Introduction:
Pulmonary arterial hypertension (PAH) is characterized by extensive vascular remodeling along the entire length of the arterial-to-microvascular vessel tree. Whilst hypertrophic remodeling in pulmonary arteries and arterioles in PAH has been extensively assessed, the mechanisms driving distal lung vessel loss and capillary rarefaction as important contributor to the changes in pulmonary hemodynamics remain scarcely studied and poorly understood. Recent studies point to the emerging role of pericytes (PC) in lung vascular remodeling in PAH. Here, we utilized a novel microvasculature-on-a-chip (MVOC) platform to assess the role of PC and their interaction with microvascular endothelial cells (MVEC) at high spatial and temporal resolution within a multicellular setting.
Methods:
MVEC and PC were isolated from healthy donors or PAH patients and suspended to a concentration of 4×107 cells/ml and 5.7×106 cells/ml, respectively. One volume of each cell type with a final ratio of 7:1 MVEC:PC and two volumes of fibrinogen were pipetted into the central chamber of the MVOC platform in different homo- or heterotypic combinations of healthy and diseased cells. Angiogenesis cocktail medium was added into the flow channels and exchanged daily. Dynamic vessel formation was monitored daily by brightfield microscopy. MVEC and PC were stained with PECAM-1 and neuron-glial antigen 2 (NG2), respectively. Vascular network morphology was assessed by AngioTool and ImageJ.
Results:
On the MVOC platforms, homotypic combinations of MVEC and PC from healthy donors spontaneously formed densely interconnected and multi-branched capillary networks with open lumina within 7 days. Unexpectedly, heterotypic networks generated by combinations of either healthy MVEC with PAH PC or PAH MVEC with healthy PC yielded equivalent results in morphological appearance as well as in quantitative assessment of total vessel length, number of junctions or endpoints. Homotypic networks of PAH MVEC and PAH PC, however, presented markedly sparser with reduced vessel length, branchings and blind endpoints as compared to all other combinations. Subsequent proximity analyses showed that in homotypic healthy or heterotypic networks, >75% of PC were adjacent to the capillary endothelium. In homotypic diseased networks, however, the number of PC located remotely from the capillary network more than doubled relative to all other groups.
Conclusion:
The present findings highlight the importance of crosstalk between MVEC and PC for the homeostasis of the human lung microvasculature. The formation and maintenance of physiologically interconnected networks as well as the process of PC attachment/detachment appear to be governed by both MVEC and PC factors. Remarkably, the interaction of MVEC and PC forms a physiological safeguard mechanism: in case of damage to one cell or cell type, the other healthy cell type is able to compensate to ensure vascular integrity. Yet, this system fails in systemic diseases affecting multiple vascular cell subsets such as in PAH. The restoration of a healthy phenotype for one of the involved microvascular cell subsets thus presents a possible target for future therapeutic strategies.
