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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: J Vasc Surg. 2010 Aug 3;52(4):1003–1014. doi: 10.1016/j.jvs.2010.05.088

THERAPEUTIC DISTANT ORGAN EFFECTS OF REGIONAL HYPOTHERMIA DURING MESENTERIC ISCHEMIA-REPERFUSION INJURY

Rachel J Santora 1, Mihaela L Lie 2, Dmitry N Grigoryev 3, Omer Nasir 2, Frederick A Moore 1, Heitham T Hassoun 1,4
PMCID: PMC2949511  NIHMSID: NIHMS210954  PMID: 20678877

Abstract

Introduction

Mesenteric ischemia-reperfusion injury (IRI) leads to systemic inflammation and multiple organ failure in both clinical and laboratory settings. We investigated the lung structural, functional, and genomic response to mesenteric IRI with and without regional intraischemic hypothermia (RIH) in rodents, and hypothesized that RIH would protect the lung and preferentially modulate the distant organ transcriptome under these conditions.

Methods

Sprague-Dawley rats underwent sham laparotomy or superior mesenteric artery occlusion (SMAO) for 60 minutes with or without RIH. Gut temperature was maintained at 15–20°C during SMAO, and systemic normothermia (37°C) was maintained throughout the study period. At 6 or 24 hours, lung tissue was collected for 1) histology, 2) MPO activity 3) bronchoalveolar lavage (BAL) fluid protein concentrations, 4) lung wet-dry ratios, and 5) total RNA isolation and hybridization to Illumina's Sentrix BeadChips (>22,000 probes) for gene expression profiling. Significantly affected genes (false discovery rate <5% and fold change ≤1.5%) were linked to gene ontology (GO) terms using MAPPFinder and hypothermia suppressed genes were further analyzed with Pubmatrix.

Results

Mesenteric IRI induced lung injury as evidenced by leukocyte trafficking, alveolar hemorrhage, increased BAL protein and wet-dry ratios, and activated a pro-inflammatory lung transcriptome when compared to sham. In contrast, rats treated with RIH exhibited lung histology, BAL protein, and wet-dry ratios similar to sham. At six hours, GO analysis indentified 232 hypothermia-suppressed genes related to inflammation, innate immune response and cell adhesion, and 33 hypothermia-activated genes related to lipid and amine metabolism and defense response. Quantitative RT-PCR validated select array changes in top hypothermia-suppressed genes lipocalin-2 (lcn-2) and chemokine ligand 1 (CXCL-1); prominent genes associated with neutrophil activation and trafficking.

Conclusions

Therapeutic hypothermia during SMAO provides distant organ protection and preferentially modulates the IRI-activated transcriptome in the rat lung. This study identifies potential novel diagnostic and therapeutic targets of mesenteric IRI, and provides a platform for further mechanistic study of hypothermic protection at the cellular and sub-cellular level.

INTRODUCTION

Mesenteric ischemia/reperfusion injury (IRI) occurs in various clinical settings including shock, sepsis, and complex aortic surgery. It incites an inflammatory response associated with local gut dysfunction as well as neutrophil (PMN) activation and remote organ injury [1–5]. Numerous effectors have been implicated in this injury cascade including cytokines, lipid mediators, nitric oxide, and cell adhesion molecules [6–10]. Unfortunately, clinical trials investigating the efficacy of pharmacological blockade of these various downstream inflammatory mediators in critically ill patients have been largely unsuccessful [11,12]. It has become clear that a more thorough top-down approach to biological discovery, as well as implementation of interventions with broader therapeutic application are needed in order to improve outcomes in these clinical settings.

Therapeutic hypothermia is cytoprotective in various IRI models, and its protective mechanisms seem to go beyond simply decreasing local metabolic demands during ischemia. Our prior work in rats demonstrated that regional intraischemic hypothermia (RIH) during superior mesenteric artery occlusion (SMAO) prevented reperfusion-induced intestinal mucosal injury and gut dysfunction, and RIH preferentially modulated the local oxidative stress transcriptional response during IRI [13]. A subsequent clinical translational study demonstrated that cold visceral perfusion during thoracoabdominal aortic aneurysm (TAAA) repair improved survival in a subset of critically ill patients (i.e. those who developed post-operative acute kidney injury) despite limited effects on the target organ [14]. These findings suggest potential systemic or distant organ therapeutic effects of visceral cooling during TAAA repair. The aim of this laboratory investigation is to gain a better understanding regarding the effects of RIH on distant organ dysfunction during mesenteric IRI.

We investigated structural, functional, and transcriptional changes in the lung during mesenteric IRI in rats, and hypothesized that regional gut hypothermia during SMAO protects the lung and preferentially modulates the IRI-activated transcriptome in the rat lung. To address this hypothesis, we utilized an established rodent model of mesenteric IRI that is known to cause both local and distant organ injury, and discovered lung microvascular injury and inflammation during mesenteric IRI that was mitigated by RIH. We then conducted global gene expression profiling of lung tissues during mesenteric IRI with and without RIH. Utilizing a candidate gene approach, we identified ischemia-specific changes in the lung transcriptome that are suppressed by hypothermia, and which include prominent genes involved in inflammation and leukocyte chemotaxis. Select genes associated with neutrophil activation and that demonstrated major changes in expression (ie. CXCL-1 and Lipocalin-2) were validated by qualitative real-time PCR. To further identify key biological processes involved in the distant organ protective effects of RIH, gene ontology analysis demonstrated early suppression of genes related to inflammation, cell adhesion and the innate immune response, and activation of genes related to the defense response. This study identifies potential novel diagnostic and therapeutic targets and provides a platform for further study of the distant organ protective effects of regional hypothermia during mesenteric IRI.

MATERIALS AND METHODS

Animal care

All procedures were approved by the Johns Hopkins Animal Care and Use Committee and were consistent with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (~300gm) were obtained from Harlan Labs (Houston, TX) and were housed under pathogen-free conditions according to NIH guidelines at least 5 days before operative procedures. The rats were fasted 18 hours prior to surgery but given free access to water. All procedures were performed using strict sterile techniques under general anesthesia with inhaled isoflurane. Assessment of adequate anesthesia was obtained by paw and tail pinching.

Surgical procedures

Figure 1 depicts the study design and animal model implemented for these experiments. Each animal underwent a midline laparotomy with isolation of the superior mesenteric artery (SMA). For rats assigned to experimental ischemia/reperfusion injury, a non-traumatic vascular clamp was placed on the SMA for 60 minutes with (IRI-H) or without (IRI) regional intraischemic hypothermia. Our method for inducing regional hypothermia has been described in detail previously [13]. Briefly, RIH was induced by exteriorizing the small intestine and placing it in moistened gauze between two cold compresses. A temperature probe was placed between the loops of bowel to monitor regional temperature, which was maintained at 15–20°C during ischemia. At the end of the allotted ischemic time period, and just before clamp release, the cold compresses were removed and the intestine irrigated with warm normal saline. The laparotomy incisions were then closed in two layers and the rats recovered from anesthesia on warming blankets. Control animals underwent the identical procedure with (Sham-H) and without (Sham) regional hypothermia. The small intestine was exteriorized for 60 min and placed between moistened gauze without placement of the vascular clamp on the SMA as described above. Each animal was placed on a heating pad and core temperature maintained at 37°C during the entire procedure. At 6 or 24 hours, the animals were sacrificed and tissue collected for the following studies described below.

Figure 1. Experimental Model.

Figure 1

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(A) Sprague-Dawley rats underwent sham laparotomy or SMAO for 60 minutes with and without regional hypothermia. Following the allotted reperfusion time, 6 or 24 hours, the animals were sacrificed and whole lung tissue was collected. The collected tissue was analyzed for 1) evidence of lung injury and inflammation, and 2) total RNA was isolated from the lung for gene expression profiling. (B) Regional intraischemic hypothermia (RIH) is illustrated in the figure above. For animals selected to undergo RIH, the intestines were exteriorized and placed between two cold compresses. A temperature probe was used to monitor RIH and animals were placed on a heating pad to maintain systemic normothermia (37°C).

Lung histology

Lung histological injury was assessed by evaluation of architecture and evidence of neutrophil influx with hematoxylin and eosin (H&E) staining as previously described [15]. After completion of the treatment period, the right main stem bronchus was identified and cross-clamped. Subsequently, a tracheostomy was performed and 0.5% low melting agarose was instilled into the left lung at a constant pressure of 25 cm H20, allowing for expansion of the lung parenchyma. The inflated lungs were fixed in 10% formalin for 48 h and embedded in paraffin blocks. Paraffin-embedded sections (5μm) were obtained and stained with H&E (Fisher Scientific, Pittsburgh PA). Representative samples were analyzed with light microscopy (40×) for evidence of injury and neutrophil influx.

MPO Assay

Whole lung tissue was homogenized using cell lysis buffer (Cell Signaling). Fifty ul of each sample was added to a 96 well plate and incubated with 100-ul of SureBlue TMB 1-Component Microwell Peroxidase (KPL, Gaithersburg, MD) at room temperature for 20 minutes. The reaction was terminated using 100-ul of 0.18 M sulfuric acid. The optical density was at 450 nm using a Kinetic Microplate Reader (Molecular Devices Corporation, Sunnyvale CA) spectrophotometer. Results are expressed as ng/mg protein.

Lung permeability

Bronchoalveolar lavage (BAL) protein concentration was performed as a surrogate for lung microvascular permeability. BAL fluid collection was obtained by delivering 3 ml of warm PBS via tracheotomy and the recovered fluid was centrifuged at 1500 rpm at 4°C for 10 min. Protein concentration was measured using BCA total protein assay (Bio-Rad, Hercules, CA) according to the manufacturer's protocol, and results are expressed as μg/ml.

Lung wet:dry ratio

As a surrogate for lung edema, lung tissues were obtained and wet weights determined before placing tissues in an oven at 60°C. After 48 hours, dry weights were measured and used to determine lung edema [(wet weight) − (dry weight)/dry weight].

Statistical Analysis

For measurements of BAL protein and lung wet-dry ratios, data are expressed as means ± SEM and were analyzed with one-way ANOVA. Individual group means were compared with a Tukey multiple comparison test and p values <0.05 were considered significant.

Microarray Methods

Transcript profiling with Illumina oligonucleotide array

To identify potential IRI-specific distant organ transcriptional changes, total RNA from lung (n=3/group) was isolated 6 or 24 hours after sham or SMAO with and without regional hypothermia. Total RNA was extracted using the Trizol Reagent method (Invitrogen, Carlsbad, CA) and additional purification was performed on RNAeasy columns (Qiagen, Valencia, CA 913555, cat. no. 74104). The quality of total RNA samples was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA samples were labeled according to the chip manufacturers recommended protocols. In brief, for Illumina, 0.5 μg of total RNA from each sample was labeled by using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin) in a two step process of cDNA synthesis followed by in vitro RNA transcription. Single stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP (Roche Diagnostics GmbH, Mannheim, Germany). 0.75 μg of biotin-labeled cRNA was hybridized (16 hours) to Illumina's Sentrix RatRef-12 v1 Expression BeadChip (>22,000 probes).

Sample quality control

The stringent quality control of the purity and integrity of the RNA was assessed using standard criteria for RNA quality. The starting material was analyzed with the Agilent 2100 Bioanalyzer (Agilent Technology) that requires nanogram quantities of RNA. All RNA samples that were used for hybridization exhibited intact 28S and 18S ribosomal RNA on denaturing agarose gel electrophoresis. 260/280nm absorbance readings for both total RNAs and biotin-cRNAs fall in the range of 1.8 to 2.1. Only samples with yields of in vitro transcribed RNA above 750 ng were hybridized to the chips. After the chips were scanned, they were inspected for possible image artifacts.

Identification of significant transcriptional changes

The hybridization signals were analyzed using BeadStudio version 1.5.0.34 software (Illumina Inc.). The resulting digitized matrix was processed by modified for Illumina platform approach described previously [16]. Briefly, the significance (BeadStudio detection>0.95) of hybridization signals was tested and “Present” and “Absent” transcripts identified. The chip background and brightness was computed using high quartile and whole set of “Absent” hybridization signals, respectively. The expression data was stratified by experimental conditions and hybridization of each transcript was evaluated for each cluster. The transcripts determined to be “Present”, produced a signal at least twice as high as that of background in at least 2 of 3 hybridizations in any given group of rat were considered expressed. The signal intensity values of these transcripts from each chip were increased by corresponding to a given chip background value (background adjustment) and divided by a chip brightness coefficient (normalization). The normalized data then were processed by Significance Analysis of Microarrays (SAM 2.20) using full permutation of 3 control and 3 IRI or IRI-H samples (720 permutations) without application of arbitrary restrictions [17, 18]. Genes with 1.5 fold change and false discovery rate (q value) less than 5% were considered significantly associated with IRI.

Hierarchical clustering

Fold change values for individual IRI and/or IRI-H samples were derived by subtraction of average control expression (log2 format) from corresponding experimental expressions (log2 format). The 300 most variable significant genes were clustered based on a gene expression pattern similarity (Pearson correlation) using MeV software.

Validation of gene expression data

Quantitative real time RT-PCR validation of selected candidate genes was conducted as previously described [19, 20]. Briefly, transcript levels of selected candidate genes in control and IRI-affected lung tissues were measured (n=3 per condition) in 96-well microtiter plates with an ABI Prism 7700 Sequence Detector Systems (Perkin-Elmer/Applied Biosystems). Three TaqMan® endogenous control genes (Gapdh, Actb, and Pgk1) were used as internal controls for normalization. Primers and probes were purchased from Applied Biosystems Inc. in a 20× mixture. All experimental protocols were based on manufacture's recommendation using the TaqMan Gold RT-PCR Core Reagents Kit (Perkin-Elmer/Applied Biosystems, P/N 402876). Experimental parameters were 48°C for 30 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Relative gene expression was calculated using the 2−ΔΔCt method, which generates fold change values for mRNA transcript levels expressed in IRI-treated lung samples relative to sham operated samples as described previously [20].

Gene ontology and PubMatrix analysis

MAPPFinder, a tool that integrates gene expression data with gene ontology data, was used to identify the significant biological trends among the array data collected from each experimental group. Compatible files were prepared using GenMAPP converting tool as we described previously [21]. Significant bioprocesses were selected by choosing GO terms containing greater than 5% of the total number of genes linked by MAPPFinder. The Z-score>2 and first GO node>0 criteria were also applied as filtering conditions. The selected gene candidates were then searched against terms of interest related to acute lung injury including, “neutrophil”, “endothelium”, “epithelium”, and “pulmonary” terms in PubMed database using the PubMatrix automated literature search engine [22].

RESULTS

Lung morphology during mesenteric IRI

To assess potential structural changes in the lung following experimental IRI with and without hypothermia, H&E-stained sections of the lung were analyzed under light microscopy and representative sections are shown in Figure 2. IRI-treated rats demonstrated persistent neutrophil infiltration and focal alveolar hemorrhage; findings that were not present following Sham, Sham-H, or IRI-H. To quantify neutrophil influx, we measured myeloperoxidase (MPO) activity in all experimental groups. At 24 hours, there was a significant increase in MPO activity in IRI compared to Sham controls (7.1±.4 vs. 5.9±.3, p<.05) during normothermia, but no difference between IRI-H and Sham-H (6.5±.4 vs. 6.3±.4,p>.05).

Figure 2. Effect of regional hypothermia on lung injury during mesenteric IRI.

Figure 2

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Representative hematoxylin and eosin (H&E)-stained sections (40 × magnification) of lung tissue at 24h after SMAO with and without hypothermia (H). IRI-treated rats demonstrate increased neutrophil influx (arrowhead) and alveolar hemorrhage (arrow) not present in sham, sham-H or IRI-H.

BAL protein during mesenteric IRI

The effect of RIH on pulmonary function during mesenteric IRI was evaluated by measuring changes in BAL protein concentrations (μg/ml) as a surrogate for microvascular permeability (Figure 3). At 24 h, BAL protein concentration was increased in IRI-treated rats (665±29) compared to sham (145±24) and IRI-H (85±58).

Figure 3. Effect of regional hypothermia on lung BAL protein during mesenteric IRI.

Figure 3

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BAL protein concentration (mg/ml) at 24 hours was greater during IRI compared to sham and IRI-H. *P≤ .05, n≥5 rats/group.

Lung weight:dry ratios during mesenteric IRI

To further evaluate the effect of RIH on pulmonary microvascular permeability, we measured weight:dry ratios as an indicator of pulmonary edema (Figure 4). Wet:dry ratios were increased in IRI compared to Sham (1.17±.01 vs. 1.21±.01, P≤ .05) and IRI-H (1.21±.01 vs. 1.13±.001, P≤ .05).

Figure 4. Effect of regional hypothermia on lung edema during mesenteric IRI.

Figure 4

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Lung edema (wet:dry ratio) was increased in IRI compared to sham and IRI-H. *P≤ .05, n≥5 rats/group.

Gene expression changes in the lung during mesenteric IRI

Global gene expression profiling is a robust tool for identification of diagnostic and mechanism-related candidate genes. To evaluate the lung genomic response to mesenteric IRI with and without hypothermia, we conducted global gene expression profiling of whole lung tissue obtained at 6 or 24 hours after Sham, Sham-H, IRI, and IRI-H. Total RNA was isolated from rat lungs (n =3/group) and hybridized to Illumina's Sentrix Expression Bead chip (>22,000 probes). At 6 hours, SAM of expression profiles identified 437 lung genes with increased expression during IRI compared with Sham, of which, 205 genes were also activated in IRI-H treated groups. Therefore, of the IRI-induced genes, we identified 232 lung candidate genes that were suppressed by regional hypothermia. In addition, we identified activation of 33 IRI-H specific genes. At 24 hours, there were only eight IRI-activated genes compared to Sham, and there were no significant lung gene changes in the IRI-H treated group when compared to sham controls.

To investigate potential differences in the lung genomic response to IRI and IRI-H, hierarchical cluster analysis of the most significantly affected genes identified groups of genes with similar as well as discordant expression patterns between the two treatment groups (Figure 5). The detailed analysis of the most differentially expressed genes between IRI and IRI-H treated animals are presented in Table 1. Of note, several prominent genes related to leukocyte activation and trafficking, such as chemokine ligand 1 (CXCL-1) and lipocalin-2 (lcn-2), were suppressed by hypothermia.

Figure 5. Hierarchical cluster analysis of lung gene expression during mesenteric IRI at 6 and 24 hours.

Figure 5

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Each column represents an experimental condition of the corresponding lung sample. Hierarchical clustering using Pearson correlation determined 5 major clusters (blue triangles) of which 2 clusters demonstrated clear differences in gene expression between IRI and IRI-H at 6 hours. Genes from these clusters are highlighted with yellow rectangles and are listed on the right. Red indicates upregulation and green indicates downregulation of gene expression with respect to corresponding controls.

Table 1.

Top differentially expressed lung genes between IRI and IRI-H.

6 hours
 24 hours
Gene Title Symbol IRI IRI-H IRI-H/IRI Ratio IRI IRI-H IRI-H/IRI Ratio
Suppressed by Hypothermia
actin alpha 1 skeletal muscle Acta1 4.12 −1.44 0.17 1.11 1.61 1.45
lipocalin 2 Lcn2 6.19 1.76 0.28 6.99 6.42 0.92
chemokine (C-X-C motif) ligand 1 Cxcl1 7.45 2.26 0.30 1.65 4.54 2.76
peripherin 1 Prph1 2.30 −1.40 0.31 −1.63 −1.11 1.47
LOC500721 LOC500721 2.45 −1.26 0.32 −1.42 1.11 1.57
Metallothionein Mt1a 9.38 3.11 0.33 2.05 7.05 3.44
chemokine (C-X-C motif) ligand 2 Cxcl2 4.67 1.69 0.36 1.19 2.54 2.13
LOC500720 LOC500720 2.64 −1.05 0.36 −1.46 1.25 1.82
nitric oxide synthase 2 inducible Nos2 4.10 1.66 0.41 1.18 1.95 1.66
ring finger protein 30 Rnf30 3.05 1.27 0.42 1.00 3.36 3.37
growth arrest and DNA-damage-inducible 45 gamma Gadd45g 4.58 1.92 0.42 1.11 1.38 1.24
ferredoxin 1 Fdx1 2.60 1.09 0.42 1.54 2.65 1.72
hyaluronan synthase 1 Has1 2.42 1.11 0.46 1.33 1.35 1.01
laminin alpha 5 Lama5 2.06 1.02 0.49 −1.04 2.18 2.27
transglutaminase 1 Tgm1 3.45 1.72 0.50 1.29 1.71 1.32
interferon induced transmembrane protein 6 Ifitm6 3.86 2.68 0.69 10.06 4.94 0.49
Recovered by Hypothermia
similar to 40S ribosomal protein S9 LOC300278 −4.35 1.04 4.53 −1.43 −1.54 0.93
similar to 40S ribosomal protein S9 LOC367102 −2.62 1.20 3.15 −1.23 −1.40 0.88
ribosomal protein S9 Rps9 −2.48 1.08 2.67 −1.24 −1.42 0.87
cytochrome P450 family 2 subfamily e polypeptide 1 Cyp2e1 −3.14 −1.22 2.56 1.44 −1.74 0.40
guanylate cyclase 1 soluble beta 3 Gucy1b3 −2.49 −1.05 2.39 −1.02 −1.40 0.73
chemokine (C-C motif) ligand 5 Ccl5 −2.41 −1.19 2.02 1.00 −1.54 0.65
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Validation of gene expression profiling findings

To validate the results of our gene expression profiling studies, quantitative real-time-PCR measurements of selected genes was conducted using the TaqMan assay as described in the Methods section. The overall agreement between gene expression by microarray analysis and by the TaqMan assay was good, and the fold changes for the selected genes (Lipocalin-2 and CXCL1) are demonstrated in Figure 6. These genes were chosen for validation based on most potentially relevant and representative significantly increased gene expression changes following IRI.

Figure 6. Expression of lipocalin-2 and chemokine ligand 1 (CXCL-1) genes in the lung by Ilumina microarray and quantitative real-time PCR.

Figure 6

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The relative fold-change (at 6 hours) identified by microarray analysis (filled bars) and RT-PCR (open bars) was calculated for IRI and IRI-H samples with respect to corresponding shams and is represented as mean ± SEM. *Significant changes in gene expression between sham and treatment groups by either unpaired t-test (RT-PCR) or significance analysis of microarrays (SAM; microarray). P ≤ .05 or q<5%, respectively, n≥3 samples/group.

Identification of ischemia-specific biological processes attenuated by intraischemic hypothermia

Once significant differences in lung genomic responses between IRI and IRI-H were established, we focused on elucidation of potential mechanistic pathways unique to mesenteric IRI-induced lung structural and functional changes. Using MAPPFinder, we performed GO analysis of the 232 IRI-specific lung candidate genes that were suppressed by hypothermia and the 33 hypothermia-specific genes activated at 6 hours. GO analysis identified hypothermia-induced suppression of genes related to inflammation,cell adhesion, and innate immune response among others, and showed predominant activation of genes related to amine and lipid metabolism and defense response (Table 2).

Table 2.

Gene ontology analysis of top differentially expressed lung genes between IRI and IRI-H.

GOID GO Name Changed Genes Measured Genes Changed Genes, % Z Score
Suppressed by Hypothermia
6928 cell motility 11 88 12.5 4.352
6954 inflammatory response 8 80 10.0 2.959
42127 regulation of cell proliferation 9 108 8.33 2.528
7155 cell adhesion 14 169 8.28 3.154
30154 cell differentiation 10 130 7.69 2.391
6955 immune response 16 230 6.96 2.619
9607 response to biotic stimulus 17 269 6.32 2.279
Activated by Hypothermia
9308 amine metabolism 4 122 3.28 3.924
6629 lipid metabolism 4 182 2.20 2.886
6952 defense response 4 250 1.60 2.142
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To further validate the biological relevance of the lung candidate genes involved in these bioprocesses, a qualitative analysis of the IRI and hypothermia-specific transcripts was conducted using PubMatrix, an automated literature search engine (http://pubmatrix.grc.nia.nih.gov). The 232 hypothermia-suppressed genes from the bioprocesses identified in the 6 hour GO analysis were matched against the terms “neutrophil”, “endothelium”, “epithelium”, and “pulmonary” terms in the Pubmed database. The most commonly cited genes related to these terms are presented in Table 3, and are listed according to weighted distribution between PMN or Tissue based on the number of Pubmed matches.

Table 3.

Qualitative analysis of hypothermia-suppressed genes using PubMatrix.

Pubmed Search Terms
Gene Title Gene Symbol Neutrophil Endothelium Epithelium Pulmonary Ratio PMN/Tissue Distribution
Neutrophil cytosolic factor 1 Ncf1 44 0 0 2 44.00 PMN
Neutrophil cytosolic factor 2 Ncf2 17 0 0 3 17.00
Lipocalin-2 Lcn2 256 5 26 39 8.26
chemokine ligand 2 Cxcl2 664 44 88 463 5.03
chemokine ligand 1 Cxcl1 761 94 162 269 2.97
biregional cell adhesion molecule-related/down-regulated by oncogenes binding protein Boc 72 7 23 57 2.40
chemokine ligand 3 Ccl3 248 55 76 249 1.89
Lipopolysaccharide binding protein Lbp 95 28 51 96 1.20 Even
CD 14 Antigen Cd14 658 300 381 533 0.97
Tumor necrosis factor receptor superfamily 12 Tnfrsf1a 19 8 13 17 0.90
MAP kinase-acitvated protein kinase 2 Mapkapk2 25 23 27 20 0.50 Tissue
CSX-associated LIM Cal 33 18 51 168 0.48
signal transducer and activator of transcription 3 Stat3 158 111 253 342 0.43
Coagulation factor 3 F3 22 15 42 69 0.39
spermidine/spermine N1-acetyl transferase Sat 18 19 40 182 0.31
inhibitor of kappaB kinase beta Ikbkb 61 80 138 166 0.28
complement component 4 gene 2 C4-2 4 3 12 7 0.27
plasminogen activator tissue Plat 10 19 19 24 0.26
desmin Des 630 755 1663 3266 0.26
guanine deaminase Gda 8 9 22 95 0.26
prostaglandin-endoperoxide synthase 2 Ptgs2 397 605 1046 1139 0.24
period homolog 1 Per1 2 1 8 18 0.22
NAD(P)H dehydrogenase quinone 1 Nqo1 10 18 29 140 0.21
ectonucleotide pyrophosphatase/phosphodiesterase2 Enpp2 2 2 8 10 0.20
runt related transcription factor 1 Runx1 12 34 38 10 0.17
gap junction membrane channel protein alpha 1 Gja1 2 5 10 8 0.13
Kirsten rat sarcoma viral oncogene homologue 2 (active) Kras2 25 44 152 265 0.13
vasodilator-stimulated phosphoprotein Vasp 13 50 54 21 0.13
secreted phosphoprotein 1 Spp1 22 43 137 79 0.12
Tissue inhibitor of metalloproteinase 1 Timp1 5 13 28 35 0.12
matrix Gla protein Mgp 5 17 34 36 0.10
oxidized low density lipoprotein (lectin-like) receptor 1 Oldlr1 3 16 15 1 0.10
elongation factor RNA polymerase II Ell 2 3 20 74 0.09
neurturin Nrtn 1 1 12 1 0.08
P450 (cytochrome) oxidoreductase Por 5 18 49 124 0.07
glutathione peroxidase 2 Gpx2 1 0 14 9 0.07
activating transcription factor 4 Atf4 1 7 14 12 0.05
Kruppel-like factor 5 Klf5 1 7 20 4 0.04
insulin-like growth factor 1 receptor Igf1r 7 59 158 161 0.03
hyaluronan synthase 1 Has1 1 12 22 17 0.03
forkhead box A2 Foxa2 1 3 45 61 0.02
wingless-related MMTV integration site 4 Wnt4 0 1 26 5 0.04
solute carrier family 7 (cationic amino acid transporter y+ system) member 1 Slc7a1 0 12 11 5 0.04
tumor-associated protein 1 (Slc7a5) Slc7a5 0 6 13 13 0.05
procollagen type VII alpha 1 Col7a1 0 0 13 0 0.08
laminin alpha 5 Lama5 0 2 10 4 0.08
low density lipoprotein receptor-related protein 6 Lrp6 0 0 10 4 0.10
huntingtin-associated protein 1 (Hap1) transcript variant 2 Hap1 0 2 7 13 0.11
surfactant associated protein B Sftpb 0 0 7 48 0.14
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Genes that had at least 10 hits in one category were retained. For calculation of PMN/Tissue ratio zeroes were substituted with 1.

DISCUSSION

Mesenteric IRI is a common clinical condition in vascular surgical patients and it is associated with significant morbidity and mortality for which there are virtually no therapeutic options. It promotes local synthesis and release of downstream inflammatory mediators that exacerbate gut injury and “prime” circulating neutrophils for enhanced superoxide anion production and subsequent remote organ injury [23, 24]. Complex thoracoabdominal aortic aneurysm (TAAA) repair results in obligatory mesenteric ischemia during repair of the visceral segment, and visceral organ IRI has been associated with distant organ injury, subsequent multiple organ failure (MOF) and death in clinical studies [25, 26]. In a recent prospective study of ten patients undergoing TAAA repair, Feezor et al. identified adverse clinical outcome by blood leukocyte genomic and plasma proteomic responses following surgery [27]. Using gene expression profiling and multiplex proteomic analysis, the authors identified time-dependent changes in gene expression that predicted subsequent organ dysfunction. This study highlights the potential utility of microarray analysis in preoperative risk assessment and as a potential tool to determine diagnostic and therapeutic targets of visceral IRI-induced MOF.

Surgical adjuncts to reduce the postoperative sequelae of visceral IRI during TAAA repair have focused on maintaining distal aortic perfusion and selective visceral cooling during repair of the visceral segment. Randomized comparisons of cold vs. warm crystalloid and cold crystalloid vs. cold blood perfusion have demonstrated the therapeutic benefits of visceral cooling for organ protection during IRI [28, 29]. In another large prospective study of 359 patients who underwent cold or warm blood visceral perfusion during TAAA repair, Hassoun et al demonstrated that while visceral cooling did not reduce the overall incidence of postoperative kidney injury during TAAA repair, it did improve survival in patients who developed renal failure [14]. These findings suggest that regional hypothermia offers systemic protection during visceral IRI, though the exact mechanisms remain unclear.

In an effort to elucidate the therapeutic potential of regional hypothermia on distant organ inflammation and injury during mesenteric IRI, our lab has developed a rodent model of regional intraischemic hypothermia to study local and remote organ injury during SMAO. Early experimental studies utilizing this model have shown that hypothermia confers local gut structural and functional protection by preserving mucosal integrity and improving intestinal transit during mesenteric IRI, and that RIH preferentially activates heme oxygenase-1 (HO-1) in the gut, while decreasing the expression of nuclear factor kappa-B (NF-κB) and deleterious inducible nitric oxide synthase (iNOS) [13,30]. Based on these observations, our lab is now focused on the potential for therapeutic hypothermia to confer protection to distant organ sites during mesenteric IRI and to develop a better understanding of its modulating effects on the inflammatory response under these conditions. Using a top-down approach, we conducted global gene expression profiling of rat lung obtained at 6 and 24 hours following mesenteric ischemia with and without RIH. To correlate genomic changes with structural and functional derangements in the lung, we assessed time-dependent changes in BAL protein concentration, wet-dry ratios and lung histology in all experimental groups. Within 24 hours of mesenteric ischemia, there was a significant increase in BAL protein concentrations and wet-dry ratios indicating increased pulmonary microvascular permeability and lung edema in IRI-treated animals, as well as histological evidence of alveolar damage and neutrophil trafficking. The structural and functional changes observed at 24 hours are likely preceded by changes in the pro-inflammatory transcriptome that return to baseline within 24 hours. These observations suggest that IRI-induced transcriptional changes occur prior to obvious functional or structural changes. Furthermore, neither transcriptional nor structural changes were present in hypothermia-treated animals suggesting prominent distant organ protection under these conditions.

After determining the IRI-specific lung molecular signature, we were able to identify and characterize the hypothermia-suppressed lung transcriptome and validated these findings with real-time PCR. Using a bioinformatic-driven approach we determined the relevant biological processes associated with the alterations in the lung transcriptome during mesenteric IRI with or without RIH. GO analysis identified hypothermia-induced suppression of genes related to inflammation, cell adhesion, and the innate immune response, and showed activation of genes related to amine and lipid metabolism and the defense response. Interestingly, among the top hypothermia-suppressed genes are CXCL-1, CXCL-2 and lipocalin-2, which all code for proteins prominently associated with neutrophil trafficking and activation. The biological relevance of these top suppressed genes was validated by using an automated literature search engine, PubMatrix (http://pubmatrix.grc.nia.nih.gov). The weighted distribution of the most commonly cited genes were matched to the terms “neutrophil” and tissue-specific components such as “epithelium” or “endothelium”, thus allowing for future mechanism-based studies of the therapeutic potential of RIH focusing on its specific effects on leukocyte or tissue-specific receptors.

Indeed, activation and migration of circulating neutrophils are central events in the development of ALI [31, 32]. Chemokines are important mediators of neutrophil recruitment and activation in the lung in response to an inflammatory stimulus. CXCL-1 and CXCL-2 are glutamic acid-leucine-arginine (ELR+) chemokines that act through a shared G-protein coupled receptor, CXCR2. Studies have highlighted the importance of CXCR2-ligand mediated lung injury and have demonstrated that increased CXCL-1 and CXCL-2 expression parallels neutrophil sequestration and progressive lung injury [33, 34]. In our current study, we identified CXCL-1 and CXCL-2 among the top lung candidate genes suppressed by hypothermia, and based on these observations, suppression of CXCR-2 ligand mediated events may confer the protective effect of RIH on distant organ dysfunction.

We also identified and validated hypothermia-induced suppression of lipocalin-2, also known as lipocalin-associated MMP-9 or NGAL. Lipocalins are small secreted proteins that form complexes with other macromolecules including neutrophil gelatinase to form NGAL, which consists of a stable complex between lipocalin and MMP-9. Matrix metalloproteinases (MMPs) are preformed granules stored in mature neutrophils that are released in response to chemokine stimulation and play an important role in extracellular matrix degradation, tissue destruction and cellular migration [35–36]. Clinical studies have implicated MMPs, particularly MMP-8 and MMP-9, as mediators of pulmonary inflammation in acute and chronic pulmonary disease states [39]. In our study, we found early suppression of lung lipocalin-2 in RIH-treated animals, making it an attractive candidate for further investigation as a potential mediator of hypothermia-induced lung protection.

A potential limitation of our study is the absence of gene expression profiling of the postischemic intestine. Without this data, it is difficult to conclude if regional hypothermia modulates the lung transcriptome in a unique fashion or if the observed gene expression changes in the lung parallel those in the gut. We identified top hypothermia-suppressed lung genes as those related to neutrophil trafficking and activation, which may also be dampened in the gut as well. Furthermore, our study utilized profound regional hypothermia (15–20° C) to achieve distant organ protection, which may have deleterious tissue consequences at later time points. The effects of more moderate hypothermia on both local and distant organ dysfunction are largely unknown and need further investigation. Another potential confounding factor is the use of topical cooling to achieve regional hypothermia. Clinical studies examining the utility of regional hypothermia have achieved this by perfusing cold blood or saline through the organ of interest. In our model, only topical hypothermia was utilized and we therefore did not monitor the potential hemodynamic effects of using cold perfusate to achieve regional hypothermia. The potential hemodynamic consequences of using a cold perfusate would provide useful information for translation into a clinical model.

In summary, clinical and experimental studies have demonstrated the promise of hypothermia as a rescue strategy following planned and unplanned ischemic events. Therapeutic hypothermia is a well-accepted post-resuscitation strategy to reduce neurological injury, improve myocardial function, and improve overall survival in patients with return of spontaneous circulation after out-of hospital cardiac arrest and in an effort to optimize the therapeutic benefit of hypothermia, preliminary studies are establishing the optimal duration, target temperature, rate and means of cooling [38–40]. While a large body of evidence exists to support the beneficial effects of systemic hypothermia, studies regarding utilization of hypothermia as a focused strategy for in vivo organ protection have been limited. Using an established model of rodent SMAO, we have identified deleterious lung structural and functional changes during IRI that were mitigated by regional intraischemic hypothermia. Furthermore, RIH preferentially modulates the IRI-activated lung transcriptome by down-regulation of select pro-inflammatory genes and leukocyte chemoattractants. This study identifies potential novel diagnostic and therapeutic targets and provides a platform for further study of the distant organ protective effects of regional hypothermia during mesenteric IRI.

Acknowledgments

Supported by grants from the American Vascular Association/American College of Surgeons Lifeline Award and NIH grant K08HL089181

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

This paper was presented at the 34th annual meeting of the Southern Association for Vascular Surgery in Paradise Island, Bahamas on January 23, 2010.

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