Interleukin-4 induces up-regulation of endothelial cell claudin-5 through activation of FoxO1: role in protection from complement-mediated injury.

Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces upregulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.

Liver metabolomic changes identify biochemical pathways in hemorrhagic shock.

BACKGROUND: Despite ongoing advances in treatment, thousands of patients still die annually from complications due to hemorrhagic shock, a condition causing dramatic physiologic and metabolic changes as cells switch to anaerobic metabolism in response to oxygen deprivation. As the shift from aerobic to anaerobic metabolism occurs in the peripheral tissues during shock, the liver must increase production of endogenous glucose as well as process excess lactate produced in the periphery. This places the liver at the center of metabolic regulation in the body during hemorrhagic shock. Therefore, we hypothesized that liver tissue from pigs during an in vivo model of hemorrhagic shock (n = 6) would reflect resultant metabolic changes. MATERIALS AND METHODS: The in vivo model of shock consisted of 45 min of shock followed by 8 h of hypotensive resuscitation (80 mmHg) and subsequent normotensive resuscitation (90 mmHg) ending 48 h after the shock period. Control groups of pigs (n = 3) (1) shock with no resuscitation, and (2) only anesthesia and instrumentation, also were included. Metabolic changes within the liver after shock and during resuscitation were investigated using both proton ((1)H) and phosphorous ((31)P) nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Concentrations of glycerylphosphorylcholine (GPC) and glycerylphosphorylethanolamine (GPE) were significantly lower at 8 h after shock, with recovery to baseline by 23 and 48 h after shock. Uridine diphosphate-glucose (UDP-glucose), and phosphoenolpyruvate (PEP) were elevated 23 h after shock. CONCLUSIONS: These results indicate that (1)H and (31)P NMR spectroscopy can be used to identify differences in liver metabolites in an in vivo model of hemorrhagic shock, indicating that metabolomic analysis can be used to elucidate biochemical events occurring during this complex disease process.

Noninvasive tissue oxygen saturation measurements identify supply dependency.

BACKGROUND: Hemorrhagic shock can lead to multiple organ failure and death. We have previously shown that noninvasive measurement of tissue oxygen saturation (StO(2)) has predictive value for outcomes in patients suffering hemorrhagic shock. Our study objectives were twofold: (1) to compare invasive and noninvasive measurements of local and systemic tissue hemoglobin oxygenation and (2) to compare the effects of various physiologic conditions seen in patients in hemorrhagic shock on tissue hemoglobin oxygenation. MATERIALS AND METHODS: We studied pigs in controlled conditions mimicking shock induced by one of the following: hypothermia, isovolemic hemodilution, or manipulations of vascular tone. We obtained both invasive and noninvasive measurements in a hind limb of StO(2), tissue hemoglobin index, femoral artery and venous flows, blood pressures, temperature, pH, pO(2), pCO(2), oxygen saturation, lactate, hemoglobin, and base excess. In all cases, we measured baseline values in both experimental and control hind limbs. RESULTS: We found that tissue hemoglobin oxygenation did not vary significantly over relevant physiologic temperatures. Under all physiologic conditions tested, we found supply-dependent oxygen consumption at oxygen levels less than 7 mL O(2)/min/kg. Similarly, we found that local oxygen delivery in animals subjected to varying degrees of isovolemic hemodilution or altered vascular tone was correlated with supply-dependent oxygen consumption, as measured by local noninvasive StO(2). CONCLUSIONS: Noninvasive StO(2) measurements are valid and durable over a wide range of physiologic conditions and correlate with invasively-measured oxygen delivery.

Inhibition of hyaluronan synthases decreases matrix metalloproteinase-7 (MMP-7) expression and activity.

INTRODUCTION: Hyaluronan (HA) and its biosynthetic enzymes hyaluronan synthases (HAS2 and HAS3) mediate Matrigel invasion by SW620 colon carcinoma cells. Because matrix metalloproteinases (MMPs) have been implicated in cancer invasion, we hypothesized that changes in HAS expression would alter MMP expression and activity in these cells. METHODS: To determine whether an MMP was involved in invasion, Matrigel invasion assays with SW620 cells were performed in the presence or absence of the MMP inhibitors GM6001 or TIMP2. HAS isozymes were inhibited by stably transfecting SW620 cells with vectors that contained antisense HAS2 and/or -3 cDNA; transfection with an empty vector served as a control. MMP-7 transcription was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-7 protein was detected by enzyme-linked immunosorbent assay (ELISA) and enzymatic activity compared using zymography. RESULTS: GM6001 and TIMP2 decreased Matrigel invasion, which confirms that an MMP played a key role in this process. MMP-7 expression was then detected in SW620 cells. Finally, MMP-7 expression, protein, and enzymatic activity were significantly lower in antisense HAS tranfectants than in SW620 or vector control cells. CONCLUSION: We have demonstrated previously that inhibition of HAS expression and HA production in SW620 colon carcinoma cells inhibits Matrigel invasion. In the studies presented here, we have demonstrated that SW620 cells express high levels of MMP-7 and that inhibition of HAS isozymes dramatically decreases MMP-7 expression, protein, and enzymatic activity. Taken together, these findings suggest that HAS and HA may mediate cellular invasion via changes in MMP-7 expression.

The effect of a hyaluronan-carboxymethylcellulose membrane vs. polyglactin 910 mesh on intra-abdominal tumor formation in mice.

PURPOSE: Hyaluronan mediates growth of SW620 colon cancer cells. Because hyaluronan is the active ingredient in Seprafilm, we hypothesized that Seprafilm would affect intraperitoneal tumor growth in a mouse model of peritoneal seeding. METHODS: Immunodeficient mice underwent laparotomy and intraperitoneal inoculation of 10(5) SW620 cells. Seprafilm (n = 22), Vicryl mesh (foreign body control; n = 24), or no material (sham; n = 19) was placed under the incision. Mice were killed after four weeks and tumors were dissected, counted, and weighed. RESULTS: Ninety-five percent of mice in the sham group and 96 percent in the Vicryl group developed intraperitoneal tumors. In contrast, only 64 percent of mice in the Seprafilm group developed tumors (P = 0.024), and these tumors were smaller than those in the sham group; (Seprafilm = 42 +/- 9 mg vs. sham = 82 +/- 17 mg; P = 0.05). In contrast, tumors in the Vicryl group were dramatically larger (349 +/- 49 mg; P < 0.001 vs. sham or Seprafilm). CONCLUSIONS: Despite previous data that suggested that hyaluronan increases colon cancer cell growth, we found that Seprafilm decreased tumor formation and tended to decrease size in this model. In contrast, Vicryl mesh increased tumor formation and size. Our results suggest that Seprafilm does not promote intraperitoneal tumor growth, especially compared with Vicryl mesh.

Comparison of endotoxin antagonism of linear and cyclized peptides derived from limulus anti-lipopolysaccharide factor.

BACKGROUND: Limulus anti-lipopolysaccharide (LPS) factor (LALF) is a 102-amino acid LPS-binding protein from the horseshoe crab, Limulus polyphemus. The peptide includes the LPS-binding domain of holoLALF, yet it lacks the loop structure stabilized by disulfide or other covalent bonds that is a common motif in the LPS-binding regions of holo- LALF and several other LPS-binding proteins. Although it neutralizes LPS and is bactericidal against Pseudomonas aeruginosa, the LALF 28-54 portion of LALF is not protective in a murine model of intraperitoneal sepsis compared with holoLALF. We examined the effects of cyclizing this linear peptide to determine if this action would recapitulate the stable loop-type structure and enhance its LPS-neutralization and bactericidal activity in vitro. METHODS: Cyclic LALF 28-54 was produced by oxidizing linear LALF 28-54. Each peptide, along with appropriate controls, was assayed for LPS neutralization using the chromogenic Limulus amebocyte lysate (LAL) assay and a bioassay to measure inhibition of LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha) by murine macrophages. Bactericidal activity against Pseudomonas aeruginosa was also assessed. Data were analyzed using a two-tailed Student t-test. RESULTS: Polymyxin B and holoLALF exhibited potent endotoxin antagonism in both assays, as well as bactericidal activity. Concordant with prior studies, the linear form of LALF 28-54 exhibited either similar or a slightly lesser degree of activity in all assays. However, cyclization was associated with significantly diminished endotoxin neutralization in both assays (p < 0.05) and decreased bactericidal activity (p < 0.05) compared with linear LALF 28-54. CONCLUSIONS: Whereas a synthetic linear peptide based on the endotoxin-binding region of holoLALF retained activity, cyclization was associated with a diminution in potency in vitro. We postulate that cyclization does not constrain the peptide in a manner that recreates the loop structure necessary for potent endotoxin antagonism.

Sp1 elements regulate transcriptional activity within the murine Toll-like receptor 4 promoter.

BACKGROUND AND PURPOSE: Toll-like receptor 4 (TLR4) mediates the innate immune response to lipopolysaccharide (LPS) by a complex intracellular signaling pathway that activates the transcription factor nuclear factor (NF)-kappaB, with subsequent production of inflammatory cytokines. The precise mechanisms involved have not been delineated. We hypothesized that specific regulatory elements exist within the TLR4 promoter. METHODS: We cloned regions of the murine TLR4 promoter (0.25 kb to 2 kb; -2000 bp to +244 bp) into the pGL3-Basic plasmid containing the firefly luciferase gene and used the resulting constructs to transfect HEK293 or RAW 264.7 cells. After 24 h, we used LPS to stimulate RAW 264.7 cells. We quantified relative light units (RLUs) of cell lysates and secreted tumor necrosis factor (TNF)-alpha. For pairwise comparison, we used Student's t-test. Sequence analysis of the promoter revealed several putative Sp1 sites. We used two constructs showing increased promoter activity, TLR4-750/-1 and TLR4-500/-1, to transfect cells in the presence of a specific Sp1 inhibitor, mithramycin A. RESULTS: Of the six promoter constructs, four showed greater transcriptional activity (p < 0.05 vs. pGL3-Basic), as measured by luciferase activity. However, we did not observe differences in transcriptional activity of the promoter in five of those six constructs when we stimulated transfected RAW 264.7 cells with 10 ng of LPS. This finding suggests that transcriptional regulation of the promoter is unaffected by cellular changes caused by LPS. Both TLR4-750/-1 and TLR4-500/-1 showed dose-dependent reductions in transcriptional activity in the presence of increasing concentrations of the specific Sp1 inhibitor mithramycin A in both HEK293 and RAW 264.7 cells (p < 0.05 vs. no mithramycin A). CONCLUSION: At least one Sp1 transcriptional regulatory element is present within the murine TLR4 promoter (range -750 bp to -250 bp). This finding holds promise for manipulating this fundamental inflammatory response.

Anti-endotoxin monoclonal antibodies are protective against hepatic ischemia/reperfusion injury in steatotic mice.

Steatotic mice are particularly susceptible to hepatic ischemia/reperfusion injury compared with their lean littermates. We have previously demonstrated that livers of mice having a spontaneous mutation in the leptin gene (ob/ob), resulting in global obesity and liver steatosis, are ATP depleted, are endotoxin sensitive, and do not survive (I/R) injury. We hypothesize that administration of an anti-LPS monoclonal antibody (mAb) prior to initiation of I/R would be protective from that insult. Steatotic mice (ob/ob) were subjected to 15 min of ischemia via complete porta-hepatis occlusion and varying lengths of reperfusion with or without pre-treatment with an anti-LPS mAb. There was 14-31% survival of isotype matched control mAb treated ob/ob mice after 15 min of ischemia and 24 h of reperfusion. In contrast, 75-83% of ob/ob mice pre-treated with an anti-LPS mAb prior to initiation of I/R survived both ischemia and 24 h of reperfusion. Furthermore, there was a decrease in ALT and circulating endotoxin levels when treated with an anti-LPS mAb compared with control antibodies. Attenuation of the endotoxin load with anti-LPS mAb, prior to initiation of I/R, was cytoprotective and improved survival. Consequently, these studies might offer a solution to the problems associated with using steatotic livers in clinical transplantation.

Structure/function studies of an endotoxin-neutralizing peptide derived from bactericidal/permeability-increasing protein.

BACKGROUND: Bactericidal/permeability-increasing protein, BPI, has a beta-turn with alternating cationic and hydrophobic residues in its lipopolysaccharide (endotoxin, LPS)-binding domain. A peptide, betapep25, was designed with 9 residues of the LPS-binding domain of BPI flanked by beta-turn-inducing elements. Thereafter, we sought to use single amino acid substitutions to identify residues that are important for the biological activities of betapep25. METHODS: Single alanine or norleucine replacement "walkthrough" peptides based on betapep25 were generated and tested for their ability to kill P aeruginosa and to neutralize endotoxin. RESULTS: Substitution of all lysines inhibited bactericidal activity. Inhibition of LPS-neutralizing activity was seen in 9 peptides in which an alanine or norleucine was substituted for each of 4 of the basic residues and 1 hydrophobic residue from the LPS-binding region of BPI and 4 hydrophobic residues from the beta-turn-inducing regions flanking the LPS-binding region on the carboxy-terminal side. Intriguingly, these last 4 substitutions resulted in peptides that exhibited increased bactericidal activity compared to betapep25. CONCLUSIONS: These results demonstrate the importance of both cationic and hydrophobic amino acid residues to bactericidal and endotoxin-neutralizing activities. These perturbations of biological activity should be considered in the design of synthetic peptide endotoxin antagonists.