A Non-Lethal Traumatic/Hemorrhagic Insult Strongly Modulates the Compartment-Specific PAI-1 Response in the Subsequent Polymicrobial Sepsis

Introduction: Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes. Methods: Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre-and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CL

A Non-Lethal Traumatic/Hemorrhagic Insult Strongly Modulates the Compartment-Specific PAI-1 Response in the Subsequent Polymicrobial Sepsis

<div><h3>Introduction</h3><p>Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes.</p> <h3>Methods</h3><p>Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre- and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP (this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice predicted to die (P-DIE) and to live (P-LIVE).</p> <h3>Results</h3><p>In experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1 mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice. Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2–4-fold higher than in SUR. PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE mice and those increases closely correlated with liver dysfunction.</p> <h3>Conclusions</h3><p>Trauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin and was independent of endothelial injury.</p> </div

Organ/tissue PAI-1 mRNA expression during the post-CLP in posttraumatic sepsis versus sepsis alone.

<p>3 week-old mice were subjected to trauma and hemorrhage (TH) followed by polymicrobial cecal ligation and puncture (CLP) sepsis or CLP alone (CLP-Only). At 0, 6, 12 and 24 h post-CLP, mice (n = 6 mice per time point) were sacrificed and organs were collected and analyzed for total plasminogen activator inhibitor type 1 (PAI-1) mRNA expression. Data as mean ± SD (TH-CLP and CLP-Only rows) representing 5–6 mice per time point. Fold PAI-1 difference between TH-CLP and CLP-Only.</p>§<p>Values obtained from a separate study (22).</p>*<p>P<0.05 versus CLP-Only. N/A, not applicable.</p

Plasma PAI-1 is increased in dying versus surviving mice.

<p>3 month-old mice (n = 42) were subjected to trauma and hemorrhage (TH) at −48 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. From the TH time point on, 20 µl of blood was collected daily and additionally at 6 h, until 96 h (day 6 post-TH/day 4 post-CLP. Animals were observed until death or day 14 post-CLP, whichever occurred first. The experimental period was divided into a pre-and post-CLP (red and black dots, respectively) phase. A) Time course of total circulating plasminogen activator inhibitor type 1 (PAI-1) of dying (DEAD, filled dots) and surviving (SUR, empty dots) mice. For SUR, n = 17 at each time point. For DEAD: n = 24 at −48 h, −24 h, 0 h and 6 h; n = 23 at 24 h; n = 15 at 48 h; n = 6 at 72 h. B) Circulating levels of PAI-1 of DIE (n = 24 at each time point) and time-matched levels of SUR (n = 24 at each time point) at the indicated time points prior to death. *p<0.05 versus SUR. CLP time point indicated by arrow.</p

Plasma thrombomodulin level during TH-CLP develops irrespective of outcome.

<p>3 month-old mice (n = 42) were subjected to trauma and hemorrhage (TH) at −48 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. The experimental period was divided into a pre- and post-CLP (red and black dots, respectively) phase. From the TH time point (−48 h) on, 20 µl of blood was collected daily, and additionally at 6 h, until 96 h (day 6 post-TH/day 4 post-CLP). Animals were observed until death or day 14 post-CLP, whichever occurred first. For SUR, n = 17 for each time point. For DEAD: n = 24 at −48 h −24 h, 0 h and 6 h; n = 23 at 24 h; n = 15 at 48 h; n = 6 at 72 h. Figure depicts the time course of circulating thrombomodulin of dying (DEAD, black-filled dots) and surviving (SUR, white-filled dots) mice. CLP time point indicated by arrow.</p

Organ-specific PAI-1 gene expression after TH-CLP.

<p>3 week-old mice were subjected to trauma and hemorrhage (TH) at −48 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. At −48 h, −42 h, −36 h, −24 h, 0 h, 6 h, 12 h and 24 h, mice (n = 6 per time point) were sacrificed and organs were collected and analyzed for expression of plasminogen activator inhibitor type 1 mRNA in the A) liver, B) heart, C) kidney, D) lung and E) left cranial vena cava (LCVC). Panels A-D show box plot diagrams representing 5–6 mice per time point, with whiskers indicating minimum and maximum and dots representing values outside of 1.5 inter-quartile range. In panel E, each LCVC dot represents a pooled collection of samples (n = 5–6 mice per time point). *P<0.05 versus −48 h; §P<0.05 versus 0 h.</p

Immunohistochemical staining of PAI-1 in the liver and lung post-TH and TH-CLP.

<p>3 week-old mice were subjected to trauma and hemorrhage (TH) at −48 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. At −48 h, −42 h and 24 h, mice were sacrificed and organs were collected and stained for plasminogen activator inhibitor type 1 (PAI-1) in the liver and lung. Representative images for 3–6 animals per time point per tissue are shown. NC, negative control for PAI-1 (incubated without primary antibody). Original magnification x100.</p

Circulating PAI-1 response to TH-CLP.

<p>3 week-old mice were subjected to trauma and hemorrhage (TH) at −48 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. At −48 h, −42 h, −36 h, −24 h, 0 h, 6 h, 12 h and 24 h, mice (n = 6 per time point) were sacrificed and blood was collected and analyzed for total plasminogen activator inhibitor type 1 (PAI-1) plasma concentration. Figure shows box plot diagrams, with whiskers indicating minimum and maximum and dots representing values outside of 1.5 inter-quartile range. *P<0.05 versus −48 h, § P<0.05 versus 6 h.</p

Circulating PAI-1 during the post-CLP phase in post-traumatic sepsis versus sepsis alone.

<p>Three week-old mice were subjected to trauma and hemorrhage (TH) followed by polymicrobial cecal ligation and puncture (CLP) sepsis or CLP alone (CLP-Only). At 0, 6, 12 and 24 h post-CLP, mice (n = 6 mice per time point) were sacrificed and blood was collected and analyzed for total plasminogen activator inhibitor type 1 (PAI-1) in plasma. Data as mean ± SD (TH-CLP and CLP-Only rows) representing 5–6 mice per time point. Fold PAI-1 difference between TH-CLP and CLP-Only.</p>§<p>Values obtained from a separate study (22).</p>*<p>P<0.05 versus CLP-Only.</p