Abstract Background Protein communication is often driven by thermodynamic interactions involving direct contact or signaling intermediates. The 14-3-3 protein family exemplifies such regulatory signaling, coordinating communication between proteins across different cells. Although these proteins are highly conserved among species, their biological roles are not fully understood. In the lung microvasculature, endothelial cells (ECs) and pericytes interact to maintain vascular stability, and disruption of this communication contributes to pulmonary hypertension (PH), a progressive and deadly disease. Our findings reveal a previously unrecognized role for 14-3-3 proteins in PH, identifying them as a promising therapeutic target. Hypothesis We hypothesized that dysregulated endothelial 14-3-3 proteins drive a pathological proliferative phenotype in pericytes. We also examined whether inhibiting 14-3-3 signaling can reverse pulmonary hypertension. Methods We used NFU1 (G206C) mutant rats, which spontaneously develop pulmonary hypertension and vascular remodeling. Single-cell RNA sequencing was performed to profile lung cell populations and their interactions. Metabolic function was assessed using the Seahorse. The 14-3-3 interaction inhibitor R18 was used to block downstream signaling. Results ECs from NFU1 rats showed markedly increased expression and secretion of 14-3-3 proteins, consistent with ECs from human PH lungs. Confocal imaging demonstrated disrupted EC-pericyte associations in NFU1 rats, accompanied by a pericyte phenotype shift characterized by enhanced proliferation and migration. scRNA-seq analysis indicated that pericytes in NFU1 lungs acquired a smooth muscle-like state, with upregulation of markers such as calponin-3 and smooth muscle myosin heavy chain. CellChat analyses further confirmed reduced EC-pericyte communication in NFU1 lungs. Exposure of pericytes to 14-3-3 proteins in-vitro caused mitochondrial fragmentation, impaired oxidative phosphorylation, and increased glycolysis, aligning with a proliferative metabolic switch. These pericytes also displayed increased proliferation and invasion. In a 3D EC-pericyte co-culture system, 14-3-3 proteins suppressed endothelial sprouting, and showed decreased EC-pericyte association, indicating functional disruption of vascular organization. Therapeutic inhibition of 14-3-3 signaling with R18 peptide significantly reduced pulmonary vascular remodeling, lowered right ventricular systolic pressure, and improved pulmonary hemodynamics in NFU1 rats. Furthermore, scRNA-seq demonstrated restoration of EC-pericyte interactions and reversal of the pathological phenotype shift. Conclusions Dysregulated 14-3-3 signaling promotes pericyte proliferation and phenotypic transformation in pulmonary hypertension. Targeting 14-3-3 proteins represents a promising therapeutic approach to reverse vascular remodeling. This abstract is funded by: NIH
James et al. (Fri,) studied this question.