Abstract Rationale Pulmonary hypertension (PH) is a fatal disease characterized by progressive pulmonary vascular remodeling, leading to increased pulmonary arterial pressures and right ventricular dysfunction. Although the lymphatic system plays an essential role in fluid clearance and immune homeostasis, the lung lymphatics have not been previously studied in experimental models of PH. This study investigated the contribution of lymphangiogenesis to the severity of PH phenotypes and the mechanistic drivers of lymphatic function in hypoxia-induced PH. Methods Prox1-CreER::Rosa26-tdT mice were generated to specifically label lymphatic endothelial cells (ECs) with tdTomato and exposed to hypoxia (Hx, 10% oxygen) for up to 6 weeks. Subsequently, Hx-exposed mice were treated with the selective Vegfr3 inhibitor MAZ51 to block lymphangiogenesis. Endpoints included functional studies of right ventricular systolic pressure (RVSP), clearance of FITC-labeled Dextran, and immunofluorescent (IF) staining of whole lung mounts and precision cut lung slices. Single cell RNA sequencing (scRNAseq) datasets from mouse Hx and human PH were analyzed. Mechanistic studies were performed in human dermal lymphatic endothelial cells (dLECs), including CD74 overexpression, trans-endothelial electrical resistance to quantify barrier integrity, and immunocytochemistry for CD74 and VEGFR3. Results In normoxia, lymphatic capillaries in mice were distributed throughout the lung parenchyma and spiraled around veins and venules. Hx induced robust proliferation of the lymphatic vasculature, yielding more dilated and more branched lymphatics that clustered around abnormally muscularized pulmonary arteries. Vegfr3 inhibition with MAZ51 blunted the lymphangiogenic response to Hx, which was associated with worsened RVSP and impaired lymphatic function, as measured by FITC-Dextran clearance. scRNAseq analysis identified CD74 as a significantly upregulated gene in lymphatic ECs in both mouse Hx and human idiopathic pulmonary arterial hypertension (IPAH) datasets. IF staining confirmed upregulation of CD74/Cd74 in lung tissue from IPAH patients and from hypoxic mice. In cultured human dLECs, CD74 overexpression reduced barrier integrity and increased monolayer permeability. Overexpression and activation of CD74 enhanced VEGFR3 expression. Conclusions CD74 is a novel mediator of lymphatic endothelial function as its activity impairs permeability, dysregulates VEGFR3 expression, and is associated with lymphatic remodeling in Hx-induced PH and human IPAH. This study provides new insights into the role of pulmonary lymphatics in experimental and human PH. Modulation of CD74 may alter lymphatic function and serve as a potential therapeutic in PH. This abstract is funded by: NIH NHLBI 5R01HL150106, 1R01HL171405, ATS/PHA Aldrighetti Research Award for Young Investigators, Bayer PHAB awards, PHA Innovation Research Award (to KY)
Moss et al. (Fri,) studied this question.