Investigation of the translocator protein (TSPO) as a novel target for pulmonary hypertension
File(s)
Author(s)
Sabrin, Farah
Type
Thesis
Abstract
Pulmonary arterial hypertension (PAH) is a fatal disease characterised by peripheral lung vessel remodelling due to the excessive proliferation of vascular cells and inflammation. Endothelial dysfunction is a major player in the development and progression of vascular pathology in PAH, contributing to an increase in mortality. The translocator protein (TSPO), a conserved mitochondrial outer membrane protein is highly expressed in inflammatory and endothelial cells (ECs). Upregulation of TSPO is associated with various cellular processes including proliferation, apoptosis, mitochondrial ROS generation and immunomodulation. Recent data has demonstrated an anti-inflammatory role for TSPO in EC activation through the regulation of mitochondrial function. My hypothesis is that aberrant expression of TSPO underlies PAH pathogenesis, and modulating TSPO by its appropriate ligands alleviates mitochondrial dysregulation and pulmonary vascular remodelling.
First, I conducted an immunohistochemical examination of lungs from idiopathic PAH (IPAH) patients and monocrotaline (MCT) pulmonary hypertension (PH) rats and demonstrated significantly increased TSPO expression in the remodelled pulmonary vasculature. Positron emission tomography (PET) imaging with an established TSPO radioligand ([11C]PBR28) was performed in MCT rats. Significantly increased PBR28 uptake was demonstrated in the MCT PH rat lungs in comparison to healthy controls, with a two-fold increase of the rate constant k3, which represents increased receptor binding. Pre-treatment with TSPO ligand XBD173 blocked the PBR28 signal, confirming radioligand specificity.
I next evaluated the efficacy of XBD173 in MCT and Sugen hypoxia (SuHx) PH rat models. In both models, XBD173 treatment attenuated the increase in pulmonary arterial pressure, right ventricular hypertrophy and pulmonary vascular remodelling. XBD173 treatment also decreased perivascular macrophage infiltration in MCT rats and alleviated peripheral small pulmonary vessel occlusion in SuHx rats. PET imaging with [18F]FDG showed that XBD173 treatment significantly attenuated lung glucose uptake, a measure of proliferative state. Cardiac magnetic resonance (CMR) demonstrated improved cardiac function with XBD173 treatment.
I investigated the role of TSPO in protecting ECs from pro-inflammatory activation under hypoxia and TNF-α stimulation. TSPO ligands including XBD173 attenuated chemical hypoxia induced by dimethyloxalylglycine (DMOG) and associated pulmonary EC apoptosis as measured by Annexin V flow cytometry and WST-1 assays. Consistent with previous reports, TNF-α induced ECs mitochondrial dysfunction is evidenced by the increased reactive oxygenase species (ROS) levels and decreased mitochondrial membrane potential (ΔΨm). Both TSPO ligand XBD173 and tspo siRNA treatments reduced TNF-α induced ROS production and maintained mitochondrial ΔΨm. Importantly, I demonstrated that TSPO engages the DNA-mediated cGAS-STING pathway in pulmonary ECs; XBD173 and tspo siRNA decreased TNF-α induced phosphorylation of IRF-3 and TBK1, key downstream molecules of cGAS-STING activation. These data are consistent with the subsequent downregulation of transcription of type 1 interferon and pro-inflammatory cytokines, leading to reduced release of cytokine and chemokines such as E-selectin, ICAM-1 and VCAM-1 from pulmonary ECs. In addition, I have also demonstrated that XBD173 exhibited anti-proliferative effects in PDGF, and hypoxia-stimulated pulmonary smooth muscle cells (SMCs).
Collectively, my studies provide the basis for further clinical investigation of TSPO as a potential biomarker and therapeutic target in PH patients.
First, I conducted an immunohistochemical examination of lungs from idiopathic PAH (IPAH) patients and monocrotaline (MCT) pulmonary hypertension (PH) rats and demonstrated significantly increased TSPO expression in the remodelled pulmonary vasculature. Positron emission tomography (PET) imaging with an established TSPO radioligand ([11C]PBR28) was performed in MCT rats. Significantly increased PBR28 uptake was demonstrated in the MCT PH rat lungs in comparison to healthy controls, with a two-fold increase of the rate constant k3, which represents increased receptor binding. Pre-treatment with TSPO ligand XBD173 blocked the PBR28 signal, confirming radioligand specificity.
I next evaluated the efficacy of XBD173 in MCT and Sugen hypoxia (SuHx) PH rat models. In both models, XBD173 treatment attenuated the increase in pulmonary arterial pressure, right ventricular hypertrophy and pulmonary vascular remodelling. XBD173 treatment also decreased perivascular macrophage infiltration in MCT rats and alleviated peripheral small pulmonary vessel occlusion in SuHx rats. PET imaging with [18F]FDG showed that XBD173 treatment significantly attenuated lung glucose uptake, a measure of proliferative state. Cardiac magnetic resonance (CMR) demonstrated improved cardiac function with XBD173 treatment.
I investigated the role of TSPO in protecting ECs from pro-inflammatory activation under hypoxia and TNF-α stimulation. TSPO ligands including XBD173 attenuated chemical hypoxia induced by dimethyloxalylglycine (DMOG) and associated pulmonary EC apoptosis as measured by Annexin V flow cytometry and WST-1 assays. Consistent with previous reports, TNF-α induced ECs mitochondrial dysfunction is evidenced by the increased reactive oxygenase species (ROS) levels and decreased mitochondrial membrane potential (ΔΨm). Both TSPO ligand XBD173 and tspo siRNA treatments reduced TNF-α induced ROS production and maintained mitochondrial ΔΨm. Importantly, I demonstrated that TSPO engages the DNA-mediated cGAS-STING pathway in pulmonary ECs; XBD173 and tspo siRNA decreased TNF-α induced phosphorylation of IRF-3 and TBK1, key downstream molecules of cGAS-STING activation. These data are consistent with the subsequent downregulation of transcription of type 1 interferon and pro-inflammatory cytokines, leading to reduced release of cytokine and chemokines such as E-selectin, ICAM-1 and VCAM-1 from pulmonary ECs. In addition, I have also demonstrated that XBD173 exhibited anti-proliferative effects in PDGF, and hypoxia-stimulated pulmonary smooth muscle cells (SMCs).
Collectively, my studies provide the basis for further clinical investigation of TSPO as a potential biomarker and therapeutic target in PH patients.
Version
Open Access
Date Issued
2022-08-28
Date Awarded
01/01/2023
License URL
Advisor
Zhao, Lan
Wilkins, Martin
Sponsor
Schlumberger Limited
Publisher Department
National Heart and Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)