Inhibition of ERK1/2 by U0126 increased animal mortality induced by intestinal ischemia/reperfusion.

<p>Mice were pretreated with U0126 or vehicle and then subjected to 75 minutes ischemia and the incision was closed. Animals were observed for 7 days for survival rates (n=10) (IR vs. IR-U0126, p<0.01). Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

Inhibition of ERK1/2 by U0126 increased the levels of proinflammatory cytokines in plasma.

<p>Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). The relatively levels of cytokines in plasma was determined using a commercial kit. A. The relatively level of cytokines TNF-α in plasma. B. The relatively level of cytokines IL-6 in plasma. C. The relatively level of cytokines IL-1β in plasma. Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

Activities of ERK1/2 and p70S6K were inhibited by U0126 in vitro and in vivo.

<p>A. IEC-6 cells were cultured in medium without FBS for 12 hours under hypoxic conditions followed treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 20 minutes (n=3). Protein expression in cells was determined by Western blot analysis. B. IEC-6 cells were transfected with/without empty plasmid or p70S6K plasmid for 24 hours and cultured in medium without FBS for 12 hours under hypoxic conditions followed treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 20 minutes (n=3). Protein expression was determined by Western blot analysis. C. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). Protein expression in the intestine was determined by Western blot analysis. U=U0126, Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

Inhibition of ERK1/2 and p70S6K by U0126 decreased cell proliferation and migration, and promoted cell apoptosis in vitro (n=3).

<p>A. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in medium without FBS for 12 hours under hypoxic conditions followed by treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 3 days. Cell proliferation was measured by MTT assay. B. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in medium without FBS in a 12-well plate for 12 hours under hypoxic conditions. The confluent cell monolayer was then scraped with a 1-ml pipette tip. Cells were then treated with U0126 or vehicle for 1 hour followed by adding 10% FBS under normoxic conditions for 24 hours. The recovery area was measured. C. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in FBS-free medium with U0126 or vehicle under hypoxic conditions for 24 hours followed by incubation in normoxic conditions for 6 hours. Cell apoptosis was determined by evaluation of DNA fragmentation. U=U0126.</p

Inhibition of ERK1/2 by U0126 increased intestinal inflammation, permeability and injury in vivo.

<p>Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). The intestinal tissue was examined. A. MPO activity by a commercial Kit. B. Permeability by the ex-vivo isolated everted ileum sac method. C. Representative images by HE staining. D. Quantitative tissue damage by Park/Chiu scoring system. Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

Inhibition of ERK1/2 by U0126 increased intestinal cell apoptosis and decreased cell proliferation in vivo.

<p>Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). A. Representative images of apoptotic cells in the intestinal tissue detected using an In Situ Cell Death Detection Kit. B. Quantitative measurement of apoptotic cells in the intestinal tissue. C. Representative images of immunohistochemical staining for Ki-67 in the intestinal tissue. Immunohistochemical staining was performed using the Dako-Cytomation EnVision + System-HRP (AEC) kit. C. Quantitative measurement of immunohistochemical staining for Ki-67 in the intestinal tissue. Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

LP and FFP increase TEER of EC monolayers.

<p>(A) Mean average ECIS generated traces of the TEER of HUVECs treated with 10% media (control), LR, FFP or LP. (B) Area under the curve analysis for thirty minutes after the addition of treatment. * = (p<0.05) compared to control via post hoc turkey tests of an unpaired one-way ANOVA.</p

FFP and LP prevent VEGF disruption of adherens junctions.

<p>Cells were pretreated with LR, FFP, LP or not treated for 1 hour before VEGF was added. Cells were fixed and stain for (A) Dapi (blue), β-Catenin (red) and VE-Cadherin (green). In (B) it is qualitatively observed that FFP and LP preserve the overlap of VE-Cadherin and β-Catenin (yellow) compare to untreated and LR controls.</p

Lyophilized plasma attenuates vascular permeability, inflammation and lung injury in hemorrhagic shock

<div><p>In severe trauma and hemorrhage the early and empiric use of fresh frozen plasma (FFP) is associated with decreased morbidity and mortality. However, utilization of FFP comes with the significant burden of shipping and storage of frozen blood products. Dried or lyophilized plasma (LP) can be stored at room temperature, transported easily, reconstituted rapidly with ready availability in remote and austere environments. We have previously demonstrated that FFP mitigates the endothelial injury that ensues after hemorrhagic shock (HS). In the current study, we sought to determine whether LP has similar properties to FFP in its ability to modulate endothelial dysfunction in vitro and in vivo. Single donor LP was compared to single donor FFP using the following measures of endothelial cell (EC) function <i>in vitro</i>: permeability and transendothelial monolayer resistance; adherens junction preservation; and leukocyte-EC adhesion. <i>In vivo</i>, using a model of murine HS, LP and FFP were compared in measures of HS- induced pulmonary vascular inflammation and edema. Both <i>in vitro</i> and <i>in vivo</i> in all measures of EC function, LP demonstrated similar effects to FFP. Both FFP and LP similarly reduced EC permeability, increased transendothelial resistance, decreased leukocyte-EC binding and persevered adherens junctions. <i>In vivo</i>, LP and FFP both comparably reduced pulmonary injury, inflammation and vascular leak. Both FFP and LP have similar potent protective effects on the vascular endothelium in vitro and in lung function in vivo following hemorrhagic shock. These data support the further development of LP as an effective plasma product for human use after trauma and hemorrhagic shock.</p></div

LP and FFP reduce that amount of leukocyte binding in vitro.

<p>Fluorescently labeled U397 cells were added to wells of HUVEC cells treated with 10(A) and 30(B) percent LR, FFP, LP or no treatment control. Unbound cells were removed after a period of one hour and the remaining cells quantified by fluorescence. All wells were normalized to control. * = (p<0.05) compared to control via post hoc turkey tests of an unpaired one-way ANOVA. N.S. clarifies that the indicated groups are Not Significantly different. Controls and FFP were featured in Wataha et al. (2013). [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192363#pone.0192363.ref016" target="_blank">16</a>].</p