Focusing on the Flood: Targeting Functional Polymorphisms in ALI Permeability Pathways

Despite considerable efforts, disappointingly few significant advances have been achieved in the management of patients with devastating acute lung injury (ALI) with the pace for novel ALI insights, biomarkers, and interventions excruciatingly slow. Over the past decade, only alterations in ventilator management have served as an impactful novel modality. In this light, the post-genome era held promise that application of high-throughput genomic and genetic technologies might accelerate ALI discovery and advantage the personalization of ALI medical care. As only a minority of patients exposed to ALI-predisposing insults manifest ALI, substantial support was generated for individual genetic variation as a key contributor to ALI susceptibility. However, in contrast to the approximately 200 complex disorders with published genome-wide association studies, large-scale genotyping has not yet been conducted in either ALI cohorts or in cohorts afflicted with the major ALI-inciting causes such as sepsis and trauma. Consequently, ALI genetic studies have employed a candidate gene approach, often leveraging genomic-intensive strategies, with case-control studies surprisingly effective in identifying single-nucleotide polymorphisms (SNPs), which confer differential risk of developing ALI or influence ALI mortality (1–3), several of which have been validated in replicate cohorts (4, 5). Unfortunately, many candidate gene studies have been difficult to replicate, either due to limited sample sizes, population stratification, variability of the control population, or heterogeneity of the ALI phenotype. Thus, while there are multiple lines of evidence supporting genetic susceptibility to ALI, many ALI candidate genes failed replication in other ALI cohorts. Furthermore, few genetic studies have been conducted in multiple ALI ethnicities, and large-scale ALI genotyping has not yet been reported. An recent alternate approach to identification of validated ALI candidate genes has focused on genes centrally involved in biological pathways relevant to the complex pathophysiology of ALI. This strategy has highlighted genes involved in epithelial and vascular barrier function, paralleling growing interest in vascular homeostatic gene products as novel ALI biomarkers. Potential ALI vascular biomarkers and targets for genetic variation include inflammatory factors (IL-1β, IL-6, IL-8, TNF-α), coagulation factors (protein C, thrombomodulin), and endothelial cell–derived gene products such as von Willebrand Factor, intercellular adhesion molecule-1, and E- or P-selectin. As marked disruption of vascular integrity, resulting in profound lung permeability and alveolar flooding, is a cardinal feature of the inflamed ALI lung, this focus would appear to be well justified, especially as ALI severity and outcome are dependent on the magnitude of alveolar epithelial and/or vascular injuries. The list of vascular-related ALI genes has grown to include angiogenic factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor, and sphingosine 1-phosphate, all agonists that influence vascular integrity (6–8), as well as cytoskeletal signaling effectors (2, 4). For example, through the potent angiogenic factor, VEGF, previously known as vascular permeability factor, lung vascular barrier function is compromised and is an important mechanism responsible for increased vascular permeability, pulmonary edema, and leukocyte migration into lung tissues resulting in persistent inflammation. In addition, angiogenic mediators such as VEGF, epidermal growth factor, and matrix metalloproteinases have been implicated in ALI pathogenesis and individually associated with ALI risk (9, 10). In this issue of the Journal, Meyer and coworkers (pp. 1344–1353) have addressed an important gap in ALI genetic studies by genotyping a cohort of critically ill trauma subjects of African and European descent utilizing a cardiopulmonary disease–centric 50K SNP array (11). These studies identified a region in the angiogenic factor gene, angiopoietin-2 (ANGPT2), to be association with increased risk for trauma-associated ALI, results validated in a multicenter European American trauma-associated ALI case-control population. While this report confirms prior report of ANGPT2 genetic variants associated with the development of ALI (12), the high merit of this strategy of SNP discovery in a cohort of African descent is supported by the rich genetic diversity and small block size; and, despite the unconventional approach of discovering in a small AA population and replicating in EA, it points to an association that is robust across ethnicities. While the African discovery cohort is relatively small (∼ 200 cases), this is comparable to the largest African descent cohorts in ALI (13), highlighting the need for accumulation of African descent–rich ALI cohorts. Angiopoietin-2 (ANG2), a naturally occurring antagonist for angiopoietin-1 (ANG1), an angiogenic factor essential for normal vascular development, is well recognized to impact lung permeability in experimental models of lung injury. ANG2 has been implicated in pulmonary vascular leak syndromes including ALI and sepsis in both animal and human studies, and elevated ANG2 levels are detected in the blood and bronchoalveolar lavage fluid of patients with ALI and conferred risk of ALI in trauma patients (14). Importantly, in the current study, Meyer and colleagues linked the ANGPT2 SNP with increased levels of a variant ANG2 isoform in plasma, suggesting that the risk polymorphism tags a splice site enhancer or novel splice site. Resequencing identified predicted novel splice sites in linkage disequilibrium with the ANGPT2 SNP and immunoblots showed higher proportion of variant ANG2 isoform associated with this SNP. While the observed ANG2 isoforms remain to be confirmed as ANG2 A and C, these results show the potential relevance of circulating ANG2 and may shift attention to the effect of variation in the coiled-coil region of ANG2 on vascular permeability regulation. In summary, the study of the genetic contributions to ALI pathogenesis, severity, and response to therapy remains a nascent, albeit exciting field that holds great promise, with defining functional relationships of genetic variants remaining a challenge. Complementing the utility of combining advanced bioinformatic techniques and multi-species gene expression profiling as a way to broaden the net for ALI-related genes, interrogation of biologically important pathways, such as permeability regulation, also serves to provide high-yield strategy for identification of novel genetic targets and biomarkers. This pathway approach is the ideal complement to more traditional, hypothesis-based testing of candidate genes, and when linked to genome-wide approaches, is anticipated to fuel additional analysis of new candidates.