IL-1β primed mesenchymal stromal cells moderate hemorrhagic shock-induced vascular permeability

Abstract

Background: Hemorrhagic shock (HS) corresponds to absolute hypovolemia creating an imbalance between oxygen supply and consumption. This causes an impaired hemostasis, a systemic inflammatory response, and microvascular permeability which can lead to multiple organ failure (MOF). There is no specific treatment for the endothelial dysfunction that plays a major role in the evolution towards MOF. Mesenchymal stromal cells (MSC) have been used in clinical trials for their immunomodulation and tissue repair capabilities for many years. Moreover, we previously showed that IL-1β-primed-MSC (MSCp) attenuated HS-induced organ injuries. The objective of the study was to determine whether MSCp could prevent the onset of MOF after HS by preventing endothelial dysfunction.

Methods: We established a rat model of HS, inducing 90 min of HS at a fixed mean arterial pressure of 35 mmHg, followed by resuscitation and transfusion. MSCp treatment was administered intravenously at the onset of resuscitation. After 6 h, we assessed plasma levels of endothelial markers, vascular permeability using Evans Blue (EB) dye, and renal and hepatic water content by measuring the wet-to-dry weight difference. Additionally, we investigated the ability of MSCp to inhibit leukocyte adhesion to activated endothelium in vitro.

Results: Our results indicate that early administration of MSCp significantly reduced the percentage of water content and EB dye in the liver but not in the kidney. These results were associated with a trend toward decreased plasma levels of Syndecan-1, ICAM-1, vWF, and VCAM-1. In vitro, MSCp reduced leukocyte-endothelial cell adhesion. Together, our results suggest that MSCp help to prevent endothelial dysfunction and vascular leakage, which, in turn, could protect the liver from injury.

Keywords: Hemorrhagic shock; Mesenchymal stromal cells; Multiple organ failure; Priming IL-1β; Vascular permeability.

Fig. 1

Characteristics of the animal model. A Experimental outline of rat HS assay. B Mean arterial pressure (MAP) tracing in sham, untreated hemorrhagic shock (HS) and HS treated with MSCp (HS + MSCp), during the phase of HS, fluid resuscitation, and re-transfusion followed by SEM (n = 12, 10, and 15 in sham, HS, and HS + MSCp groups respectively). The three phases of the protocol were analyzed were analyzed by non-parametric ANOVA for Longitudinal data followed by a pair.comparison test (# for p < 0.05 and #### for p < 0.0001)

Fig. 2

Vascular leakage. Tissue water content (% of H₂O in tissue) and Evans Blue dye content (µg.g⁻¹ dry weight) are expressed with median and interquartile range. Kruskal–Wallis statistical analysis with Dunn’s correction (adjust for multiplicity) were used to assess differences between all groups, significance is defined as § p < 0.05, §§ p < 0.01 and §§§ p < 0.005. Mann–Whitney statistical analysis was used to test the effect of Control vs HS or sham vs HS group and HS versus HS + MSCp: significance is defined as *p < 0.05, **p < 0.01 and *** p < 0.005. For liver and kidney: sham, HS, and HS + MSCp groups respectively: n = 12, 11, and 15

Fig. 3

Circulating concentrations of endothelial markers. Kruskal–Wallis statistical analysis with Dunn’s correction (adjust for multiplicity) were used to assess differences between all groups, significance is defined as § p < 0.05 and §§ p < 0.01. Mann–Whitney statistical analysis was used to test the effect of Control vs HS group and HS versus HS + MSCp: significance is defined as *p < 0.05, **p < 0.01, *** p < 0.005 **** p < 0.0001. For Control, Sham, HS, and HS + MSCp groups respectively: n = 6, 10, 14 and 9 for Syndecan-1; n = 6, 8, 12 and 12 for vWF; n = 6, 10, 14 and 12 for ICAM-1; n = 6, 9, 12 and 12 for VCAM-1

Fig. 4

Leukocyte binding assay. PBMC adhesion values are reported at the expression level of the LPS condition which had the value 100%. Data are expressed with median and interquartile range. A Gating strategy. B Percentage of adherent PBMC on endothelial cells. Data are expressed with median and interquartile range. Kruskal–Wallis statistical analysis with Dunn’s correction (adjust for multiplicity) were used to assess differences between all groups (Non intox, LPS and treated groups: LPS + MSC at ratios MSC:PBMC 2:1, 1:1 or 1:2), significance is defined as §§ p < 0.01. Mann–Whitney statistical analysis was used to test the effect of PBMC Non intox versus PBMC + LPS and LPS versus LPS + PBMC:MSCp 2:1, significance is defined as *p < 0.05 and ***p < 0.005. For Non intox, and LPS conditions n = 7 PBMC donors. For treated groups, n = 5 MSC donors