selp -/- mice had significantly higher mortality rates post HS and 70% PH, as none of these animals survived up to 48h postoperatively. The survival curve was restored in Selp -/- mice pre-treated with rP-sel-Fc. In the HS followed by 50% PH experimental arm, liver remnant growth ratios were significantly higher in Selp -/- mice (15.7 ± 3.1 vs 11.7 ± 4.9, P = 0.040). The elevated serum concentrations of alanine aminotransferase post-PH were significantly reduced in Selp -/- mice. Hepatocyte proliferation indices (CYP7a1 and PCNA) expression were enhanced and myeloperoxidase activity in the regenerated remnant liver was reduced in the Selp -/- mice.

Conclusion: In critical conditions induced by HS and PH, P-selectin mediates two distinct phenotypic characteristics. Soluble-form circulating P-selectin improves survival in the acute stage of HS and extensive loss of liver parenchyma; membrane-bound P-selectin induces regional pro-inflammatory reactions in the remnant liver after the acute stage of two insults, thereby inhibiting hepatic regeneration. The results of this pre-clinical study may provide molecular mechanistic insight and clinical therapeutic applications of P-selectin in the acute and regenerative phases of traumatic hepatic injury.

Keywords: Hemorrhagic shock; Inflammation; Liver regeneration; P-selectin; Trauma.

Fig. 1

(A). Kaplan-Meier plots for estimating the overall survival rates of wild-type (WT) and P-selectin deficient (Selp −/−) mice up to 72h after induced hemorrhagic shock (HS) and 70% partial hepatectomy (PH). In some Selp −/− mice, recombinant P-sel IgG-Fc fusion protein (rP-sel-Fc, 1.5 mg/kg) was administered intraperitoneally before HS to increase circulating levels of soluble P-selectin. Time-to-event analysis showed that none of the Selp −/− mice survived up to 48h (^#P = 0.018 vs WT), but the survival rate was restored in Selp−/− mice that were pre-treated with rP-sel-Fc. Survival curves were compared with log-rank test, n = 5–6 different animals in each group. (B). Remnant liver tissues were harvested from mice receiving HS and 70% PH that survived more than 72h for analysis of tissue concentrations of 3-nitrotyrosine (3-NT). Enhanced 3-NT in the remnant livers of Selp−/− mice were significantly suppressed by intraperitoneal injection of rP-sel-Fc. *P = 0.013, n = 5–6 different animals in each group; data were analyzed using the Kruskal-Wallis test by rank. Results are presented as box-and-whisker plots, in which the horizontal lines of color boxes indicate the 75th percentile, median and 25th percentile of the distribution, and the upper and lower whiskers indicate the maximal and minimal values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Clinical outcomes at 72h after induced hemorrhagic shock (HS) and 50% partial hepatectomy (PH). (A). Gross autopsies of the regenerated remnant liver lobes at 72h after HS and 50% PH in wild-type (WT) and P-selectin deficient (Selp −/−) mice. (B). Physiological stress in experimental mice was determined by changes in body weight at the time of sacrifice. Body weight of wild-type animals that received the 2-hit injury was significantly lower than wild-type animals that received only PH (^P = 0.028 vs WT with PH only). (C). The regeneration of remnant liver lobes was assessed by the liver growth ratio and was normalized by the body mass at the time of sacrifice. The liver growth ratio was significantly reduced in WT mice received HS and 50% PH in comparison to PH only groups and Selp −/− mice. *P = 0.010 vs Selp −/− with HS + PH; ^#P = 0.026 vs Selp −/− with PH only. n = 6 different animals in each group; data were analyzed using the Kruskal-Wallis test by rank. Results are presented as box-and-whisker plots, in which the horizontal lines of color boxes indicate the 75th percentile, median and 25th percentile of the distribution, and the upper and lower whiskers indicate the maximal and minimal values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Liver function at 72h after 50% partial hepatectomy (PH) with or without hemorrhagic shock (HS) was assessed by analysis of serum concentrations of alanine aminotransferase ALT (liver enzyme), total bilirubin (liver metabolism), and albumin (liver biosynthesis) in wild type (WT) and P-selectin deficient (Selp −/−) mice. *P = 0.002 vs Selp −/− with HS + PH; ^P < 0.05 vs the PH only within groups; ^#P = 0.020 vs Selp −/− with PH only. n = 6 different animals in each group; data were analyzed using the Kruskal-Wallis test by rank. Results are presented as box-and-whisker plots, in which the horizontal lines of color boxes indicate the 75th percentile, median and 25th percentile of the distribution, and the upper and lower whiskers indicate the maximal and minimal values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

A-C. Tissue concentrations of pro-inflammatory cytokines in the regenerated remnant liver at 72h after 50% partial hepatectomy (PH) with or without hemorrhagic shock (HS) in wild type (WT) and P-selectin deficient (Selp −/−) mice. IL-6: interleukin-6; TNF-α: tumor necrosis factor-α; MCP-1: monocyte chemoattractant protein-1. 4D. Representative histologic sections of the regenerated liver. Arrows indicate the expression of myeloperoxidase. 4E. Enzymatic activity of myeloperoxidase in the liver homogenates of mice exposed to PH with or without HS. *P < 0.05 vs PH only (within groups); ^P < 0.05 vs PH only (between groups); ^#P < 0.05 vs Selp −/− with HS + PH. n = 5 different animals in each group; data were analyzed using the Kruskal-Wallis test by rank. Results are presented as box-and-whisker plots, in which the horizontal lines of color boxes indicate the 75th percentile, median and 25th percentile of the distribution, and the upper and lower whiskers indicate the maximal and minimal values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Proliferation indices of the regenerated remnant liver at 72h after 50% partial hepatectomy (PH) with or without hemorrhagic shock (HS) in wild type (WT) and P-selectin deficient (Selp −/−) mice. A. Western blotting analysis of the protein expressions of vascular endothelial growth factor receptor (VEGFR)-2, hepatic growth factor (HGF), cytochrome P450 7a1 (CYP7a1) and proliferating cell nuclear antigen (PCNA) in the regenerated liver homogenates. B. Immunohistochemical expressions of Ki67 and PCNA in the regenerated remnant liver sections. *P < 0.05 vs Selp −/− with HS + PH; ^#P < 0.05 vs WT with PH only; ^P<0.05 vs Selp −/− with PH only. n = 5 different animals in each group; data were analyzed using the Kruskal-Wallis test by rank. Results are presented as box-and-whisker plots, in which the horizontal lines of color boxes indicate the 75th percentile, median and 25th percentile of the distribution, and the upper and lower whiskers indicate the maximal and minimal values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Graphical abstract of the study. Wild-type (WT) and P-selectin deficient (Selp −/−) mice were randomized assigned to receive hemorrhagic shock resuscitation (HSR) and 70% or 50% partial hepatectomy (PH). After 70% PH and HSR, the overall mortality was significantly increased in Selp −/− mice and the survival rate was improved in Selp −/− mice received pre-treated with recombinant P-sel IgG-Fc fusion protein (rP-sel-Fc), suggesting that soluble form P-selectin (sP-sel) in the systemic circulation mediates “self-rescue” responses that are essential for host survival from the acute phase of major traumatic liver injury. In the other “2-hit” model with 50% PH, Selp −/− mice were associated with enhanced liver regeneration and attenuated regional inflammatory reactions in the regenerated remnant liver, suggesting that membrane-bound P-selectin mediates infiltration of pro-inflammatory neutrophils that may impair hepatic growth during the proliferative phase of liver regeneration. MPO: myeloperoxidase; PMN: polymorphonuclear leukocytes; 3-NT: 3-nitrotyrosine; ROS: reactive oxygen species.