Genome wide expression analysis suggests perturbation of vascular homeostasis during high altitude pulmonary edema

Abstract

Background: High altitude pulmonary edema (HAPE) is a life-threatening form of non-cardiogenic edema which occurs in unacclimatized but otherwise normal individuals within two to four days after rapid ascent to altitude beyond 3000 m. The precise pathoetiology and inciting mechanisms regulating HAPE remain unclear.

Methodology/principle findings: We performed global gene expression profiling in individuals with established HAPE compared to acclimatized individuals. Our data suggests concurrent modulation of multiple pathways which regulate vascular homeostasis and consequently lung fluid dynamics. These pathways included those which regulate vasoconstriction through smooth muscle contraction, cellular actin cytoskeleton rearrangements and endothelial permeability/dysfunction. Some notable genes within these pathways included MYLK; rho family members ARGEF11, ARHGAP24; cell adhesion molecules such as CLDN6, CLDN23, PXN and VCAM1 besides other signaling intermediates. Further, several important regulators of systemic/pulmonary hypertension including ADRA1D, ECE1, and EDNRA were upregulated in HAPE. We also observed significant upregulation of genes involved in paracrine signaling through chemokines and lymphocyte activation pathways during HAPE represented by transcripts of TNF, JAK2, MAP2K2, MAP2K7, MAPK10, PLCB1, ARAF, SOS1, PAK3 and RELA amongst others. Perturbation of such pathways can potentially skew vascular homeostatic equilibrium towards altered vascular permeability. Additionally, differential regulation of hypoxia-sensing, hypoxia-response and OXPHOS pathway genes in individuals with HAPE were also observed.

Conclusions/significance: Our data reveals specific components of the complex molecular circuitry underlying HAPE. We show concurrent perturbation of multiple pathways regulating vascular homeostasis and suggest multi-genic nature of regulation of HAPE.

Figure 1. Work flow of gene expression analysis.

The figure represents scheme of extraction of differentially expressed genes (up regulated: 420, down regulated: 308) from the raw microarray data.

Figure 2. Hierarchical clustering of gene expression data obtained from acclimatized controls (CN) and HAPE (PE) individuals.

Hierarchical clustering distinctly separated the two groups of individuals (CN and PE) indicating unique gene expression signatures. Expression values of specific genes are represented by color intensities shown in the reference color key.

Figure 3. Comparison of relative expression (log2 values) of selected genes obtained by microarray and real time PCR experiments (TLDA).

17 out of 20 genes show similarity of trend on both the platforms.

Figure 4. Clustering of GO terms related to biological processes.

GO terms were clustered utilizing software BINGO and individual clusters indicated as shown in the figure.

Figure 5. Intergenic Network.

The figure represents an integrated network of multiple pathways interacting through common gene products. Specific sub-networks (gene associations within individual pathways) constituting the integrated network are highlighted in Supplementary Figure S3 (A-R). The major sub-network pathways include regulation of actin cytoskeleton, calcium signaling, MAPK pathway, immune cell signaling, cell-cell communication, VEGF signaling amongst others. The network shown was extracted from the list of differentially expressed genes utilizing ‘Pathway Miner’. Relationships between the genes (nodes connected by edges) suggest co-occurrence in a biological pathway. Weight of a specific edge is a relative representation of the number of pathways in which the associating nodes (gene products) co-occur in the KEGG pathway resource. The nodes are labeled by gene names and colored based on the respective fold changes for specific genes in the original data set.

Figure 6. Schematic representation of possible events occurring during HAPE.

The pathways suggested by the current data set have been integrated with established phenomenon such as pulmonary vasoconstriction, elevated pulmonary artery pressure which are known to precede HAPE. Perturbation of pathways such as those regulating vasoconstriction, inflammation, gap junctions and adhesion molecules can dysregulate vascular homeostasis leading to fluid leak and edema formation.