Objective—: To determine whether pulmonary arterial hypertension is associated with endothelial cell (EC)–Cav-1 (caveolin-1) depletion, EC-derived extracellular vesicle cross talk with macrophages, and proliferation of Cav-1 depleted ECs via TGF-β (transforming growth factor-β) signaling. Approach and Results—: Pulmonary vascular disease was induced in Sprague-Dawley rats by exposure to a single injection of VEGFRII (vascular endothelial growth factor receptor II) antagonist SU5416 (Su) followed by hypoxia (Hx) plus normoxia (4 weeks each–HxSu model) and in WT (wild type; Tie2.Cre ⁻ ; Cav1 ^lox/lox ) and EC- Cav1 ^−/− (Tie2.Cre ⁺ ; Cav1 ^fl/fl ) mice (Hx: 4 weeks). We observed reduced lung Cav-1 expression in the HxSu rat model in association with increased Cav-1+ extracellular vesicle shedding into the circulation. Whereas WT mice exposed to hypoxia exhibited increased right ventricular systolic pressure and pulmonary microvascular thickening compared with the group maintained in normoxia, the remodeling was further increased in EC- Cav1 ^−/− mice indicating EC Cav-1 expression protects against hypoxia-induced pulmonary hypertension. Depletion of EC Cav-1 was associated with reduced BMPRII (bone morphogenetic protein receptor II) expression, increased macrophage-dependent TGF-β production, and activation of pSMAD2/3 signaling in the lung. In vitro, in the absence of Cav-1, eNOS (endothelial NO synthase) dysfunction was implicated in the mechanism of EC phenotype switching. Finally, reduced expression of EC Cav-1 in lung histological sections from human pulmonary arterial hypertension donors was associated with increased plasma concentration of Cav-1, extracellular vesicles, and TGF-β, indicating Cav-1 may be a plasma biomarker of vascular injury and key determinant of TGF-β–induced pulmonary vascular remodeling. Conclusions—: EC Cav-1 depletion occurs, in part, via Cav-1+ extracellular vesicle shedding into the circulation, which contributes to increased TGF-β signaling, EC proliferation, vascular remodeling, and pulmonary arterial hypertension.

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B₄ (LTB₄) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB₄ involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3^-/-, ASC^-/-, Casp-1/11^-/-, gp91^{phox-/- ,} and IL-1R^-/- mice treated with ATP or LTB₄ did not decrease parasitic load as was observed in WT mice. When ASC^-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R^-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11^-/- macrophages, neither ATP nor LTB₄ were able to reduce parasitic load, and Casp-11^-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7^-/- L. amazonensis-infected mice locally treated with exogenous LTB₄ showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7^-/- mice. A similar observation was noted when infected P2X7^-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7^-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB₄ was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. Leishmania spp. is a protozoan parasite that infects human and causes several diseases. Leishmania amazonensis causes cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Leishmania parasites preferentially infect macrophages. In macrophages, several mechanisms have been described as controlling L. amazonensis infection. Here, we showed that P2X7 receptor and LTB₄ eliminated L. amazonensis in macrophages by a pathway dependent on non-canonical NLRP3 inflammasome activation and IL-1β signaling.