Shock/sepsis/trauma/critical careMetalloproteinase inhibition reduces lung injury and improves survival after cecal ligation and puncture in rats☆
Introduction
Acute respiratory distress syndrome (ARDS) is a form of acute lung injury characterized by refractory hypoxemia and high permeability pulmonary edema 1, 2. Despite significant advances in surgical technique and therapy, mortality from ARDS remains high [3]. The current treatment of ARDS is predominately supportive. A thorough understanding of the pathogenesis of ARDS is required to develop effective treatment strategies.
The development of ARDS can be separated into two phases: the initiator stage followed by an effector stage (Fig. 1). The initiator phase of ARDS involves the release of inflammatory mediators (i.e., cytokines, complement, coagulation factors, and arachidonic acid metabolites), which promote systemic inflammation resulting in pulmonary neutrophil sequestration 4, 5, 6. The results of clinical trials testing efficacy of antiinflammatory agents targeting these initiators have been dismal 7, 8. Ware and Matthay have offered several explanations for these failures including the timing of treatment intervention and the inherent redundancy of the inflammatory cascade [9]. We further define the argument of Ware and Matthay by highlighting that redundancy occurs during the initiator phase. The second stage, the effector phase, involves the activation of neutrophils with subsequent release of toxic oxygen radicals and proteolytic enzymes, specifically neutrophil elastase (NE) and matrix metalloproteinases (MMPs). Both NE and MMPs are known to mediate lung injury 10, 11, 12. The effector phase represents a “bottleneck” in the development of ARDS and offers a therapeutic target that, unlike pharmaceuticals targeting initiators, may effectively reduce the morbidity and mortality associated with sepsis-induced ARDS [13].
Neutrophil elastase has the capacity to injure pulmonary endothelial cells [14] and degrade products of the extracellular matrix, such as elastin, collagen, and fibronectin, which comprise the lung basement membrane [15]. Several experimental animal models of acute lung injury have demonstrated elevated levels and activity of NE in bronchoalveolar lavage (BAL) fluid 16, 17. Furthermore, increases in NE concentration and activity have been demonstrated in both plasma and BAL fluid of patients with ARDS 18, 19. Experimental studies 20, 21, 22, 23, 24, 25 have also demonstrated reduction of lung injury using a wide array of NE inhibitors, however, all experiments except one [25] have used acute models of pulmonary injury that do not parallel the development of ARDS clinically.
MMPs are a family of proteolytic enzymes known to degrade type 4 collagen of the alveolar basement membrane and possess the ability to act synergistically with NE [26]. Recent data emphasizes the role of MMPs (specifically MMP-2 and MMP-9) in the development of acute lung injury both experimentally 27, 28, 29, 30, 31 and in clinical ARDS 32, 33, 34. Furthermore, recent evidence from Carney et al. [13] demonstrated that inhibition of MMP-2 and MMP-9 prevented lung dysfunction in an acute animal model of pulmonary injury.
Chemically modified tetracycline 3 (COL-3) is a synthetic derivative with no antimicrobial activity that has recently demonstrated efficacy in the prevention of acute lung injury in several acute animal models 13, 35. Unlike specific protease inhibitors 17, 20, 36, COL-3 has the capability of neutralizing both NE and MMPs. Additionally, by targeting the terminal effectors of tissue damage, as opposed to the initiators, COL-3 as a treatment strategy in acute lung injury eliminates the concern of the redundant inflammatory mediators (Fig. 1). Early success of COL-3 in acute studies was, in part, attributed to prophylactic administration before lung injury was established 13, 35. Although the ability of COL-3 to prevent lung injury in these acute studies is encouraging, validation of the efficacy of COL-3 in a more clinically relevant model of sepsis-induced acute lung injury is necessary. The purpose of this study was to test the hypothesis that COL-3 given prophylactically could reduce pulmonary injury and improve survival in a rat model of sepsis-induced acute lung injury from cecal ligation and puncture. Furthermore, we theorize that repeat doses of COL-3 would further reduce pathologic lung injury and enhance survival benefit.
Section snippets
Surgical procedure
Male Sprague-Dawley rats weighing between 250 and 300 g were acclimatized to the laboratory environment for 1 week prior to surgery. Free access to food and water was available for this time period. Rats were anesthetized with intraperitoneal (IP) ketamine (90 mg/kg)/xylazine (10 mg/kg). Sepsis was produced using a modification of the cecal ligation and puncture (CLP) technique described by Chaudry et al. [37]. After the abdominal fur was shaved, a 2-cm midline incision was made through the
Survival
Mortality in the CLP + CMC group was 70% at 168 h (7 days). Mortality was significantly reduced (54%) with a single prophylactic administration of COL-3 in the CLP + COL-3 (SD) group (Fig. 2). Additionally, a repeat dosing with COL-3 at 24 h after CLP (24 h after the first dose), further reduced mortality (33%) in the CLP + COL-3 (MD) group (Fig. 2). All animals in the sham groups (sham CLP + CMC and sham CLP + COL-3) survived.
Histology
Cecal ligation and puncture without treatment (CLP + CMC group)
Discussion
The most significant finding in this study is that the modified tetracycline COL-3 improves survival of rats in a dose-dependent fashion in a clinically applicable model of sepsis-induced acute lung injury. Specifically, improvement in survival correlated with reduction of lung injury and decreased pulmonary tissue MMP-2 and MMP-9 levels. These data correlate and support findings from our previous studies that demonstrated reduction of lung injury with COL-3 in acute models 13, 35. However,
Acknowledgements
We thank Andrew Paskanik and Kathy Snyder for their expert technical assistance.
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