Background.
Pulmonary arterial hypertension (PAH) is an unmet clinical need where the progressive narrowing and vasoconstriction of pulmonary arteries increase pulmonary arterial pressure. Loss-of-function of bone morphogenetic protein receptor 2 (BMPR2) increases susceptibility to PAH, which can be initiated by other factors, such as hypoxia and inflammation. Endothelial dysfunction characterised by abnormal apoptosis, proliferation, angiogenesis, inflammation, and endothelial to mesenchymal transition (EndMT) is widely considered to be a pathological trigger in the onset of PAH development but mechanisms involved in the vascular pathology are complex and not well understood.
Current pre-clinical models do not fully reproduce the arteriopathy observed in human PAH, significantly hampering identification and evaluation of new drug targets. Organ-on-chip technologies emerged in the last decade with huge potential to build new in vitro models more closely resembling the structure and function of human organs and tissues. These new 3D cell culture platforms open new avenues for modelling of human diseases and personalised medicine.

Hypothesis.
Organ-on-chip technologies can be used to study human pulmonary endothelial and smooth muscle cell responses to pathological triggers of PAH, to provide new mechanistic insights into the process of pulmonary vascular remodelling and allow evaluation of drug effects.

Methods.
A pulmonary artery-on-a-chip (PA-on-a-chip) containing two parallel channels separated by a porous flexible membrane, was produced by soft-lithography. Human pulmonary endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) were cultured on either side of the membrane to create an endothelial-smooth muscle cell interface. The organ-on-a-chip model of PAH, named the “two hit” model, was created by combining BMPR2-deficient HPAECs or late outgrowth endothelial progenitor cells (ECFCs) from patients with disabling BMPR2 mutations with healthy HPASMCs and culturing them under hypoxic conditions and under flow in a pulmonary artery-on-a-chip. Considering potential applications in drug screening, the model focused on acute cell responses, measurable within 24 hours of cell activation. Functional studies, involving evaluation of endothelial barrier function, endothelial and smooth muscle cell proliferation and changes in HPAEC and HPASMC transcriptomic profile, were studied. Following RNA sequencing, differentially expressed genes (DEGs) were subjected to pathway analysis and compared against known PAH datasets. The model was used to evaluate effects of novel and clinically approved drugs on HPASMC proliferation.

Results.
Following optimization of the design, device fabrication and cell culture conditions, HPAECs and HPASMCs cultured in PA-on-a-chip under flow conditions showed adaptational changes consistent with arterial phenotype in vivo. shRNA-mediated BMPR2 knockdown in HPAECs or BMPR2 mutations in PAH ECFCs did not affect endothelial barrier function or endothelial cell proliferation under normoxic or hypoxic conditions. A significant increase in HPASMC proliferation was observed under the “double hit” conditions, combining the endothelial loss of BMPR2 with hypoxia (p<0.01 in the HPAEC PAH model; p<0.05 in the PAH ECFC model), consistent with observations in animal models of PAH.
Transcriptomic analyses revealed genotype- and oxygenation-specific gene clustering and changes in key PAH pathways such as angiogenesis, apoptosis, and proliferation. Important similarities between HPAECs and ECFCs were also observed, including a novel link between BMPR2 and the recently identified PAH gene SOX17. The BMPR2-dependent regulation of SOX17 expression was confirmed in cultured HPAECs and knock-in BMPR2C118W mice. Compensatory overexpression of SOX17 in BMPR2-deficient HPAECs significantly reduced HPASMC proliferation (p<0.05). Administration of Imatinib and Ambrisentan reduced smooth muscle cell proliferation to control levels in the “two-hit” organ-on-a-chip HPAEC model. Novel pre-clinical compounds BRD4i and FLAPi also demonstrated anti-proliferative potential in this model.

Conclusions:
The “double hit” organ-on-a-chip PAH model, combining natural and induced endothelial BMPR2 dysfunction with hypoxia, captures vascular cell responses likely to reflect changes seen in early PAH. The model reinforces the view of an important role of pulmonary endothelium in the regulation of pulmonary vascular smooth muscle proliferation in response to triggers of PAH. The model can potentially be used as both an analytical tool in the investigation of disease mechanisms and in the evaluation of novel PAH therapeutics.