Hypoxia Induces Adaptive Lymphangiogenesis via Cd74 and Vegfr3 to Modulate Pulmonary Hypertension

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by excessive remodeling of the distal arterioles and arteries, driven by endothelial cell (EC) apoptosis and uncontrolled mural cell proliferation. Increasing evidence suggests immune dysregulation in PAH, but one crucial part of the immune system, the pulmonary lymphatics, has been largely overlooked. Patients with idiopathic PAH (IPAH) often develop abnormal tertiary lymphoid structures adjacent to remodeled arteries, yet the role of lymphatics in PAH pathogenesis and vascular remodeling remains unclear. METHODS: Mice with lymphatic EC-specific fluorescent label (Prox1-CreERT2::Rosa26-LSL-tdT) or lymphatic EC-specific deletion of Vegfr3 (vascular endothelial growth factor receptor 3; Prox1-CreERT2::Vegfr3 fl/fl ) were used. Pulmonary hypertension (PH) was induced in mice via exposure to hypoxia (10% FiO 2 ) for 1, 2, 3, or 6 weeks and in Sprague-Dawley rats via a single monocrotaline injection. MAZ51 was administered to inhibit Vegfr3. Right ventricular systolic pressure, right ventricular hypertrophy, and echocardiography were assessed. Immunofluorescent staining was performed in rodent and in IPAH lung sections for lymphatic EC analysis. Publicly available single-cell RNA sequencing data sets from human IPAH and mouse PH were reanalyzed to identify differentially expressed genes in lymphatic ECs. RESULTS: Pulmonary lymphatic vessels proliferated, branched, and dilated in response to hypoxia. Vegfr3 inhibition using MAZ51 or lymphatic EC-specific Vegfr3 deletion reduced hypoxia-induced lymphangiogenesis and was associated with worsened PH, right ventricular hypertrophy, and impaired drainage. Comparative single-cell RNA sequencing analysis revealed upregulation of CD74/Cd74 in lymphatic EC clusters from human IPAH and hypoxia-induced PH mouse lungs. In IPAH lymphatic ECs, increased CD74 expression was associated with increased FLT4 and activation of the VEGFR3-downstream MEK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) signaling pathways. Consistently, increased CD74 expression was found in lymphatic ECs from IPAH tissues, whereas CD74 overexpression in human lymphatic ECs enhanced VEGFR3 expression and was associated with impaired barrier permeability. CONCLUSIONS: These findings identify adaptive lymphangiogenesis as a protective response in experimental PH and reveal a previously unrecognized CD74-VEGFR3 signaling axis in lymphatic ECs. Targeting lymphatic dysfunction may represent a novel therapeutic strategy to improve outcomes in PAH.