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Angiopoietin-2 (Ang-2) is a potent regulator of vascular permeability and inflammation in acute lung injury and acute respiratory distress syndrome (ARDS). Genetic variants in the Ang-2 gene may lead to altered activities of Ang-2 (or ANGPT2) gene. The aim of this study was to assess if genetic variants of Ang-2 are associated with the risk of ARDS.
Unmatched, case-control study nested within a prospectively enrolled cohort.
Intensive care units (ICU) of an academic medical center.
1529 critically ill patients with risk factors for ARDS consecutively admitted to the ICUs from 1999 to 2006. Cases were 449 patients who developed ARDS and controls were 1080 subjects who did not developed ARDS.
Nine tagging SNPs (tSNPs) spanning the entire Ang-2 gene were genotyped in all patients. The results were analyzed using logistic regression models, adjusting for covariates. The variant T allele of one tSNP (rs2515475) was significantly associated with increased risk of ARDS (ORadjusted = 1.28; p = 0.042). This association was stronger in subjects with extrapulmonary injuries (ORadjusted = 1.79; p = 0.004). Haplotype TT containing the T allele of the rs2515475 was also significantly associated with higher risk of ARDS (ORadjusted = 1.42; p = 0.009), particularly in subjects with extrapulmonary injuries (ORadjusted = 1.90; p = 0.004).
Common genetic variation in the Ang-2 gene may be associated with increased risk of ARDS, especially among patients with extrapulmonary injuries.
Acute respiratory distress syndrome (ARDS) is characterized by non-cardiogenic pulmonary edema and acute respiratory failure in seriously ill patients . The pathological alterations of ARDS include diffuse endothelial and epithelial damage, with neutrophils, macrophages, erythrocytes, hyaline membranes, and protein rich edema fluid in the alveolar spaces, as well as capillary injury, and disruption of the alveolar epithelium [2, 3]. Endothelial activation and dysfunction has been shown to play a major role in the development of organ injuries thereby representing an independent parameter for worse clinical outcome in critically ill patients [4, 5]
Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are vascular growth factors that bind with similar affinity to the endothelial cell-specific receptor, Tie-2 [6, 7]. Ang-1 activates the endothelial Tie-2 receptor thereby maintaining a quiescent resting state of the endothelium. Ang-1 may have an anti-inflammatory action by signaling the down-regulation of surface-adhesion molecules such as VCAM-1 and E-selectin . Ang-1 can also protect the vasculature against plasma leakage . Conversely, Ang-2 binds to the same site of Tie-2 receptor but functions as an antagonist ligand of the Tie-2 . Therefore, Ang-2 is able to destabilize blood vessels, to enhance vascular leakage, to induce vascular regression, and to prime the endothelium to respond to VEGF and others angiogenetic and inflammatory cytokines [11, 12]. Ang-2 is stored in endothelial cell-specific storage granules (Weibel-Palade bodies) and is rapidly released upon stimulation of the endothelium, serving as a potential marker of endothelial activation and dysfunction . In hyperoxic acute lung injury, Ang-2 gene is overexpressed in lung epithelial cells, where it has critical roles in oxidative injury, epithelial cell death, and inflammation . Increased circulating Ang-2 levels have been observed in patients with acute lung injury  and sepsis [15, 16]. Circulating Ang-2 levels were correlated with mortality of ARDS and acute lung injury.
Human Ang-2 (ANGPT2) gene is located on chromosome 8 and has been found to be highly polymorphic [18, 19]. Genetic variants in the Ang-2 gene may affect Ang-2 gene expression or vascular angiogenesis [20, 21]. Studies of a single nucleotide polymorphism (SNP) in angiogenesis-associated diseases suggest that the effects of individual SNPs of Ang-2 on disease susceptibility are limited [22, 23]. However, no studies have been addressed the impact of multiple genetic variants of Ang-2 on disease risk.
In this study, we evaluated common genetic variations across the entire Ang-2 gene using a haplotype tagging SNP approach to test the hypothesis that variations in Ang-2 gene are associated with ARDS risk in patients with clinical risk factors for ARDS. Since pulmonary and extrapulmonary acute lung injury are known to express different morphological and inflammatory patterns [26, 27], and Ang-2 is selectively expressed by endothelial cells at sites of pathologic angiogenesis [24, 25], we further hypothesized that the associations between Ang-2 polymorphisms and ARDS risk are related to the sites of lung injuries-pulmonary or extrapulmonary.
This study is part of an ongoing molecular epidemiology project investigating the influences of genetic factors on the development and outcomes of ARDS. Details of the study have been described previously . Briefly, study subjects in the present study were selected from patients admitted to the intensive care units (ICU) at Massachusetts General Hospital (MGH, Boston, MA) from September 1999 to November 2006. Patients with clinical risk factors for ARDS such as sepsis, septic shock, trauma, pneumonia, aspiration, or multiple transfusions were eligible for inclusion (Supplemental Table 1). Exclusion criteria included age <18, diffuse alveolar hemorrhage, chronic lung diseases other than COPD or asthma, directive to withhold intubation, immunosuppression not secondary to corticosteroid, and treatment with granulocyte colony-stimulating factor. Baseline characteristics and Acute Physiology and Chronic Health Evaluation (APACHE) III scores were recorded on ICU admission. Baseline clinical and laboratory information were collected in the first 24 hrs of ICU admission. Patients who fulfilled the American-European Consensus Committee (AECC) criteria for ARDS  upon ICU admission or during the daily follow-up were considered as ARDS cases, whereas at-risk patients who did not meet the criteria for ARDS were considered as controls. The MGH Human Subjects Committee approved the study and informed written consent was obtained from all subjects or surrogates.
Tagging SNPs were selected based on HapMap phase II release . A genomic region of 66.6 kbp on chromosome 8p23.1 containing the entire Ang-2 gene plus about 3 kbp each upstream and downstream was selected. Nine haplotype tagging SNPs were identified using the following tagging criteria: pairwise tagging of the HapMap CEU Population with r2 ≥ 0.8 and a minor allele frequency (MAF) ≥ 10% (supplement Table 2). Pairwise linkage disequilibrium (LD) between the 9 tagging SNPs in Ang-2 was measured by D′ and r2, and was calculated based on the genotypes of 1529 study subjects using the Haploview software. Genomic DNA was extracted from whole blood using Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN) and/or the AutoPure LS® Workstation (Qiagen, Valencia, California)
Genotyping was performed using TaqMan® SNP Genotyping Assay and ABI Prism® 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Laboratory personnel were blinded to case-control status and 10% of randomly selected samples were interspersed in the plates for quality controls. All genotyping results were reviewed by two investigators independently.
We compared baseline variables using χ2 test, Fisher’s exact test, Student’s t-test, or Wilcoxon test, as appropriate. The Hardy-Weinberg equilibrium was evaluated using χ2 test. The LD between SNPs, haplotypes and their frequencies were estimated using the expectation maximization algorithm . Haplotypes were coded as an additive fashion. Logistic regression model was used to assess the associations of Ang-2 polymorphisms with ARDS in both dominant and additive models, adjusting for potential confounding factors including age, gender, APACHE III score, diabetes, history of steroid use.
To adjust for multiple comparisons in SNP association analyses, the false discovery rate (FDR) was assessed using SAS procedure PROC MULTTEST . To account for multiple comparison in estimating haplotype association, we conducted global tests of association by simultaneously including all of the haplotypes in the logistic regression model and then comparing it with a null model without any of the SNPs or haplotypes . This multiloci global test automatically adjusts for multiple testing based on the df of the corresponding χ2 test.
All statistical analyses were performed using the SAS statistical software package (version 9.1, SAS, Cary, NC). A p value <0.05 was considered to be statistically significant.
Although individuals of all races were screened for this study, we restricted our analysis to Caucasians since 92% of ICU admissions at MGH were Caucasians during the study period. A total of 1529 consecutive ICU admissions meeting the study criteria and with no exclusion criteria were screened. Among them 449 patients were diagnosed as ARDS cases and 1080 subjects were classified as non-ARDS controls. The baseline characteristics of the study population on admission to the ICU are shown in table 1. Compared with controls, patients who developed ARDS had higher APACHE III scores, higher serum bilirubin levels, and lower platelets counts. To further investigate if genetic associations are related to the sites of injuries, subjects were divided into two subgroups based on the sites of clinical injuries: pulmonary injury (n = 856; including subjects with pneumonia, aspiration or pulmonary contusions) and extrapulmonary injury (n = 673, consisting of patients with sepsis from extrapulmonary sources, trauma without pulmonary contusion and multiple transfusion). Patients with both pulmonary and extrapulmonary injuries were classified into the subgroup of pulmonary injury. Extrapulmonary injury and diabetes were less frequent in patients with ARDS.
The genotyping success rates ranged from 98% to 99% and did not diverse significantly from Hardy-Weinberg equilibrium. Genotype frequencies did not differ between cases and controls for all tagging SNPs. When subjects were stratified by pulmonary and extrapulmonary injury, genotype frequencies of one tagging SNP rs2515475 were significantly different between cases and controls among subjects with extrapulmonary injury (p = 0.017). In overall analyses, variant genotypes of rs2515475 (p = 0.042) and rs2959811 (p = 0.028) in LD block 2 showed borderline significant associations with increased risk of ARDS development. In stratified analysis, these associations were stronger in patients with extrapulmonary injury (OR = 1.79; 95% CI, 1.21–2.65; p = 0.004 for rs2515475; OR = 1.36; 95% CI, 1.02–1.81; p = 0.040 for rs2959811). The association of rs2515475 remained significant even after adjusting for multiple comparisons (FDR p = 0.004). But the associations between rs2959811 and ARDS risk became insignificant after adjusting for multiple comparison (FDR p = 0.180). No significant associations were observed for the other 7 tagging SNPs.
We divided the genotyped tSNPs into two haplotype blocks using Haploview, a LD-based partitioning algorithm (Fig. 1). Block 1 contains 5 tSNPs (rs2916702, rs2442468, rs2442635, rs2515435, and rs2515466) spanning 25 kb on the upstream region. Block 2 includes 2 tSNPs (rs2515475 and rs2959811) spanning 3 kb on the down stream region. We further conducted haplotype analyses based on LD blocks to determine whether regions of gene and/or combinations of SNPs were associated with ARDS risk. Reconstruction of haplotypes in LD block 1 generated 5 common haplotypes (≥5%) in our study population. In global test, haplotypes in LD block 1 were not significantly associated with ARDS risk (p = 0.115), although the individual haplotype CCTGG was significantly associated with increased risk of ARDS (OR = 1.69, 95% CI, 1.15–2.48; p = 0.008). In LD block 2, haplotypes were globally associated with ARDS development (p = 0.04). Specifically, haplotype TT was significantly associated with ARDS risk in overall analysis (OR = 1.42; 95% CI, 1.09–1.85; p = 0.009). Similar with the results from individual SNP analysis, the trend of haplotype association was stronger in patients with extrapulmonary injury (OR = 1.90; 95% CI, 1.23–2.92; p = 0.004) (Table 3).
In this study, we investigated the associations between genotypes and haplotypes of 9 tagging SNPs in the Ang-2 gene and the risk of ARDS. The variant T allele of one tSNP (rs2515475) was identified to be significantly associated with increased risk of ARDS, particularly in patients with extrapulmonary injury. These associations remained significant after adjusting for variables that were associated with ARDS development. Consistent with findings in genotype analyses, a haplotype harboring the T allele of rs2515475 was also associated with higher risk of ARDS, specifically among subjects with extrapulmonary injury.
The association of common genetic variation in Ang-2 with ARDS risk is biologically plausible for several reasons: (1) Ang-2 has been identified as a critical factor in pulmonary vascular leak required for the development of ARDS ; (2) Circulating Ang-2 and alveolar edema fluid are increased in patients with acute lung injury and pulmonary edema [14, 33, 34]; (3) Serum Ang-2 levels are associated with disease severity and inflammatory mediators in sepsis [11, 16]; (4) In vitro study has suggested that variations in the Ang-2 gene alter gene expression . It has been shown that genetic variations in the Ang-2 gene affected vascular development and angiogenesis .
Although only one tagging SNP (rs2515475) was associated with increased risk of ARDS, it is worth noting that the associations of rs2515475 with ARDS development are more likely to depend on linkage disequilibrium between rs2515475 and rs2959811 than on associations of individual SNPs. In support of this, haplotype TT that contains the variant allele of rs2515475 and rs2959811 is significantly associated with increased risk of ARDS in the same direction as that of rs2515475 or rs2959811 variants. Consistent with the effect of rs2515475, the association between haplotype TT and ARDS was also stronger in patients with indirect lung injury, suggesting that the effects of rs2515475 and TT haplotype were similarly. Moreover, these two tagging SNPs are probably representative genetic markers rather than causative genetic polymorphisms in ARDS development. Both the rs2515475 and rs2959811 are located in LD block 2 that capture other 15 tagging SNPs across a genomic region of about 2 kbp (Fig. 1 and Supplemental Table 2). Since these 15 tSNPs are in highly LD with each other, the associations of rs2515475 and rs2959811 with ARDS development may reflect the combined contributions of genetic variants in the LD block 2 to ARDS development.
The associations between the Ang-2 polymorphisms and ARDS susceptibility are more evident in subjects with extrapulmonary injury. Such effect modification by the type of lung injury may suggest a possible gene-environment interaction in ARDS. Similar heterogeneous effects on ARDS risk have been seen with candidate genes in inflammatory pathways, such as Inhibitor kappa B-alpha (IκB) gene and tumor necrosis factor (TNF) gene [28, 35]. The mechanisms underlining the different response to genetic associations are not clear. However, it has been observed that pulmonary insult primarily affects the alveolar epithelium with a local alveolar inflammatory response while the extrapulmonary insult affects the vascular endothelium by inflammatory mediators through blood stream . The inflammatory response, histological and mechanical properties of the lung differ depending on whether the etiology of acute lung injury or ARDS is pulmonary or extrapulmonary [26, 27, 37, 38]. Since extrapulmonary injury is characterized with systematic vascular endothelium damage , and Ang-2 is mainly stored in endothelial cell-specific storage granules  and contributes to pulmonary endothelial barrier disruption , it is possible that the Ang-2 variants affects ARDS development by regulating endothelial cell Ang-2 expression and vascular stability .
The strengths of our study include a comprehensive approach toward characterizing the Ang-2 gene by leveraging the linkage disequilibrium that exists at this locus, carefully adjusting for potential confounders, as well as stringent laboratory quality-control procedures. Despite these strengths, we acknowledge some limitations of our study. First, although our results have been adjusted for multiple comparisons, we underscore the need for replication of our findings given the large number of false positive generated in genetic association studies . Second, we did not re-sequence the gene and instead used publicly available SNP databases. Thus, some variation could have been missed due to incompleteness of these databases. Third, we did not examine the functions of Ang-2 genotypes and haplotypes and Ang-2 levels, thus the functional significance of Ang-2 variations in ARDS risk remains to be defined. Fourth, due to the study design, the results may not be generalized to the community setting, to patients with different risk factors for ARDS. In addition, the analyses were restricted to Caucasians, which reduced the possibility of confounding from different genetic make-up, but the extrapolation of the results to other ethnic groups might not be applicable.
In summary, we observed significant associations of genetic variants in Ang-2 with increased risk of ARDS. We also found that the contributions of Ang-2 polymorphisms to ARDS risk were associated with the etiology of lung injury. However, as this was the first study to comprehensively analyze the genetic variation in Ang-2 and ARDS risk, further studies are needed to replicate these interesting findings.
The authors would like to thank Weiling Zhang, Kelly McCoy, Thomas McCabe, Julia Shin, and Hanae Fujii-Rios for patient recruitment; Andrea Shafer and Starr Sumpter for research support; Maureen Convery for laboratory expertise; Janna Frelich for data management; and the patients and staff of ICUs at Massachusetts General Hospital.
Supported by grants from National Institute of Health (HL60710, ES00002) and Flight Attendant Medical Research Institute (062459-YCSA).