The acute respiratory distress syndrome (ARDS) affects nearly 200,000 patients per year. Despite intensive research and improved mechanical ventilation strategies, ARDS claims the lives of nearly 30% of the afflicted individuals (1). Acute respiratory distress syndrome can be caused by either primary or secondary lung injury. Primary ARDS is caused by pulmonary processes such as aspiration or pneumonia, whereas secondary ARDS is caused by systemic inflammation following severe injury such as trauma, hemorrhage, or sepsis (1, 2).
The pathophysiology of secondary ARDS can be conceptually divided into an initiating inflammatory phase followed by secondary tissue damage phase (2). In the inflammatory phase, trauma, hemorrhage, and severe sepsis cause a massive release of multiple inflammatory mediators that cause the clinical manifestations of acute shock (3). In the tissue damage phase, these inflammatory mediators trigger organ damage by promoting a variety of proteolytic and oxidative effectors including matrix metalloproteinases (MMPs), reactive oxygen species (4), and damage-associated molecular patterns (5). Experimental pharmacotherapies that target single mediators in the inflammatory phase of ARDS pathophysiology have not met with success in the clinical realm. This failure of single-mediator pharmacotherapy in ARDS is attributed primarily to the complexity and redundancy of the inflammatory phase of shock and also to limitations of the small-animal and cell culture models in which these therapies were initially tested (6).
Pleiotropy is a pharmacologic term given to medications that work on multiple targets rather than a specific one. We hypothesized that a pleiotropic drug, acting on multiple targets in both the inflammatory and tissue damage phases of secondary ARDS pathophysiology, could succeed where prior drugs targeted at individual mediators have failed. In theory, such a drug should not only attenuate the release of inflammatory mediators, but also inhibit the proteolytic and oxidative effectors that cause end-organ damage as well. Chemically modified tetracycline (6-demythyl-6-deoxy-4dedimentylamino-tetracycline; CMT-3) is a nonantimicrobial, pleiotropic, anti-inflammatory agent that inhibits multiple inflammatory and proteolytic mediators including MMPs, tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) (7, 8). In a previous study using the same “2-hit” porcine peritoneal sepsis + ischemia/reperfusion (PS + IR) model described herein, we demonstrated that CMT-3 administered prophylactically before PS + IR injury did prevent ARDS (9). In the present study, we hypothesized that CMT-3 would attenuate the lung injury associated with our porcine PS + IR model and prevent the development of ARDS when administered 1 h after the injury. In terms of clinical time frame, CMT-3 can be delivered to trauma patients at high risk for developing ARDS during the initial resuscitation in the emergency department. Thus, the current study demonstrates the proof of concept that CMT-3 can be used in a clinically relevant time frame in patients at risk of developing ARDS secondary to trauma or sepsis (9, 10).
The differences between the CMT-3–treated and placebo-treated animals were dramatic. Chemically modified tetracycline 3 treatment completely prevented ARDS as measured by PaO2/FIO2 ratio criteria as well as by quantitative histology. Chemically modified tetracycline 3 significantly lowered plasma concentrations of IL-1β, TNF-α, IL-6, IL-8, and IL-10, suggesting that the mechanism of lung protection may have been the drug’s pleiotropic reduction of systemic inflammation. The placebo group developed severe ARDS, coagulopathy, and histological injury to the bowel. These data clearly support our hypothesis that CMT-3 prevents the development of ARDS in this clinically relevant model of sepsis. Interestingly, in developing this porcine model of sepsis and IR, we found that the injury not only caused ARDS but also caused injury to other organ systems (11), specifically, coagulopathy and histopathologic injury in the intestine. These injuries were prevented by CMT-3 in the present study as well.
All techniques and procedures described have been fully approved by the Committee for the Human Use of Animals at Upstate Medical University.
Detailed methods describing this model have been published elsewhere (12, 13). Briefly, healthy female Yorkshire pigs (21–38 kg) were pretreated with glycopyrrolate, tiletamine hydrochloride and zolazepam hydrochloride, and xylazine. After intubation, anesthesia was maintained using a continuous infusion of ketamine/xylazine to maintain a comfortable plane of anesthesia. All animals were continuously monitored and cared for by the investigators for the full 48-h duration of the experiment.
Tracheostomy and mechanical ventilation
An open tracheostomy was performed, and the animal was connected to a Galileo ventilator (Hamilton Medical, Reno, Nev) with initial settings during the surgical preparation as follows: tidal volume of 12 mL/kg, respiratory rate of 15 breaths/min, FIO2 of 21%, and positive end-expiratory pressure of 3 cmH2O. Respiratory rate was titrated to maintain PaCO2 within the reference range (35–45 cmH2O); FIO2 was titrated to maintain O2 saturation of greater than 88%. Low-tidal-volume protective mechanical ventilation was not used because we did not want to “protect” the lung with the ventilator but rather measure the development of lung injury if it occurred.
Under sterile conditions, a carotid arterial catheter and two external jugular central venous catheters were placed. A Foley catheter was inserted directly into the bladder for measurement of urine output and collection of urine samples. All animals received a regimen of intravenous fluids and antibiotics at a dose and quantity established in our initial experiments (see Fluids and antibiotic management).
After placement of arterial and venous catheters, a midline laparotomy was performed for placement of a gastric tube (Bard, Covington, GA) and induction of a 2-hit injury, PS + IR, described in detail in prior publications from this model (12, 13). The superior mesenteric artery (SMA) was clamped for 30 min to induce intestinal ischemia. During the 30-min ischemic time, 0.5 mL/kg of feces was harvested from a cecotomy and mixed with 2 mL/kg of blood to create a fecal clot. After releasing the clamp on the SMA, the clot was implanted into the lower portion of the abdominal cavity. The abdomen was then closed with a running monofilament fascial suture and skin staples.
Baseline (BL) measurements were taken following vascular access before injury. Time 0 (T0) measurements were taken immediately after the induction of injury (i.e., removal of SMA clamp and placement of fecal clot) upon closure of the abdomen. (For a full timeline, see Fig. 1.)
One hour after injury, the animals were randomized into two groups: group 1, CMT-3 treated (n = 7), the animals received an orally active dose (200 mg/kg) of the modified tetracycline CMT-3 (6-demythyl-6-deoxy-4dedimentylamino-tetracycline; CollaGenex Pharmaceuticals Inc, Newton, Pa) delivered per gastrostomy; group 2, placebo (n = 9), the animals received the same dose of a vehicle (carboxymethylcellulose) for CMT-3 delivered per gastrostomy.
Fluids and antibiotic management
Ringer’s lactate was used to for fluid resuscitation and maintenance. Maintenance fluid requirements were calculated on a per-kilo basis according to clinical guidelines. Fluid boluses were given as indicated by deteriorations in hemodynamic parameters or decreases in urine output less than 0.5 mL/kg per hour. Broad-spectrum antibiotics were delivered intravenously following closure of the abdomen (ampicillin 2 g i.v. [Bristol Myers Squibb, Princeton, NJ] and metronidazole 500 mg i.v. [Baxter, Deerfield, Ill]). This antibiotic regimen was repeated at 12, 24, and 36 h after injury.
Physiologic measurements and pulmonary mechanics
Hemodynamic parameters were measured (CMS-2001 System M1176A, with Monitor M1094B; Agilent, Böbingen, Germany) using Edwards transducers (Pressure Monitoring Kit [PXMK1183]; Edwards Lifesciences, Irvine, Calif). Pulmonary parameters were measured or calculated by the Galileo ventilator (Hamilton Medical).
Plasma and bronchoalveolar lavage fluid cytokine measurements
Blood was drawn every 6 h and spun at 3,500 revolutions/min at 15°C for 10 min. The concentrations of TNF-α, IL-1β, IL-6, IL-8, and IL-10 were determined in the plasma and bronchoalveolar lavage fluid (BALF) using enzyme-linked immunosorbent assay according to the manufacturer’s recommendations.
Measurement of blood gases and chemistries was made with a Roche blood gas analyzer (Cobas b221; Basel, Switzerland). Clinical pathology and blood cultures were performed on arterial blood samples by the Upstate Medical University pathology laboratory facility.
Bronchoalveolar lavage procedure
At necropsy, the right middle lobe was lavaged with 60 mL of normal saline (three injections of 20 mL flushed into the right middle lobe bronchus and aspirated out), and the volume collected was recorded. The BALF was spun for 10 min at 3,500 revolutions/min at 15°C for 10 min.
Immediately following early mortality or killing at 48 h, necropsy was performed to recover tissue for analysis. Lungs were inflated to 25 cmH2O using stepwise increases of continuous positive airway pressure on the ventilator and clamped to maintain a consistent lung volume history in all animals. The lung was then unclamped, and the airway filled with formalin to a consistent filling pressure of 25 cmH2O reclamped and submerged in formalin. Following 24 h in the fixative, the lungs were removed, cut, and sent to histology for processing. The following procedure was performed on the last six animals (three from each treatment group): the laparotomy was subsequently reopened, and 20-cm specimens of small bowel were removed from the proximal jejunum, distal jejunum, and distal ileum. These specimens were preserved in 10% neutral buffered formalin for 48 h before histological sectioning.
Histological analyses (hematoxylin-eosin) were performed on all tissue from necropsy. The histologist was blinded to animal group during morphometric measurements. A detailed description of our histological methods has been previously described (12, 13).
The quantitative histological technique described in Methods allows an unbiased comparison of the degree of tissue injury to the lungs between the two treatment groups. As previously described (12, 13), the quantitative histological assessment of the lung was based on image analysis of 120 photomicrographs (10 per pig), made at high-dry magnification following an unbiased, systematic sampling protocol. Each photomicrograph was scored using a four-point scale for each of five parameters: atelectasis, fibrinous deposits, and blood in airspace; vessel congestion; alveolar wall thickness; and leukocyte infiltration.
Survival rates between the two treatment groups were compared using the event time distribution functions and a log-rank test. A repeated-measures analysis of variance (RM ANOVA) with pig number and treatment as random effects was performed to compare differences within and between treatment groups for continuous parameters over time. Significance in the RM ANOVA is expressed as P for group × time meaning that there was a difference between groups over time. A criterion of the repeated-measures analysis is that there can be no missing data points. This study produced severe critical illness, and certain animals suffered early mortality. To account for animals that failed to survive the full course of the experiment and therefore incorporate all animals randomized in the study, a least squares regression model was used to calculate the intra-animal predicted value for the repeated-measures analyses. Significant results from the random-effects RM model were further examined at each post-BL time point following a Bonferroni correction to adjust for multiplicity. Quantitative histology data were analyzed using a Mann-Whitney U test after testing for normality. P < 0.05 was considered significant. All analyses were performed using JMP version 5.1.1 (Cary, NC).
Animals in both groups developed polymicrobial bacteremia as assessed by qualitative blood cultures. The bacteria found in these cultures were Escherichia coli, streptococcal species, staphylococcal species, Serratia marcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Aeromonas hydrophila.
The mean arterial pressure (MAP) for the placebo group at BL was 117 ± 1 mmHg, and by T48, it had dropped to 53.2 ± 5.4 mmHg. The CMT-3 group’s BL MAP dropped from 135 ± 5.2 mmHg to 69 ± 7.6 mmHg at T48. These differences were not statistically significant. Central venous pressure and cardiac output did not differ significantly between the two treatment groups (Table 1).
The CMT-3 group had significantly higher urine output, lower resuscitative fluid requirements, and correspondingly lower positive fluid balance over the 48-h study (P < 0.05).
Circulating levels of TNF-α, IL-1β, and IL-6 all increased significantly and progressively over the 48-h course of the experiment (Table 2) in the placebo group. Chemically modified tetracycline 3 significantly reduced circulating cytokine levels versus placebo.
Bronchoalveolar lavage cytokines
Levels of IL-1β, TNF-α, IL-6, IL 10, and IL-8 were lower in the BALF of the CMT-3–treated animals than that of the placebo group; however, these data did not show statistical significance (Table 3).
Levels of MMP-2, MMP-9, and neutrophil elastase were not different between CMT-3 and placebo group s in this study (Table 4).
The placebo group experienced 40% survival at 48 h, whereas the CMT-3 group experienced 85.7% survival at 48 h. However, this difference was not statistically significant (Fig. 2).
There was no statistical difference between treatment groups in tissue edema of the lungs.
Peak airway pressures (Ppeak) were initially similar in the two groups (CMT-3: 22.4 ± 2.4 cmH2O vs. placebo: 21.1 ± 0.8 cmH2O). However, by 30 h into the experiment, the placebo group had Ppeaks of 36.0 ± 4.5 cmH2O, whereas the CMT-3 group was nearly unchanged at 25.4 ± 1.6 cmH2O. At the end of the experiment, the protective effect of CMT-3 on peak airway pressures was clearly demonstrated, as the CMT-3 group had Ppeaks of 28.6 ± 2.5 cmH2O, whereas the placebo group was extremely difficult to ventilate at a Ppeak of 34.5 ± 5.6 cmH2O. Plateau and mean airway pressures correlated to the trend seen in the peak airway pressures (Table 5). In addition, the static compliance changes (Fig. 3) corroborate the changes in lung pressures. The PaO2/FIO2 ratio (Fig. 4) demonstrated that, by 30 h into the experiment, the placebo group met the criteria for acute lung injury, and by 48 h, these animals were well within the range of ARDS. In contrast, ARDS did not develop in the CMT-3 group.
The quantitative histological technique described in Methods allows a bias-free comparison of the degree of tissue injury between the two treatment groups. The specific histological lesions analyzed are considered pathognomonic for ARDS when in the context of the appropriate clinical course (14). Atelectasis, fibrin deposits, leukocyte infiltration, and alveolar wall thickness were all significantly lower in the animals treated with CMT-3. Dramatic differences were observed between groups in alveolar wall thickness, which was nearly quadrupled in the placebo group versus the CMT-3 group (placebo: 2.41 ± 0.3, CMT: 3 0.49 ± 0.10) (Table 6). Dramatic differences were also observed in the degree of fibrinous deposits and leukocyte infiltration, which were both doubled in the placebo group as compared with the CMT-3 group (Table 6). The histological lesions of ARDS mentioned above are visually represented in Figure 5, supporting the impression of protective effects of CMT-3 on the lung. Figure 5A shows the lung of a placebo-treated animal with classic stigmata of ARDS atelectasis, intra-alveolar hemorrhage, fibrinous exudates, and leukocytic infiltrates. Figure 5B shows the lung of a CMT-3–treated animal, with preservation of nearly normal pulmonary architecture.
Placebo-treated animals became progressively and profoundly leukopenic, which is consistent with severe sepsis in humans. Baseline white blood cell counts were significantly higher in the CMT-3 group than in the placebo group (Table 7). This difference between groups persisted throughout the course of the experiment. There was a predominance of neutrophils in both groups at T12. As placebo animals became leukopenic, all subpopulations (neutrophils, eosinophils, basophils, lymphocytes) decreased equally. Chemically modified tetracycline 3 preserved all subpopulations of white blood cells equally.
Chemically modified tetracycline 3 exerted a significant, protective effect on the coagulation system. Placebo-treated animals became progressively coagulopathic, whereas CMT-3–treated animals did not as evidenced by international normalized ratio (INR) and partial thromboplastin time data (Table 7) (P < 0.05). Chemically modified tetracycline 3 prevented the profound thrombocytopenia observed in the placebo group (Table 7); these data were statistically significant.
The placebo group (n = 3; Fig. 6: center panel “Fecal clot”) exhibited grossly shortened and denuded villi with significant sloughing of surface epithelium into the bowel lumen. Intestinal glands showed areas devoid of cellular definition, suggesting a loss of the epithelial barrier. In contrast, the CMT-3 group (n = 3; (Fig. 6: far right panel “Fecal clot + CMT-3”) exhibited villi of normal length with only focal denudation of the surface epithelium; intestinal glands were normal in this group. The far left panel of Figure 6 (“Naive pig”) is a nonmanipulated healthy pig used as a histological control for comparison.
The central finding of the present study is that administration of CMT-3 immediately following injury in a clinically relevant porcine model of lung injury prevents the development of ARDS, supporting our hypothesis. Severe sepsis and its complications have eluded attempts at single mediator targeted therapies (15). Only activated protein C has gained clinical relevance, but pharmacotherapy with activated protein C is extremely limited in postsurgical patients because of hemorrhagic complications (16). A major cause for the failure of single-mediator–targeted medications is thought to be the extreme redundancy of the inflammatory cascade (17). Chemically modified tetracycline 3 is known to work through a variety of mechanisms to reduce inflammation; it is a truly pleiotropic anti-inflammatory agent (18). It is possible that CMT-3 treatment produced clinically relevant improvements in our large animal model because the pleiotropy of the drug’s effects overcame the redundancy of the inflammatory response.
The present study is the culmination of a series of experiments demonstrating the efficacy of CMT-3 in preventing ARDS (9, 10). Early studies using small animal models (rats) and acute pig studies demonstrated the potential for CMT-3 use in systemic inflammatory injury caused by cecal ligation and puncture, cardiopulmonary bypass, and endotoxin (9, 10). Steinberg et al. (9) established proof of concept in a similar chronic PS + IR porcine model of ARDS, where CMT-3 was given before the 2-hit injury was induced. The current study advances the therapeutic potential of CMT-3 because we demonstrated that CMT-3 is efficacious when delivered 1 h after injury in a translational model. This suggests that patients at risk of ARDS secondary to sepsis or other acute inflammatory insults could be given CMT-3 in the emergency room or intensive care unit to effectively prevent ARDS.
An intriguing corollary to the main finding of the present study was that CMT-3 also reduced sepsis-associated coagulopathy, thrombocytopenia, and the histologically observed bowel injury that were recently shown to be features of this animal model (12, 13). In a subset of the animals in this study, intestine was harvested for histological analysis. There was a significant reduction in mucosal and submucosal inflammation in the small bowel of the CMT-3 group. Our study suggests that the pleiotropic anti-inflammatory properties of CMT-3 have a protective effect on multiple organ systems. We discuss those effects below.
Effects of CMT-3 on inflammatory cytokines and proteases
Chemically modified tetracycline 3 caused marked reductions in TNF-α and IL-1β (Table 2), both of which are implicated in the early sepsis-induced systemic inflammatory response, and may be the mechanism of protection to lung and bowel in this study. Treatment with soluble TNF receptor–binding protein prevented lung injury and ARDS in a porcine model of postpump syndrome (19). Treatment with IL-1β receptor antagonist prevented lung injury in a rat model (20). In that study, the investigators also demonstrated that IL-1β blockade reduced alveolar epithelial injury, pulmonary neutrophil sequestration, alveolar wall thickness, and inflammatory infiltrate; these effects are similar to those shown here with CMT-3 treatment. Because blockade of either TNF-α or IL-1β can prevent ARDS, the fact that CMT-3 blocks the production of both suggests that this may be a mechanism of action offering lung and bowel protection in this study.
The majority of studies on the anti-inflammatory effects of CMT-3 have shown that the drug’s efficacy is due in part to inhibition of both MMP-2 and MMP-9 (7, 8). The current study did not find any significant differences in plasma and BALF concentrations of MMP-2, MMP-9, and elastase between groups (Table 4), although prior studies from our laboratory demonstrated a sepsis-induced increase in MMP activity that was reduced with CMT-3 (9, 21). In the current study, MMP activity was not examined. Chemically modified tetracycline 3–induced reduction of MMP activity may be responsible for the positive outcomes observed and will require further investigation.
Another explanation for the lack of change in MMP concentration between the CMT group and the placebo group may be related to the use of ketamine in this study. Ketamine has been shown to mitigate the effects of cecal ligation and puncture in rats via inhibition of nuclear factor κB (22). Nuclear factor κB signal blockade reduced the expression of MMP-9 in a murine lung cancer cell line (23). Therefore, ketamine may have acted via nuclear factor κB blockade to reduce overall MMP levels. Lastly, it is possible that the absence of MMP activity may not be related to ketamine but rather to a CMT-3–induced reduction in the overall inflammatory status, such that MMPs were not upregulated and released in the same concentrations as in previous studies (9).
Effects of CMT-3 on hematologic and coagulation function
The coaguloprotective effect of CMT-3 was unanticipated (Table 7). Previous studies have noted a connection between coagulopathy and ARDS in sepsis (24). A recent review observed that disordered coagulation likely underlies the alveolar fibrin deposition that is characteristic of ARDS (25). In the present study, CMT-3 decreased alveolar fibrin deposition (Table 6), suggesting that modulating the coagulation system may be yet another mechanism by which CMT-3 reduces lung injury.
The most important mechanism of sepsis-associated coagulopathy is the direct alteration of the coagulation cascade and attendant thrombocytopenia (26); CMT-3 prevented the direct alteration in the coagulation cascade, evidenced by normalization of prothrombin time and INR and the prevention of thrombocytopenia (Table 7). The mechanisms underlying these novel actions of CMT-3 are unknown, and further studies are necessary.
Adequate platelet number and function are both necessary for normal coagulation (27). Therefore, the ability of CMT-3 to prevent thrombocytopenia may be the central mechanism by which this drug preserves normal coagulation. Additional mechanisms may also underlie the coaguloprotective effects of CMT-3. Three enzymes essential for platelet activation are cyclooxygenase 2, thromboxane A2, and phospholipase A2 (27). Chemically modified tetracycline 3 is known to act on cyclooxygenase 2 to inhibit both nitric oxide and prostaglandin E2 (28), which are essential to platelet activation and degranulation.
Effects of CMT-3 on the gut
Kubiak et al. (11) recently showed that this animal model causes injury not only to the lung but also to the kidney, liver, and intestine. Chemically modified tetracycline 3 reduced bowel histopathology as compared with placebo (Fig. 6). In our animal model, the intestines are the organ system receiving the most serious insult. The sepsis and IR injuries both target the abdomen; the severe intestinal histopathology seen in the placebo group reflects this. The prevention of detectable bowel damage by CMT-3 provides compelling preliminary evidence for this drug’s efficacy as an anti–systemic inflammatory response syndrome–induced organ damage pharmacotherapy.
The mechanism by which CMT-3 protects the bowels is as yet unknown, but might be related to the known effects of CMT-3 on TNF-α (29). Monoclonal anti–TNF-α antibody (Remicade) is the standard of care for reduction of intestinal inflammation in the management of inflammatory bowel disease (30). The CMT-3–induced reduction in plasma TNF-α could be responsible for the protection of the bowel. Further studies are needed to determine if the reduction of systemic or local TNF-α by CMT-3 is the mechanism for bowel protection by this drug.
In summary, this study demonstrates that the modified tetracycline CMT-3, given 1 h after injury, will prevent the development of ARDS and intestinal histopathology in a clinically relevant porcine model of established sepsis and gut IR. The mechanism of this protection may involve a reduction in the circulating levels of TNF-α and IL-1β. However, CMT-3 also prevented thrombocytopenia and reduced the coagulopathy associated with this animal model, mechanisms that may also play a role in the protective effect of CMT-3 to the lung and gut. Thus, CMT-3 or related compounds may represent novel therapies for prevention of ARDS.
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