VEGF-A blockade reduces reperfusion edema but favors arterial thromboembolism in a rat model of orthotopic lung transplantation.

BACKGROUND: Ischemia-reperfusion edema is a common early complication after lung transplantation where the hypoxia-induced vascular endothelial growth factor (VEGF)-A plays a pivotal role. It remains unclear whether a VEGF blockade is beneficial in lung transplantation. METHODS: VEGF-A blockade was investigated in an orthotopic rat model of lung transplantation. VEGF-A antibody was added into the preservation solution alone (α-VEGF D/-), in the preservation solution and systemically to the recipient before reperfusion (α-VEGF D/R), or applied to the recipient alone before reperfusion (α-VEGF -/R). Forty-eight hours after lung transplantation, left lungs were collected and wet-to-dry ratio, Western blotting, RT-PCR, and immunohistology were performed. RESULTS: VEGF-A blockade in α-VEGF D/-, α-VEGF D/R, and α-VEGF -/R resulted in neutralization of tissue VEGF-A. Reperfusion edema was only reduced in α-VEGF D/R and α-VEGF D/- groups versus Perfadex controls. Some α-VEGF -/R rats showed a hyperinflammation leading to increased pro-inflammatory cytokine expressions as well as increased edema. Whereas generally the α-VEGF D/- group showed decreased inflammation, the combination with anti-VEGF treatment to the recipient resulted in a pro-inflammatory and a pro-apoptotic phenotype. Short-term survival, however, was not significantly different in all groups as compared to the controls. In the α-VEGF (D/R) or (D/-) groups, animals mainly died from arterial thromboembolisms and in the α-VEGF (-/R) group, hyperinflammation was the main cause of death. CONCLUSION: VEGF-A directly contributes to the formation of a reperfusion edema, which might be reduced by its blockade. However, the α-VEGF effect on the endothelial integrity might also favor arterial thrombosis formation.

Prednisolone as preservation additive prevents from ischemia reperfusion injury in a rat model of orthotopic lung transplantation.

The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/ß and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.

Cecum ligation and dissection: a novel modified mouse sepsis model.

BACKGROUND: The fact that many sepsis therapeutics failed to be translated into the human indicates that there is still a serious need to reassess our models of sepsis research. We aimed to develop a novel modified model of sepsis in the mouse, which simulates the clinical situation more accurately. MATERIALS AND METHODS: Sepsis was induced in C57Bl/6 mice by dissecting the cecum and placing the discontinued organ back into the abdomen (cecum ligation and dissection [CLD]). Septic animals were relaparotomized after 6 h, followed by peritoneal lavage, and antibiotic treatment. Results were compared with shams or the classic colon ligation and puncture (CLP) model. The postoperative lung impairment was assessed using neutrophil invasion as a surrogate. Proinflammatory cytokines were measured by either real-time polymerase chain reaction or Luminex technology, and liver damage was evaluated by aspartate transaminase and alanine transaminase measurements. RESULTS: In CLD animals with relaparotomy after 6 h, lung interleukin (IL) 6, monocyte chemoattractant protein (MCP)-1 messenger RNA levels, and neutrophil invasion were significantly increased. Liver messenger RNA expression in CLD animals was significantly upregulated for IL-6, tumor necrosis factor alpha, IL-10, and MCP-1 compared with sham and CLP animals. Significantly higher levels of alanine transaminase were observed in CLD animals. Finally, systemic inflammation as measured by plasma IL-6, tumor necrosis factor alpha, IL-1ß, IL-10, and MCP-1 was significantly increased in all CLD animals compared with shams, whereas CLP animals only showed an insignificant increase in the latter molecules. CONCLUSIONS: Our modifications to the classic CLP model significantly produced organ inflammation, liver damage, and a similar mortality compared with a clinical setting, with a reliable onset of sepsis.

The fibrinopeptide bß15-42 reduces inflammation in mice subjected to polymicrobial sepsis.

Sepsis is still a leading cause of death on intensive care units. Despite intensive research, only few new therapies have been developed and used in the clinical setting. The fibrin fragment Bß15-42 was already shown to preserve endothelial barrier function by binding to VE-cadherin and thus stabilize the interendothelial junctions. This was accompanied by reduced inflammation. Now we show that treatment with Bß15-42 reduces inflammation in a murine polymicrobial sepsis model. Administration of Bß15-42 reduced proinflammatory cytokine levels in the lung, liver, and blood and decreased neutrophil infiltration into the lung. Analysis alanine aminotransferase and aspartate aminotransferase further indicated reduced liver damage following polymicrobial sepsis. In vitro experiments using endothelial cells and macrophages further revealed that Bß15-42 had no direct effect on Toll-like receptor-mediated inflammation. Therefore, we assume that attenuated inflammation is rather due to sustained vascular integrity and thus suppresses vascular leakage and subsequently leukocyte infiltration during sepsis.