Mechanisms of Hypoxia-Induced Pulmonary Arterial Stiffening in Mice Revealed by a Functional Genetics Assay of Structural, Functional, and Transcriptomic Data

Abstract

Hypoxia adversely affects the pulmonary circulation of mammals, including vasoconstriction leading to elevated pulmonary arterial pressures. The clinical importance of changes in the structure and function of the large, elastic pulmonary arteries is gaining increased attention, particularly regarding impact in multiple chronic cardiopulmonary conditions. We establish a multi-disciplinary workflow to understand better transcriptional, microstructural, and functional changes of the pulmonary artery in response to sustained hypoxia and how these changes inter-relate. We exposed adult male C57BL/6J mice to normoxic or hypoxic (FiO2 10%) conditions. Excised pulmonary arteries were profiled transcriptionally using single cell RNA sequencing, imaged with multiphoton microscopy to determine microstructural features under in vivo relevant multiaxial loading, and phenotyped biomechanically to quantify associated changes in material stiffness and vasoactive capacity. Pulmonary arteries of hypoxic mice exhibited an increased material stiffness that was likely due to collagen remodeling rather than excessive deposition (fibrosis), a change in smooth muscle cell phenotype reflected by decreased contractility and altered orientation aligning these cells in the same direction as the remodeled collagen fibers, endothelial proliferation likely representing endothelial-to-mesenchymal transitioning, and a network of cell-type specific transcriptomic changes that drove these changes. These many changes resulted in a system-level increase in pulmonary arterial pulse wave velocity, which may drive a positive feedback loop exacerbating all changes. These findings demonstrate the power of a multi-scale genetic-functional assay. They also highlight the need for systems-level analyses to determine which of the many changes are clinically significant and may be potential therapeutic targets.

Application and reduction of a nonlinear hyperelastic wall model capturing ex vivo relationships between fluid pressure, area, and wall thickness in normal and hypertensive murine left pulmonary arteries

“…Our analysis considers two hypertensive cases (HPH a and HPH b ). While one case (HPH b ) is based on the only known measurements of collagen fiber angles in a (right) pulmonary artery, 34 we note that their hypoxic exposure was for a much longer and more variable duration (3–6 weeks) across the mice. Our data is from the LPA and based on a less variable hypoxic exposure of 10 days per mouse (Section 2.4).…”

“…Since detailed histology for the murine LPA is not available, we use a recent literature value estimating a mean diagonal collagen fiber angle of 35.55° (measured from the axial direction) in the right pulmonary artery (RPA) of normoxic mice, 34 corresponding to a value of in our control animal model (CTL). In addition, from the same study, 34 we assume that the media occupies 63% of the vessel wall thickness in the stress‐free reference state for our CTL model.…”

“…Because our data set is based on 10‐days of hypoxic exposure, these values are calculated as a 30% perturbation in the direction of the hypertensive values reported in Ref. 34, where murine RPA was exposed to 3–6 weeks of hypoxic conditions; the values in Ref. 34 are a mean diagonal fiber angle of 28.45° (measured from the axial direction) and a media thickness percentage of 53%.…”

“…34, where murine RPA was exposed to 3–6 weeks of hypoxic conditions; the values in Ref. 34 are a mean diagonal fiber angle of 28.45° (measured from the axial direction) and a media thickness percentage of 53%. For both hypertensive cases that we consider, the opening angle value ( α = 94.2 27 ) is chosen to be the same as in the CTL model, based on the only known measurements of this quantity in a similar vessel and species after 10‐days of hypoxic conditions 26 .…”

Computation and visualization of cell–cell signaling topologies in single-cell systems data using Connectome

“…Here we describe a computational package in R called Connectome which facilitates each of these tasks . Connectome has been used to explore native signaling in human lung 9 and to identify aberrant signaling in pulmonary arterial hypertension (PAH) 10 , chronic obstructive pulmonary disease (COPD) 11 , and COVID-19 12 .…”