Covalent targeting of IP receptor and PTEN mediates pyrrolizidine alkaloid-induced pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disorder characterized by obstructive pulmonary vascular remodeling and right ventricular dysfunction. Exposure to pyrrolizidine alkaloids (PAs), including monocrotaline (MCT), is a risk factor for PAH, yet the molecular targets of their reactive metabolite, dehydropyrrolizidine (DHP), remain incompletely defined. Using a rat model of MCT-induced PAH combined with untargeted proteomic profiling, covalent docking, targeted mass spectrometry, pharmacological intervention, and platelet-pulmonary arterial smooth muscle cell co-culture, we investigated DHP-mediated protein adduction in disease progression. Untargeted proteomic analysis identified multiple DHP-adducted proteins in platelets and lung tissues, with functional enrichment implicating platelet activation-related and proliferation-associated pathways. Targeted analyses demonstrated covalent modification of the platelet prostacyclin (IP) receptor at H237 in humans (H266 in rats), accompanied by reduced platelet cAMP levels and sustained platelet activation. In lung tissues, DHP covalently modified PTEN, including residue K125, consistent with disruption of its inhibitory control over PDGFR/PI3K signaling. In vivo, selexipag attenuated MCT-induced pulmonary vascular remodeling and reduced pulmonary PDGF-B accumulation and vascular cell proliferation. In parallel, dehydro-monocrotaline (the reactive form of MCT) stimulation of co-cultures containing platelets and rat pulmonary arterial smooth muscle cells (RPASMCs) increased P-selectin and PDGF levels, enhanced RPASMC proliferation, and activated PDGFR-β/PI3K/AKT signaling, whereas ACT-333679 attenuated these responses. Pharmacological upregulation of PTEN with rosiglitazone further suppressed PDGFR/PI3K signaling and pulmonary vascular remodeling. Collectively, these findings support a dual-target model in which DHP-mediated covalent modification of platelet IP receptor and pulmonary PTEN is associated with platelet dysregulation and proliferative vascular signaling in PA-induced PAH, and identify both pathways as potential therapeutic targets.