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Expression Profiling and Disseminated Intravascular Coagulation

Finding Genes Gone Wild

The Texas Lung Injury Institute, The University of Texas Health Science Center at Tyler, Tyler, Texas

In this issue of the Journal (pp. 602–609), Slofstra and colleagues demonstrate the power of gene expression profiling to identify new candidate genes that may contribute to the pathogenesis of disseminated intravascular coagulation (DIC) (1). The authors applied the technique in mice and used a two-hit endotoxin challenge to induce the generalized Shwartzman reaction (2). They interrogated hepatic gene expression in these mice under baseline conditions as well as after endotoxin priming and a subsequent endotoxin challenge.

The gene profiling technique the authors used is a remarkable strength of the study. Briefly, they identified 3,787 sequences that were differentially regulated across the 26-hour period from baseline to 2 hours after the second endotoxin challenge. Of these genes, 1,097 sequences were found that exhibited a greater than twofold change. Next, the genes were sorted by ascending, descending, or no response to the two endotoxin treatments. Because both endotoxin challenges are required to induce the generalized Shwartzman reaction, particular attention was paid to the 39 genes that were up-regulated by both injections. Some of these genes, such as plasminogen activator inhibitor (PAI)-1 or KC (the murine IL-8 analog), are established marker/effectors linked to sepsis (3, 4). However, several new candidate potential effectors were also identified. The transcription factor C/EBP was among this last subset of differentially regulated genes. Its role in endotoxin-induced DIC was then pursued because C/EBP has previously been implicated in the pathogenesis of inflammation (5).

The authors showed that C/EBP expression was increased in lung, liver, and kidney tissues of mice with endotoxin-induced DIC and that plasma tumor necrosis factor (TNF)- and IL-6 were attenuated in C/EBP^–/– mice. Interestingly, the survival advantage of C/EBP^–/– mice to a lethal generalized Schwartzman reaction was modest compared with wild-type mice. Based on renal and hepatic biomarker analyses, it is likely that the survival response involved the contributions of renal protection and concurrent incremental liver injury in C/EBP^–/– mice. Although the mechanisms that underlie these responses remain to be elucidated, renal protection was also conferred in C/EBP^–/– mice in a second model of ischemia–perfusion renal injury. These findings indicate that C/EBP expression contributes to the pathogenesis of endotoxin-induced DIC in mice via a complex interplay of differential effects in different organ systems, some salutary and others deleterious.

Apart from the nice application of gene expression profiling technology, this study raises a number of questions. First and foremost, what happens in the lungs of C/EBP versus wild-type mice subjected to the generalized Schwartzman reaction? Because differential changes in the lungs could have importantly influenced survival, this question assumes particular importance. The design of this study does not address the issue aside from the inclusion of data that show that the pulmonary response to the generalized Schwartzman reaction incorporates increments of C/EBP, which closely approximates that of the kidney. Recently, the authors addressed part of the puzzle in another study in which they show that pulmonary thrombi progressively occurred in their model during the 6 hours after repeat endotoxin challenge, that little thrombus formation occurred after priming, and that the appearance of pulmonary thrombi correlated with those in the liver over time (2). No assessment of lung histology was presented, so the presence or extent of acute lung injury (ALI) at the intervals interrogated in the present study remains at issue.

Although DIC is a prominent feature of the generalized Schwartzman reaction, the authors remind us that tissue damage also occurs in association with microvascular injury, vascular occlusion, inhibition of fibrinolysis, and microvascular apoptosis (2, 6). These observations suggest that end-organ tissue damage encompasses ALI, which, in fact, has been reported to occur in the generalized Shwartzman reaction (7). The relationship between coagulopathy and ALI is of particular clinical relevance given the long-established association between DIC and adult respiratory distress syndrome (ARDS) (8), extravascular fibrin formation and ALI (9), and the occurrence of pulmonary vascular thrombi in association with severe ARDS (10). Extravascular fibrin deposition and aberrant fibrin turnover contribute to the pathophysiology of most forms of ALI (11–14), and aberrant remodeling of the transitional fibrinous neomatrix can promote the subsequent development of accelerated pulmonary fibrosis (9, 15, 16). Whether C/EBP exerts a protective or deleterious effect on lung inflammation, fibrinous alveolitis, epithelial cell apoptosis, or remodeling is unresolved and this question represents an opportunity for future investigation. The effects of C/EBP deficiency on the histology of the kidneys and liver also remain at issue, especially as to the temporal appearance and extent of the microvascular apoptotic response.

Another fascinating aspect of the study relates to the timing and contribution of the coagulopathy to the thrombotic response and organ dysfunction associated with the generalized Schwartzman reaction itself. End-organ dysfunction as a response to endotoxin challenge in this model is associated with prominent tissue microthombus formation that characterizes DIC and multiple organ failure in humans (6). The authors had previously shown that at 6 hours after endotoxin challenge, there was clear evidence of microthrombi in the liver and kidneys in the same model (2). They now show that a systemic inflammatory response occurs in this interval, with systemic increases in TNF- and IL-6 expression. Yet, there was no actual evidence of a systemic coagulopathy during this interval, and plasma D-dimers and thombin–antithrombin complexes remained undetectable then and after administration of the priming dose of endotoxin. The literature is unclear about the progression of coagulopathy in the generalized Schwartzman reaction, but the present findings are curious. Assuming that technical factors were not responsible, a definitive analysis of the time course for the appearance of such biomarkers would allow us to better understand the temporal contribution of the coagulation and fibrinolytic systems to tissue damage in this model. The authors' speculation that earlier changes underlie tissue thrombus formation may well prove to be correct, but should be tested.

This study offers a new approach to the identification of genes that go awry in disease. Gene expression profiling represents a potentially powerful addition to the investigative armamentarium. Broader application of the technique could accelerate better understanding of the key players in a broad range of diseases, including those of the lung.

FOOTNOTES

Conflict of Interest Statement: The author has no financial relationship with a commercial entity that has an interest in the subject of this manuscript.

REFERENCES

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Gene Expression Profiling Identifies C/EBP as a Candidate Regulator of Endotoxin-induced Disseminated Intravascular Coagulation

Sjoukje H. Slofstra, Angelique P. Groot, Maartje H. P. Obdeijn, Pieter H. Reitsma, Hugo ten Cate, and C. Arnold Spek
AJRCCM 2007 176: 602-609. [Abstract] [Full Text]  

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