Abstract Body (Do not enter title and authors here): Introduction: Vascular inflammation is a major component in the pathogenesis of pulmonary arterial hypertension (PAH). Inflammatory mediators initiate a pro-inflammatory state in capillary-resident pericytes resulting in dysfunctional endothelial-pericyte interactions, reduced pericyte coverage, and microvessel loss in severe PAH. However, molecular mechanisms driving excessive proinflammatory responses in pericytes remain elusive.
Hypothesis: Lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (LITAF) perpetuates pro-inflammatory state in pericytes that consequently exacerbate tissue inflammation, reduces pericyte coverage, compromises vascular integrity leading to loss of microvasculature in PAH.
Methods: Transcriptomic and mechanistic studies were conducted on isolated human pericytes from healthy donors and patients with PAH. Impact of LITAF ablation and overexpression on the secretion of proinflammatory mediators was assessed using cytokine arrays, and protein interactions using mass-spectrometry. Pericyte-specific Litaf deletion was generated using NG2-CreERT2 and Litaf^flox/flox transgenic mouse lines.
Results: LITAF transcription and protein levels are significantly elevated in PAH pericytes, which correlated with the increased levels of cytokines including TNFα and monocyte chemoattractant protein-1 (MCP1) as compared to healthy pericytes. Comparatively, PAH pericytes exhibited rapid stabilization and increased nuclear translocation of LITAF and MCP1 upon exposure to cytokines (TNFα, IL1β, IL6). LITAF proteins also colocalized to late endosomes and lysosomes. Secretome analysis from PAH pericytes confirmed increased LITAF and MCP1 secretion, and coregulation of ALG-2-Interacting Protein-X (ALIX), which is a bona fide marker of exosomes. Our observations confirm the positive feedback loop between TNFα signalling in the transcriptional activation of LITAF promoter, and the function of LITAF as a proinflammatory transcription factor in the regulation of TNFA transcription. Pericyte-specific LITAF deletion protects against development of chronic hypoxia-induced PH via reduction in the serum levels of proinflammatory cytokines, inflammatory cell infiltration, capillary loss, and vascular remodeling in vivo.
Conclusions: LITAF activation mediates pro-inflammatory state in pericytes via regulation of endolysosomal and secretory pathways that amplifies pro-inflammatory mediator release, endothelial dysfunction, and vascular inflammation in PAH.