To determine the association between the clinical scores and the most significant upregulated and downregulated genes over time, we used proportional Venn diagrams as shown in Figure 2. A total of 88 genes were identified that were significantly increased over control expression levels within any time interval, with a criterion value of P < 0.001; this domain of 88 genes is depicted by the large circle in Figure 2, A to C. Figure 2A is a Venn diagram that illustrates the overlap between this domain of 88 genes with genes that were significantly correlated with the Acute Physiology and Chronic Health Evaluation II (APACHE II) scores. The APACHE II score is a clinical intensive care unit (ICU) score that is applied within 24 h of patient admission to an ICU (24, 25). Two genes, CYP51A1 and CYP4F3, were significantly upregulated and correlated with APACHE II scores. A pathway analysis of these genes by www.GeneMania.org indicates that these two genes are highly coexpressed with genes that support sterol biosynthesis (false discovery rate [FDR], 5.05e−28). Figure 2B compares the domain of 88 significantly changed genes with genes that correlated with the Max Denver 2 score over time. The Denver 2 is a clinical multiorgan failure scoring system that is computed based on the pulmonary, renal, hepatic, and cardiac systems (26). There was a common overlap of five genes that were significantly changed from control and that correlated with Max Denver scores greater than 2. These five genes were CYP19A1, HMOX2, NCF1, NCF2, and PTGER2. The pathway analysis of these genes (www.GeneMania.org) indicates that they are highly coexpressed with genes that provide an oxidative respiratory burst (FDR, 2.49e−9) and superoxide anion generation (FDR, 6.71e−8). Figure 2C compares the overlap between the 88 significantly changed genes and genes that correlated with Injury Severity Score (27) over time with a value of P < 0.001, as demonstrated by the blue circle. There was a common overlap of 10 genes that were significantly changed from control that also correlated with the Injury Severity Score. These 10 genes are CYP19A1, CYP20A1, CYP2A7, CYP2B7P1, CYP2C19, CYP2E1, CYP4A11, CYP8B1, ESR2, and NOX1. The pathway analysis of these genes indicates that they are highly coexpressed with genes that provide a cellular response to xenobiotic stimulus (FDR, 1.16e−23) and monooxygenase activity (FDR, 2.91e−20).
Oxidized lipids synthesized from linoleic and arachidonic acid are known to be elevated after injury (1–3) or inflammation (11–14). Recent studies have demonstrated that the CYP, LOX, and COX enzymes likely mediate oxidation of PUFAs after tissue injury (9, 10, 28). Given the substantial effect of oxidized lipids on hemodynamic, pulmonary, immune, and neuronal systems (29–31), it is likely that these lipids play major roles in initiating and integrating pathophysiologic responses to many forms of injury. However, it is not known whether the enzymatic machinery capable of oxidizing these bioactive lipids is upregulated after major blunt trauma. Here, we present clinical data that this effect is substantial, selective, and persists long after the traumatic event. Interestingly, neither COX1 nor COX2 were upregulated after trauma, suggesting a more dominant role for either CYP or LOX isozymes under these conditions. It is important to note that gene expression does not necessarily correlate to protein or enzyme activation. This study is focused on changes in the expression of leukocyte oxidative genes after traumatic injury, and these results could be extended to future proteomic studies.
The existence of a selective alteration in expression of transcripts encoding oxidative enzymes reveals a complex molecular response to major blunt trauma in circulating leukocytes. Evidence for the selectivity of this pattern of genetic changes is based on the finding of highly significant increased, as well as decreased, changes in gene expression. Nonetheless, the mechanisms mediating this response are unclear. It can be speculated that endocrine responses to stress and trauma might alter oxidative enzyme expression, although no previous published studies have reported this pattern of transcriptional events. Alternatively, it is possible that trauma itself, via introduction of foreign antigens or altered interactions with damaged cells or extracellular matrices, might lead to the observed changes. These alternatives should be evaluated in subsequent studies. Because the clinical samples consisted of circulating leukocytes, these oxidative enzymes are capable of generating biologically active substances at a systemic level, including both vascular and interstitial compartments. The physiologic significance of this finding is supported by the observed association of several of these transcripts with clinical trauma indices. This association provides a novel means for integrating pathophysiologic responses to trauma that is distinct from classical endocrine, paracrine, or neuronal forms of signaling.
Evaluation of the time course reveals that many of the sampled transcripts display persistent changes in expression out to the last time point measured, at 28 days. The full time-response curve is undefined but clearly extends to at least a month after trauma. Persistent changes in gene expression may signify epigenetic mechanisms regulating (32) the expression of these transcripts. If epigenetic mechanisms are involved, it is possible that patients with a history of major trauma would be predisposed to an altered response to any subsequent traumatic event. These responses could be enhanced or suppressed, depending on changes in gene expression and the function of the translated proteins. Nonetheless, such epigenetic changes may play an important role in patients with a history of polytrauma-related events.
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