TdTomato Transgenic Reporter Rat Reveals Endothelial‐Specific Changes in Progression of PAH

Abstract

Background Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance and elevated pulmonary artery pressure, leading to right heart failure. Vasoconstriction and complex vascular remodeling contribute to progressive vasculopathy. The Sugen-hypoxia (SuHx) protocol generates arterial pathology in rats that recapitulates key features of PAH including vascular remodeling. Previously, a transgenic rat with endothelial cell-specific tdTomato expression was developed. This study aims to demonstrate the application of this transgenic rat as a novel tool for assessing the role of the pulmonary vascular endothelium in occlusive remodeling in PAH. Methods A cohort of CDH5-iCre/tdTomato reporter rats received an injection of the vascular endothelial growth factor receptor inhibitor Sugen5416, followed by 3 weeks of hypoxia, then 2-10 weeks of normoxia. Rats underwent echocardiography prior to tissue harvest, and the agarose/gel-infused lung preparation was used to evaluate tdTomato fluorescence, Hoechst nuclear dye, and autofluorescence. The pulmonary artery was cannulated through the right ventricle, and HBSS, NucBlue, then gelatin solution were perfused through the pulmonary circulation. Agarose solution was then infused through the trachea to inflate the lungs. Solidified lung tissue was cut into ~300 μm-thick serial lung slices for confocal imaging. Images were stitched together using the ImageJ Grid/Collection stitching plugin, generating up to 4.6 trillion-pixel high-content composite segments of tissue. Fulton Index measurements were obtained at 6 time points between weeks 5 and 13 of the SuHx protocol. Results Severe occlusive lesions were formed by week 5, consistent with the expected time course of SuHx in Sprague-Dawley rats. Plexiform lesions were observed by 9 weeks. TdTomato expression increased in grade II-IV lesions from 5 to 9 weeks, and abruptly decreased beyond 9 weeks. Echocardiography and Fulton Indices indicated right heart hypertrophy as PAH progressed from weeks 5 to 13. A peak RV/LV+S ratio of 0.68 was recorded at the 12-week time point, an approximate three-fold increase from normal values, before plateauing at 13 weeks. High-content images revealed marked differences in fluorescence intensity and visible lesion formation between early and late stage PAH. Conclusion Biphasic tdTomato endothelial fluorescence intensity at mid-late stages of PAH may indicate endothelial cell proliferation followed by cell death. Alternatively, time-dependent regulation of CDH5 may explain the time course of reporter expression, though further investigation is needed. In summary, the present study has demonstrated that the tdTomato transgenic reporter rat is a valuable tool for investigating endothelial cell dynamics, and high-content montage images offer a unique, comprehensive perspective of endothelial-specific remodeling over the progressive course of PAH.