Identification of VEGFA-centric temporal hypoxia-responsive dynamic cardiopulmonary network biomarkers

Hypoxia, a pathophysiological condition, is profound in several cardiopulmonary diseases (CPD). Every individual's lethality to a hypoxia state differs in terms of hypoxia exposure time, dosage units and dependent on the individual's genetic makeup. Most of the proposed markers for CPD were generally aim to distinguish disease samples from normal samples. Although, as per the 2018 GOLD guidelines, clinically useful biomarkers for several cardio pulmonary disease patients in stable condition have yet to be identified. We attempt to address these key issues through the identification of Dynamic Network Biomarkers (DNB) to detect hypoxia induced early warning signals of CPD before the catastrophic deterioration.

The human microvascular endothelial tissues microarray datasets (GSE11341) of lung and cardiac expose to hypoxia (1% O₂) for 3, 24 and 48 h were retrieved from the public repository. The time dependent differentially expressed genes were subjected to tissue specificity and promoter analysis to filtrate the noise levels in the networks and to dissect the tissue specific hypoxia induced genes. These filtered out genes were used to construct the dynamic segmentation networks. The hypoxia induced dynamic differentially expressed genes were validated in the lung and heart tissues of male rats. These rats were exposed to hypobaric hypoxia (simulated altitude of 25,000 or PO₂ - 282 mm of Hg) progressively for 3, 24 and 48 h.

To identify the temporal key genes regulated in hypoxia, we ranked the dominant genes based on their consolidated topological features from tissue specific networks, time dependent networks and dynamic networks. Overall topological ranking described VEGFA as a single node dynamic hub and strongly communicated with tissue specific genes to carry forward their tissue specific information. We named this type of VEGFAcentric dynamic networks as "V-DNBs". As a proof of principle, our methodology helped us to identify the V-DNBs specific for lung and cardiac tissues namely V-DNB^L and V-DNB^C respectively.

Our experimental studies identified VEGFA, SLC2A3, ADM and ENO2 as the minimum and sufficient candidates of V-DNB^L. The dynamic expression patterns could be readily exploited to capture the pre disease state of hypoxia induced pulmonary vascular remodelling. Whereas in V-DNB^C the minimum and sufficient candidates are VEGFA, SCL2A3, ADM, NDRG1, ENO2 and BHLHE40. The time dependent single node expansion indicates V-DNB^C could also be the pre disease state pathological hallmark for hypoxia-associated cardiovascular remodelling. The network cross-talk and expression pattern between V-DNB^L and V-DNB^C are completely distinct. On the other hand, the great clinical advantage of V-DNBs for pre disease predictions, a set of samples during the healthy condition should suffice. Future clinical studies might further shed light on the predictive power of V-DNBs as prognostic and diagnostic biomarkers for CPD.