Loss of Syndecan-1 Abrogates the Pulmonary Protective Phenotype Induced by Plasma After Hemorrhagic Shock

Abstract

Syndecan-1 (Sdc1) is considered a biomarker of injury to the endothelial glycocalyx following hemorrhagic shock, with shedding of Sdc1 deleterious. Resuscitation with fresh frozen plasma (FFP) has been correlated with restitution of pulmonary Sdc1 and reduction of lung injury, but the precise contribution of Sdc1 to FFPs protection in the lung remains unclear. Human lung endothelial cells were used to assess the time and dose-dependent effect of FFP on Sdc1 expression and the effect of Sdc1 silencing on in vitro endothelial cell permeability and actin stress fiber formation. Wild-type and Sdc1 mice were subjected to hemorrhagic shock followed by resuscitation with lactated Ringers (LR) or FFP and compared with shock alone and shams. Lungs were harvested after 3 h for analysis of permeability, histology, and inflammation and for measurement of syndecan- 2 and 4 expression. In vitro, FFP enhanced pulmonary endothelial Sdc1 expression in time- and dose-dependent manners and loss of Sdc1 in pulmonary endothelial cells worsened permeability and stress fiber formation by FFP. Loss of Sdc1 in vivo led to equivalency between LR and FFP in restoring pulmonary injury, inflammation, and permeability after shock. Lastly, Sdc1 mice demonstrated a significant increase in pulmonary syndecan 4 expression after hemorrhagic shock and FFP-based resuscitation. Taken together, our findings support a key role for Sdc1 in modulating pulmonary protection by FFP after hemorrhagic shock. Our results also suggest that other members of the syndecan family may at least be contributing to FFP's effects on the endothelium, an area that warrants further investigation.

Conflict of interest statement

Conflict of Interest: The authors declare no competing conflicts of interest.

Figures

Fig. 1. FFP increases cell surface Sdc1 in a time- and dose-dependent manner

Human lung microvascular endothelial cells were cultured in low serum medium (0.5% FBS) for 2 hours and then incubated with serum-free media containing the indicated percentages of FFP. Cells were stained with anti syndecan-1 antibody and images were captured with Nikon E800 fluorescence microscope. Original magnification, × 600. The relative fluorescence intensity was quantified using Quantity One (Bio-RAD) after converting the images to greyscales and reported as relative fluorescence units (RFU). A. Dose dependent increase in sdc1 and B. Time dependent increase in sdc1. Results are presented as means ± SEM, n=3/group.

Fig. 1. FFP increases cell surface Sdc1 in a time- and dose-dependent manner

Human lung microvascular endothelial cells were cultured in low serum medium (0.5% FBS) for 2 hours and then incubated with serum-free media containing the indicated percentages of FFP. Cells were stained with anti syndecan-1 antibody and images were captured with Nikon E800 fluorescence microscope. Original magnification, × 600. The relative fluorescence intensity was quantified using Quantity One (Bio-RAD) after converting the images to greyscales and reported as relative fluorescence units (RFU). A. Dose dependent increase in sdc1 and B. Time dependent increase in sdc1. Results are presented as means ± SEM, n=3/group.

Fig. 2. FFP increases monolayer permeability and stress fiber formation in Sdc1-deficent endothelial cells

Human lung microvascular endothelial cells were transfected with Sdc1 siRNA (siR) or scrambled RNA (scR) as control. A. In vitro permeability was assessed by fluoresceine-isothiocyanate cells treated with 10% LR or 10% FFP in serum free medium. B. Stress fiber formation, a marker of endothelial disruption, was examined by staining cells with Texas red-X phalloidin. Top panel: Images were captured with Nikon E800 fluorescence microscope. Original magnification, × 600. Middle panel: Sdc1 protein expression was assessed by Western blot. Bottom panel: the relative fluorescence intensity was quantified using Quantity One (Bio-RAD) after converting the images to greyscales and reported as relative fluorescence units (RFUs). Results are presented as means ± SEM, n=4/group.

Fig. 2. FFP increases monolayer permeability and stress fiber formation in Sdc1-deficent endothelial cells

Human lung microvascular endothelial cells were transfected with Sdc1 siRNA (siR) or scrambled RNA (scR) as control. A. In vitro permeability was assessed by fluoresceine-isothiocyanate cells treated with 10% LR or 10% FFP in serum free medium. B. Stress fiber formation, a marker of endothelial disruption, was examined by staining cells with Texas red-X phalloidin. Top panel: Images were captured with Nikon E800 fluorescence microscope. Original magnification, × 600. Middle panel: Sdc1 protein expression was assessed by Western blot. Bottom panel: the relative fluorescence intensity was quantified using Quantity One (Bio-RAD) after converting the images to greyscales and reported as relative fluorescence units (RFUs). Results are presented as means ± SEM, n=4/group.

Fig. 3. Plasma lessens hemorrhagic shock-induced lung hyperpermeabilty

Wild-type and Sdc1 null mice underwent laparotomy and hemorrhagic shock followed by resuscitation with either LR or FFP. After 3 hours Evans blue dye extravasation was measured in lungs. Data are expressed as mean ± SEM, n=8 per group with significance indicated by lines over the respective groups. FFP, fresh frozen plasma; LR, lactated Ringer’s; WT, wild type; KO, Sdc1 knockout; HS, hemorrhage shock.

Fig. 4. Plasma mitigates lung inflammation and injury after hemorrhagic shock in wild-type but not syndecan-1 −/− mice

Wild-type and syndecan-1 null mice underwent laparotomy and hemorrhagic shock followed by resuscitation with either LR or FFP. After 3 hours lungs were harvested for assessment of A. Myeloperoxidase (MPO) enzyme by immunofluorescent staining. Results are reported as relative fluorescence units (RFUs) or B. Lung histopathologic injury. Lungs sections were stained with hematoxylin and eosin and scored on alveolar thickness, capillary congestion, and cellularity. Representative images are shown in the upper panel and injury scores in the lower panel. Data are expressed as mean SEM, n=8 per group with significance indicated by lines over the respective groups. FFP, fresh frozen plasma; LR, lactated Ringer’s; WT, wild type; KO, Sdc1 knockout; HS, hemorrhage shock.

Fig. 4. Plasma mitigates lung inflammation and injury after hemorrhagic shock in wild-type but not syndecan-1 −/− mice

Wild-type and syndecan-1 null mice underwent laparotomy and hemorrhagic shock followed by resuscitation with either LR or FFP. After 3 hours lungs were harvested for assessment of A. Myeloperoxidase (MPO) enzyme by immunofluorescent staining. Results are reported as relative fluorescence units (RFUs) or B. Lung histopathologic injury. Lungs sections were stained with hematoxylin and eosin and scored on alveolar thickness, capillary congestion, and cellularity. Representative images are shown in the upper panel and injury scores in the lower panel. Data are expressed as mean SEM, n=8 per group with significance indicated by lines over the respective groups. FFP, fresh frozen plasma; LR, lactated Ringer’s; WT, wild type; KO, Sdc1 knockout; HS, hemorrhage shock.

Fig. 5. Pulmonary syndecan4, but not syndecan2, expression is increased by FFP in syndecan1 −/− mice

Wild-type and syndecan-1 −/− mice underwent laparotomy and hemorrhagic shock followed by resuscitation with either LR or FFP. After 3 hours lungs were homogenized for measurement of A. syndecan2 and B. Sdc4 by ELISA. Data are expressed as mean SEM, n=8 per group with significance indicated by lines over the respective groups. FFP, fresh frozen plasma; LR, lactated Ringer’s; WT, wild type; KO, Sdc1 knockout; HS, hemorrhage shock.

Fig. 5. Pulmonary syndecan4, but not syndecan2, expression is increased by FFP in syndecan1 −/− mice

Wild-type and syndecan-1 −/− mice underwent laparotomy and hemorrhagic shock followed by resuscitation with either LR or FFP. After 3 hours lungs were homogenized for measurement of A. syndecan2 and B. Sdc4 by ELISA. Data are expressed as mean SEM, n=8 per group with significance indicated by lines over the respective groups. FFP, fresh frozen plasma; LR, lactated Ringer’s; WT, wild type; KO, Sdc1 knockout; HS, hemorrhage shock.